key: cord-126419-u61qc8ey authors: qi, chong; karlsson, daniel; sallmen, karl; wyss, ramon title: model studies on the covid-19 pandemic in sweden date: 2020-04-03 journal: nan doi: nan sha: doc_id: 126419 cord_uid: u61qc8ey we study the increases of infections and deaths in sweden caused by covid-19 with several different models: firstly an analytical susceptible-infected (si) model and the standard susceptible-infected-recovered (sir) model. then within the sir framework we study the susceptible-infected-deceased (sid) correlations. all models reproduce well the number of infected cases and give similar predictions. what causes us deep concern is the large number of deaths projected by the si and sid models. our analysis shows that, irrespective of the possible uncertainty of our model prediction, the next few days can be critical for determining the future evolution of the death cases (updated april 02). the fast spread of covid-19 (nearly 1m infected cases till april 2nd, 2020) has caused wide concern. within the basic research community, quite a few mathematical and physical models have been proposed [1] [2] [3] [4] to study the evolution of the infected cases, aiming to make reliable predictions and to help the governments to make proper strategic preparedness and response plans. we deem it is of special importance to study the covid-19 spreading in sweden where, unlike other countries, the government is taking a rather relaxed strategy with no massive testing on suspected individuals and no strict lockdown in her most affected regions. we start by introducing the sir (susceptible-infectedrecovered) model which is widely used for virus spreading predictions [5, 6] . it consists of a system of three time-dependent variables: • infected cases (number of total infected individuals at given time), i(t). • susceptible cases (number of individuals susceptible of contracting the infection), s(t). • recovered cases (cumulative number of recovered individuals), r(t). one has in total n = s(t) + i(t) + r(t). above quantities satisfy the following non-linear differential equations where λ is the transmission rate, β the recovery rate. they can be parametrized using known infection data. it should be straightforward to extend the models to include deceased cases in the form dd dt = νi (2) and the exposed cases (individuals who are already infected but asymptomatic) if the recovery rate is very low during the pandemic time interval (as it is indeed the case for covid-19 upto now), we can well approximate the infected cases by with initial value i(0) = 1. for such si model, the solution can be derived analytically, this function is known as woods-saxon form in nuclear physics and is widely used describing nuclear potential and matter distribution. we can write it in a more general form by introducing one more parameter as for such si model, the solution can be derived analytically, where d describes the diffuseness. we apply above formula to study the reported cases in sweden as a function of time in fig. ii . we firstly assume that the death cases follow the same woods-saxon form where the three parameters can be fitted separately to reported data by taking into account the time interval between infection and death dates. the result is given in fig. 2 . we should emphasize that the uncertainty in the model is very large due to the limited data available. we were very optimistic when we first derived the curve from data back by two days which was very different with modest increases, as can be seen in fig. 3 . the above woods-saxon function seems to agree rather well with the data on reported covid-19 death cases from china where the pandemic period may be expected to be over. it may, however, not expected to work well within the late stage of the pandemic period when the total infected cases show a saturation behavior. therefore we insert eq. (6) to eq. (3) to see if we can get a better estimation on d. the integral of i(t) reads therefore, we can propose a second form for the evolution of d d(t) = a ln e t 0 −t d the result is given in fig. 4 where a modest increase is predicted. however, again, the data from the last two days show significant deviation from the predicted curve. new data from the next few days can be critical in pining down the uncertainty in the predicted behavior. we now include the recovery cases in above si model. there is no analytical solution but the evolution of the sir quantities can be done numerically. the result as of april 01 is given in fig. 5 what we can see from above simulation is that the recovery rate will remain very low during the expected pandemic time interval. instead we now include the reported death cases in above sir model by replacing the quantity r with d. the result as of april 01 is given in fig. 6 . the projected death cases are again very large and are more than 3000. our simulations show that all si, sir, sid models describe well the reported infected cases show rather modest increase in the near future which is very promising. however, the projected deceased cases in both si and sid models are extremely high even though the uncertainty is astonishingly large. we deem it urgent to explore the uncertainty of our model. the new data from the future few days can be critical for confining the predicted curve on decreased cases. if the model is correct, one should worry that the infected cases in sweden may be much much higher than it is reported today and a massive testing on exposed and suspicious cases may be urgent. estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries a contribution to the mathematical theory of epidemics the mathematical theory of infectious diseases and its applications key: cord-029410-m19od0wj authors: scatti-regàs, aina; aguilar-ferrer, marta carmen; antón-pagarolas, andrés; martínez-gómez, xavier; gonzález-peris, sebastià title: clinical features and origin of cases of parotiditis in an emergency department() date: 2020-07-19 journal: an pediatr (engl ed) doi: 10.1016/j.anpede.2019.11.007 sha: doc_id: 29410 cord_uid: m19od0wj nan to the editor: the mumps virus (muv), or myxovirus parotiditis, continues to cause sporadic cases and outbreaks of disease. this is associated to the progressive waning of immunity against the mumps component of the measles, mumps, rubella (mmr) vaccine in absence of a natural booster (especially from 10 years after administration of the second dose), the use in the 1993---1999 period of a vaccine that had the rubini strain, which proved to be less effective, and the presence of pockets of unvaccinated people in the population. 1 in spain, 10 260 cases were notified in 2017 and 8996 in 2018, a significant increase compared to previous seasons. 2 some of the infectious agents other than muv that may be involved in parotitis as a general clinical presentation include influenza a virus, parainfluenza virus, epstein-barr virus (ebv), adenovirus, coxsackievirus, cytomegalovirus (cmv), parvovirus b19, herpesvirus and lymphocytic choriomeningitis virus, as well as gram-positive bacteria, atypical mycobacteria and bartonella species. 3---5 in the paediatric population, these pathogens are probably more frequent causative agents compared to muv. this, combined with the benign course of most presentations, leads many paediatric health care facilities to make the diagnosis without an aetiological investigation. the aim of our study was to establish the viruses involved in cases of parotitis in our area. we carried out a retrospective study through the collection of data corresponding to 2 full years (2016 and 2017), including all patients given a diagnosis of parotitis (with swelling of the parotid glands being a requirement for inclusion) in the paediatric emergency department of a tertiary ଝ please cite this article as: scatti-regàs a., aguilar-ferrer m.c., antón-pagarolas a., martínez-gómez x., gonzález-peris s. caracterización clínica y etiológica de los casos de parotiditis en un servicio de urgencias. an pediatr (barc). 2019. https://doi.org/10. 1016/j.anpedi.2019.11.004 care hospital in barcelona that manages patients up to age 16 years and based on diagnostic judgment of the paediatrician in charge of the patient. per hospital protocol, polymerase chain reaction (pcr) tests for detection of muv in saliva and urine samples were performed in patients with parotitis. serologic tests were added if blood tests were requested by the paediatrician in charge based on his or her clinical judgment. when it came to serologic testing, in case of negative results of the test for detection of muv in saliva, molecular methods were used for detection of influenza a and b virus, respiratory syncytial virus a/b, adenovirus, metapneumovirus, coronavirus nl63/oc43/229e, enterovirus, rhinovirus, parainfluenza virus, ebv and cmv. mump viruses were characterised by partial sequencing of the small hydrophobic (sh) gene. we identified 169 cases of symptomatic acute parotitis (0.21% or paediatric emergency visits). the median age of the patients was 7.7 years (range, 11 months-16.8 years). the rate of adherence to the protocol for the ordering of tests for aetiological diagnosis was 79.3%, so we were able to obtain data on testing of saliva samples from 134 patients. fig. 1 summarises the results of pcr testing of these samples. another 5 patients received an aetiological diagnosis of parotitis due to muv by serologic testing (positive igm test), adding up to a total of 18 cases caused by muv. the median age of patients with muv infection (in all cases muv genotype g) was 14.3 years (range, 18 months-16.8 years), with a predominance of the male sex (72.2%). in 3 cases (16.7%) there was no known history of contact with a case of parotitis. all patients were correctly vaccinated save for 2 children that had not received any dose of mmr by parental choice and 1 adolescent that had only received 1 dose of vaccine. there were no documented complications, except for 1 patient that developed guillain-barré syndrome with onset the week after the initial visit, who had a favourable outcome. the management of 19.1% of the patients included empiric antibiotherapy despite there being no evidence confirming bacterial infection. table 1 presents the demographic and clinical characteristics of cases of parotitis in which testing was performed for investigation of the aetiology. patients with muv infection were significantly older compared to children with a different aetiological agent (median age, 14.3 vs 6.5 years; p = .005). the findings in our study, despite the limitations intrinsic to its retrospective design, were consistent with those of other authors, and showed that a significant proportion of cases of parotitis in the paediatric age group may be caused by viruses other than muv (such as ebv, cmv and common respiratory viruses). 3---5 the high frequency of cases with negative results in all tests can be explained by the involvement of other viruses that were not included in the testing (such as human herpesvirus 6), technical factors affecting the yield of microbiological diagnosis and the potential presence of non-infectious parotitis cases, among others. viral coinfection was also frequent. lastly, we ought to underscore that muv continues to be a frequent cause of parotitis in our area (especially in older children), even in correctly vaccinated patients, and our findings confirmed that the causative virus continues to circulate in the community with a well-known pattern characterised by incidence peaks every 3---5 years. the aetiological diagnosis and notification of cases can alert the health care authorities of potential outbreaks at an early stage, allowing implementation of containment measures such as administration of a third dose of vaccine in selected patients. 6 waning immunity against mumps in vaccinated young adults non-mumps viral parotitis during the 2014-2015 influenza season in the united states letter to the editor: there is a need to consider all respiratory viruses in suspected mumps cases viral etiology of mumps-like illnesses in suspected mumps cases reported in catalonia effectiveness of a third dose of mmr vaccine for mumps outbreak control hospital infantil vall d'hebron, barcelona, spain b unidad de virus respiratorios, servicio de microbiología, hospital vall d'hebron key: cord-006328-0tpj38vb authors: dass hazarika, rashna; deka, nayan mani; khyriem, a. b.; lyngdoh, w. v.; barman, himesh; duwarah, sourabh gohain; jain, pankaj; borthakur, dibakar title: invasive meningococcal infection: analysis of 110 cases from a tertiary care centre in north east india date: 2012-07-22 journal: indian j pediatr doi: 10.1007/s12098-012-0855-0 sha: doc_id: 6328 cord_uid: 0tpj38vb objectives: to report an outbreak of invasive meningococcal disease from meghalaya, in the north east india, from january 2008 through june 2009. methods: retrospective review of case sheets was done. one hundred ten patients with invasive meningococcal disease were included for the study. results: of the total patients, 61.8 % were boys and 38.2 % were girls (boy to girl ratio = 1.62:1). the average age of presentation was 8.48 ± 5.09 y. meningococcal meningitis was seen in 61.8 % of cases, meningococcemia in 20 % and 18.2 % had both. fever was the most common manifestation (100 %) followed by meningeal signs (78.2 %), headache (56.4 %), vomiting (53.6 %), shock (38.2 %), low glasgow coma scale (gcs) (25.5 %), purpura and rashes (23.6 %), seizures (9.1 %), abdominal symptoms (4.5 %), irritability and excessive crying (4.5 %) and bulging anterior fontanalle (23 %) in those below 18 mo of age. raised intracranial pressure (icp) was the most common complication (28.2 %) followed by coagulopathy (16.4 %), hepatopathy (10 %), herpes labialis (9.1 %), syndrome of inappropriate adh secretion (siadh) (8 %), pneumonia (7 %), arthritis (6 %), purpura fulminans, respiratory failure, sixth nerve palsy and diabetes insipidus in 4.5 % each, subdural empyema, optic neuritis, ards and arf in 1.8 % each, cerebral salt wasting syndrome, third nerve palsy, cerebritis and hearing impairment in 0.9 % each. culture was positive in 35.5 %. patients were treated initially with ceftriaxone and dexamethasone but later on with chloramphenicol due to clinical drug resistance. mortality was 6.4 %. conclusions: this is the first epidemic report of invasive meningococcal disease from the north east india. chloramphenicol acts well in areas with penicillin or cephalosporin resistance. mortality reduces significantly with early diagnosis and prompt intervention. meningococcal disease is a global problem. it has a rapid onset with varied presentations and wide regional variation in disease pattern. the endemic disease is rare but epidemic form occurs commonly in many regions of the world especially described in the 'meningitis belt' in sub-saharan africa, parts of asia and also in india. meningococcal disease mostly affects children in the school going age and adults working in close contact such as in military barracks. the disease requires early and prompt antibiotic treatment and supportive therapy. the outcome of the disease depends on the time required to seek medical help i.e., the 'house to hospital time' and also on the rapidity of administration of the first antibiotic dose i.e., the 'door to needle time'. meghalaya situated at an altitude of 1,961 m above sea level has a predominantly rural tribal population. an epidemic of meningococcal disease occurred in this region during [2008] [2009] . the present study documents the occurrence of the disease in this part of the world, and also highlights the various clinical manifestations, laboratory findings and management outcome. this descriptive retrospective study over the period of the epidemic, january 2008 through june 2009 is being reported from the department of pediatric disciplines, nei-grihms, shillong. one hundred ten children diagnosed as either 'meningococcemia' or 'meningococcal meningitis' or 'meningococcemia with meningitis' during the study period were identified from discharge summaries and inpatient records. their charts were retrieved and reviewed thoroughly. at admission, blood and cerebrospinal fluid (csf) were sent to the laboratory immediately for culture and sensitivity testing, cytology and gram staining. complete blood count (cbc) and peripheral blood smear for malarial parasite, random blood sugar (rbs), liver function test (lft), coagulation profile, renal function test (rft), serum electrolytes and chest x-ray (cxr) were done on the day of admission in all patients and repeated periodically if necessary. mri brain was done when clinically indicated. the cases of meningococcal meningitis and meningococcemia in the present case series were labelled as probable meningococcal meningitis, confirmed meningococcal meningitis, probable meningococcaemia and confirmed meningococcaemia as per standard guidelines [1] . patients were treated for the first 6 mo with injection ceftriaxone but later with parenteral chloramphenicol due to observation of clinical drug resistance in the form of delayed or no response to ceftriaxone in 48-72 h. antibiotics were administered for a minimum of 7 d in all patients along with supportive care and monitoring. injection dexamethasone was used in all cases with meningitis for 2 d. shock was treated with normal saline and inotropes (dopamine and dobutamine), whenever indicated and hydrocortisone. a total of 110 children were diagnosed as having either 'meningococcemia' or 'meningococcal meningitis' or 'meningococcemia with meningitis'. the demographic profile and clinical presentations are outlined in table 1 . among these cases, 61.8 % were boys and 38.2 % were girls (boys:girls01.62:1). the mean age of presentation was 8.48 ± 5.09 y (4 mo-18 y). fever was the most common symptom (100 %) followed by headache (56.4 %), vomiting (53.6 %), altered sensorium (25.5 %), purpura and rashes (23.6 %), seizures (9.1 %), abdominal symptoms (4.5 %), irritability and excessive crying (4.5 %). meningeal signs were present in 86 cases (78.2 %) and bulging anterior fontanalle in 3 out of 13 cases (23 %) below the age of 18 mo. shock was seen in 42 cases (38.2 %) (29 compensated and 13 decompensated). the average number of isotonic saline boluses required was 40 ml/kg (range: 20 ml/kg to 100 ml/kg). fifteen cases (13.6 %) required inotropic support and hydrocortisone singly or in combination. the average duration of inotropic support was 24-72 h. the laboratory investigations of all the cases are summarized in table1. culture (either blood or csf) was positive in 39 cases (35.5 %) (csf: 29, blood: 13). in three cases (2.7 %), growth was seen both in the blood and csf. gram negative diplococci in csf was seen in 27 cases; of which 8 cases were culture negative. all cases were identified as serogroup a and were susceptible to ceftriaxone and chloramphenicol by in-vitro antimicrobial testing. the mean blood leukocyte count was 16,071±14525/cumm. the csf cell count ranged from 5 to 60,000/cu mm and hypoglycorrhacia were seen in 67.3 % of the cases. ten percent of the cases had deranged lft and 16.4 % had coagulopathy. majority of the cases were seen in the months of december 2008 and january to march 2009 (fig. 1) . sixty nine percent of the cases were seen in children above 5 y of age (fig. 2) . meningococcal meningitis and meningococcemia were diagnosed in 68 cases (61.8 %) and 22 cases (20 %) respectively with a corresponding mortality of 2.9 % (2/68) and 18.2 % (4/22). twenty children (18.2 %) presented with both meningococcemia and meningitis with 1 death. there was no difference in mortality or morbidity between the culture positive or culture negative cases. of the 68 children with meningococcal meningitis, 44 had probable meningitis while 24 were confirmed. of the 22 children with meningococcemia, 15 had probable meningococcemia while 7 were confirmed. of the 20 children with meningococcemia and meningitis, 12 were probable while 8 were confirmed (fig. 3) . the important complications have been summarized in table 2 . raised icp was the most common (28.2 %) and was diagnosed clinically by the presence of bulging anterior fontanelle, bradycardia/tachycardia, papilledema and hypertension. herpes labialis was observed in 9.1 % of cases. three important metabolic complications of meningococcal infection observed in the present case series were siadh (8 cases, 7.3 %), diabetes insipidus (5 cases, 4.5 %) and cerebral salt wasting syndrome (1 case, 0.9 %). all the cases with diabetes insipidus and cerebral salt wasting syndrome expired. meningococcal purpura fulminans were seen in 5 cases (4.5 %) whereas 6 cases (5.5 %) developed arthritis, and 2 cases each had subdural empyema and optic neuritis. mortality was 6.4 %. epidemic meningococcal disease was first described by vieusseaux in 1805 from switzerland [2] . meningococcal infections are commonly found in developing countries such as in the african meningitis belt and occasionally in developed countries like the united states. serogroup a is more prevalent in developing countries whereas, in the developed countries the disease is mostly caused by serogroup b and c [3] . in india, meningococcal disease is endemic in delhi with sporadic cases reported in the past [1] . isolated cases of meningococcal meningitis were also reported from several states of india involving haryana, uttar pradesh, rajasthan, sikkim, gujarat, jammu & kashmir, west bengal, chandigarh, kerala and orissa in 1985 [4] . most of these outbreaks have been caused by serogroup a [5] . n. meningitidis was the dominant pathogen isolated in surat between 1985 and 87 [6] . in early 2005, spurt of cases of neiserria meningococcemia and meningitis due to serogroup a have been reported from delhi and adjoining areas [7] . no previous reports exist from north east india. approximately 69 % were above 5 y of age. maximum cases reported were below 1-2 y of age from usa for endemic disease [8] . in epidemic outbreaks a shift to higher age occurs [9] . in sudan, 58 % were above 5 y in a group a -n. meningitidis outbreak [10] . in ghana however the peak incidence was found in 10-14 y old children [11] . neonatal meningococcal meningitis is rare and there was no case of neonatal meningitis in the present study. meningococcal infection is characteristically fulminant presenting with fever, severe headache, vomiting, neck stiffness, positive meningeal signs, photophobia, drowsiness and confusion. deterioration and death can occur in hours. the disease spectrum usually ranges from meningococcal meningitis to meningococcemia. meningitis may or may not be present with rash. seizures occur in 40 % of cases. meningococcemia is more abrupt presenting with chills, nausea, vomiting, myalgias and the classical purpuric or petechial rash with or without bullae formation. absence of meningitis is a poor prognostic factor. septicaemia was found in 20 % cases. urmila et al. from delhi reported that 67 % children had meningococcal meningitis, 20 % had meningococcemia and 13 % had both with mortality of 4.5 %, 25 % and 69 %, respectively [12] . this is similar to findings in the present study. shock was the presenting symptom in 38 % of the index cases. of these, 69 % had compensated and 31 % had decompensated shock compared to 26 % in other reports [12] . shock is endotoxin mediated and due to factors such as widespread capillary leak, loss of vasomotor tone and maldistribution of intravascular volume, impaired myocardial function and impaired cellular function. early recognition of shock is crucial for early intervention and improved outcome [13] . tachycardia may be the only sign present in the early phase of the disease and is enough to mandate fluid resuscitation. circulatory management aims to maintain tissue perfusion and oxygenation. repeated fluid boluses with 20 ml/kg of isotonic saline are to be given initially till shock resolves. in case shock persists after 60 ml/kg of fluid, central venous pressure (cvp) line is inserted and fluid resuscitation continued with addition of dopamine and/or dobutamine. some children require as high as 100-200 ml/kg of fluid resuscitation but such patients also require mechanical ventilation. about 13.6 % of the index cases required inotropic support either alone or in combination for an average duration of 24-72 h. some studies have shown that 4.5 % albumin is more useful as a resuscitating fluid [14] . albumin is routinely used in the uk with significant reduction in mortality in the last 20 y (decrease up to 2 %) in patients with meningococcal disease and albumin use may play a role along with other factors [15] . the authors do not have any personal experience of using albumin. survival rate reaches 94 % when shock is reversed within 75 min of presentation [13] . rash was observed in 23.6 %, while this sign ranged from 7.3 % to 100 % in other studies [16, 17] . meningococcal purpura fulminans is a hemorrhagic condition associated with meningococcal septicemia with features of hypotension, disseminated intravascular coagulation (dic), and purpura leading to tissue necrosis and small vessel thrombosis. in the present study, 5 cases (4.5 %) presented with purpura fulminans and of them 3 died. schaad ub [18] has described arthritis in 10 % of patients with meningococcal disease. in the present study, 5.5 % presented with arthritis involving big joints. arthritis may occur early in the disease due to direct bacterial seeding of the joints or in the sub-acute or convalescent phase of the illness secondary to immune-complex reactions. treatment of bacterial arthritis consists of analgesics, antibiotics and drainage of joint fluid if needed. immune complex reactions are usually treated with non-steroidal anti-inflammatory drugs or steroids. some may require intravenous immunoglobin [19] . reactivation of latent herpes simplex virus infections (primarily herpes labialis) is common during meningococcal infection as observed in the present study with good response to local acyclovir. coagulopathy is frequent and multifactorial, and was seen in 16.4 % of the present cases. mild clotting abnormalities are well tolerated. in severe cases fresh frozen plasma (ffp) is recommended. the authors have used intravenous vitamin k and if required ffp with good results. currently the best treatment for meningococcal related coagulopathy is the optimal management of shock. dodge and swartz [20] reported seizures in 10 % in the acute stage of the disease, focal cerebral signs in 10 %, and 15 of 39 patients had cranial nerve involvement early in the course of disease. in the present study, 9.1 % of the cases presented with seizures in the acute stage and cranial nerve involvement was present in 7.3 % cases. siadh was detected in 4 of 39 patients by dodge and swartz [20] . in the present study, siadh was found in 8 cases (7.3 %) and was managed with fluid restriction and low dose diuretic (furosemide) therapy. five cases (4.5 %) had diabetes insipidus (di), requiring aggressive management with hypotonic fluids, vasopressin and mechanical ventilation and one had cerebral salt wasting (csw). all the index patients with di and csw had 100 % mortality. although pollard rb [21] has reported that deafness has not been a common complication of meningococcal meningitis in the antibiotic era, there was one case with bilateral sensorineural hearing defect in the present study. pneumonia, epiglotitis and otitis media can occur. pneumonia is seen in 5 to 15 % of invasive meningococcal disease cases, particularly with serogroups y and w-135 [22] . in the present study, pneumonia was present in 6.4 % cases. recovery may be complicated by ards, anuria and multi organ failure. in some cases ards develops within a few hours after admission and in the present study 2 cases each developed ards and arf. in a study from punjab (ludhiana), 56.5 % were culture positive and all isolates were sensitive to most of the common antibiotics [23] . urmila j et al reported 26 positive cultures (13/98 blood cultures and 13/89 csf cultures) [12] . low rate of culture positivity in the present study (35.5 %) may be due to prior use of antibiotics outside or delay in transporting the specimen. antibiotic therapy remains the cornerstone of therapy in meningococcal disease. three factors that influence the success of antibiotic therapy are timing of the antibiotic, tissue penetration and antibiotic resistance. broad spectrum antibiotics like penicillin g, ceftriaxone and cefotaxime remain widely used. increasing resistance to penicillin is being reported and ceftriaxone remains the recommended first line therapy in the present scenario. however in the authors' experience they had patients with good response to ceftriaxone in the beginning of the epidemic. after about 6 mo of the epidemic, there was poor clinical response to ceftriaxone and the unit antibiotic policy was revised to intravenous chloramphenicol for 7 d with good response. they now routinely use parenteral chloramphenicol as the first line therapy in meningococcal disease. there are other reports of ciprofloxacin as well as ceftriaxone resistance from india [24, 25] . the second line therapy consists of vancomycin and azithromycin. the nice guidelines recommend dexamethasone therapy for suspected or confirmed bacterial meningitis above 3 mo of age [26] . the authors used injection dexamethasone in all meningococcal meningitis cases for 2 d. steroids are not indicated in meningococcal shock unless there is suspicion of hypoadrenalism. overall fatality rate of invasive meningococcal infection is 5-16 %, although these rates are difficult to assess as some studies only take into account meningococcal meningitis, while others reflect overall fatality from meningococcal disease [27, 28] . reported mortality from meningococcemia ranges from 18 % to 35 % [29] . for overall invasive meningococcal infection, the fatality rate in the present study was low (6.4 %). for meningococcemia, fatality rate in the present study was 18.2 % which is similar to other studies [29] . low mortality in the present study can be explained by the fact that patients reached the hospital fast due to good information, education and communication activities by the local health authorities, combined with a low threshold for diagnosis and aggressive management of shock, rapidity of administration of the first antibiotic dose (door to needle time) and continuous monitoring in a well equipped pediatric intensive care unit. this is the first epidemic report of invasive meningococcal disease from north east india. although the majority of patients had meningitis, the full range of manifestations were also seen. this study highlights that clinical resistance to commonly used antibiotics such as ceftriaxone can be seen where chloramphenicol is an alternative effective choice. mortality reduces significantly with early diagnosis and prompt interventions like early shock management, antibiotic therapy and frequent monitoring in an intensive care set up. although invasive meningococcal infection did not have much impact on the morbidity and mortality of children from this region compared to other parts of the world, it remains one of the major causes of life threatening infections requiring continuous vigilance. contributions rd conceived the idea of the study and approved the final manuscript and will act as guarantee of the paper; nmd, hb, sgd, pj and db were involved in data retrieval, analysis and writing of the paper; abk and wvl were involved in the laboratory diagnosis and analysis of the microbiological data. meningococcal disease, need to remain alert. cd alert mémoire sur la maladie qui a regné a genêve au printemps de 1804 meningococcal meningitis outbreak control strategies meningococcal meningitis in delhi and other areas group b meningococcal meningitis in india meningococcal meningitis in an industrial area adjoining surat citysome clinic-epidemiological aspects meningococcal disease: history, epidemiology, pathogenesis, clinical manifestations, diagnosis, antimicrobial susceptibility and prevention multicenter surveillance of invasive meningococcal infections in children update on meningococcal disease with emphasis on pathogenesis and clinical management clinical features and complications of epidemic group a meningococcal disease in sudanese children meningococcal meningitis in northern ghana: epidemiology and control measures clinical profile of group a meningococcal outbreak in delhi early reversal of pediatric-neonatal septic shock by community physicians is associated with improved outcome albumin: saint or sinner treatment of meningococcal infection meningococcal disease among children who live in large metropolitan area review of management of purpura fulminans and two case reports arthritis in disease due to neisseria meningitides immune complex reaction after successful treatment of meningococcal disease: an excellent response to ivig bacterial meningitis-a review of selected aspects. ii. special neurologic problems, post meningitic complications and clinicopathological correlations early bilateral eight nerve involvement in meningoccal meningitis connecticut, and selected areas meningococcal meningitis in ludhiana emergence of non-ceftriaxonesusceptible neisseria meningitidis in india ciprofloxacin-resistant neisseria meningitidis management of bacterial meningitis and meningococcal septicaemia in children and young people: summary of nice guidance epidemiology of bacterial meningitis meningococcal infection in children: a review of 100 cases prognostic factors in acute meningococcaemia role of funding source none. key: cord-144860-a4i9vnjz authors: nason, guy p. title: rapidly evaluating lockdown strategies using spectral analysis: the cycles behind new daily covid-19 cases and what happens after lockdown date: 2020-04-16 journal: nan doi: nan sha: doc_id: 144860 cord_uid: a4i9vnjz spectral analysis characterises oscillatory time series behaviours such as cycles, but accurate estimation requires reasonable numbers of observations. current covid-19 time series for many countries are short: preand post-lockdown series are shorter still. accurate estimation of potentially interesting cycles within such series seems beyond reach. we solve the problem of obtaining accurate estimates from short time series by using recent bayesian spectral fusion methods. here we show that transformed new daily covid-19 cases for many countries generally contain three cycles operating at wavelengths of around 2.7, 4.1 and 6.7 days (weekly). we show that the shorter cycles are suppressed after lockdown. the preand post lockdown differences suggest that the weekly effect is at least partly due to non-epidemic factors, whereas the two shorter cycles seem intrinsic to the epidemic. unconstrained, new cases grow exponentially, but the internal cyclic structure causes periodic falls in cases. this suggests that lockdown success might only be indicated by four or more daily falls in cases. spectral learning for epidemic time series contributes to the understanding of the epidemic process, helping evaluate interventions and assists with forecasting. spectral fusion is a general technique that is able to fuse spectra recorded at different sampling rates, which can be applied to a wide range of time series from many disciplines. what the impact of measures that came in on march 23 will be". the measures that professor mclean referred to were the widespread uk social distancing and lockdown interventions made in the face of the covid-19 threat. at the time of writing, few countries have experienced in excess of 70 days of covid-19 cases and most only have around 50 days. professor mclean is correct in that many scientific inferences require longer time series than those currently available. however, we show that there are considerable and useful similarities in the underlying cyclic (spectral) behaviours of the numbers of new daily covid-19 cases for a range of different countries (see extended data figures). we use recent bayesian spectral fusion methods [3] (regspec) to pool spectral information across countries, which provides significantly more accurate estimates of cyclic behaviour than provided by a typical spectral analysis of a single country alone. the bayesian principles underlying our fusion method handle mean that uncertainty is treated coherently, producing rational uncertainy assessment for our cycle (spectral) estimates. our methods produce cycle estimates using the equivalent of over nine hundred daily observations, compared to the fifty or so that a typical standard spectral analysis might use. using data [2] from all of the countries we considered, our results show that transformed new daily covid-19 cases have three underlying cycles: one operating at a wavelength of 2.7 days, a second at 4.1 days and a third at 6.7 days, which we take to be a weekly effect. we conducted separate analyses for the uk and groups of countries with similar spectra and note some variation in those cycles. for some purposes it is not reasonable to compare or pool the number of new daily cases from one country to another [14] . for example, different countries might use different definitions of the number of daily cases and they record cases through different national structures and this is even the case for countries with political, geographical or cultural similarities. however, as long as the method of recording cases is broadly unchanged over the period in question for a particular country, the spectral properties across countries are comparable. the transformed cases' spectrum quantifies the internal oscillatory structure within the series and is largely unaffected by the overall level of cases, the different start times of epidemics in different countries (phase) and country-specific internal delays due to reporting requirements (also phase). in addition, the demonstration of the presence three consistent cycles across all countries, with some variation, provides supporting evidence for the suitability of the transformed new daily cases as a target of analysis, and comparisons between and across countries, another topic of great current interest is to ascertain whether and how a lockdown will influence the number of new daily covid-19 cases. we consider this question for the group consisting of the uk, italy, france, germany, spain, switzerland, belgium and the netherlands. the number of days (with cases) be-fore lockdown is, on average, 22 for this group of countries, and, after lockdown, is 26 (except the uk, which started its lockdown later). the averages just quoted include allowance for a seven day incubation period. our analysis compares the spectral properties before and after lockdown. a spectrum based on about 25 days worth of data would provide a very poor and highly uncertain estimate. however, our spectral fusion methods [3] permit effective sample sizes for the group of 192 days worth of data prior to the lockdown, and 196 after, resulting in highly accurate spectral estimates for these periods. we learn that, after lockdown, the weekly cycle remains strong, but the cycles operating around 2.7 and 4.1 days become suppressed. this indicates that the weekly cycle is due, at least in part, to administrative recording effects, which are not effected by the lockdown, whereas the 2.7 and 4.1 day cycles might be related to virus dynamics, which is certainly affected by lockdown. the discovery of how the high-frequency cycles are disrupted by full lockdown suggests that they could be monitored during partial lockdowns. for example, if schools are reopened and the 2.7 and 4.1 day cycles do not reappear, then this might indicate the effectiveness of that strategy. given the similarity of the cycles across countries, this indicates that cases could be monitored and pooled across regions, over a short number of days to be fused into longer effective samples using the methods described here. a more difficult problem is that of forecasting transformed new daily covid-19 cases. such information would be of great interest, e.g., to those planning health provision over a short timescale. knowledge of the three cycles is helpful and we have had moderate success in forecasting daily cases. however, with individual country series, with smaller number of days, it is unrealistic to expect too much and, in particular, the transformed cycles experience both a degree of time-modulation and possible frequency changes. more useful perhaps, are not daily forecasts, but the knowledge that the number of cases will increase and decrease over a period of three/four days. this means that if one observes a decrease in the number of daily covid-19 cases after lockdown, that does not necessarily mean the peak has been reached, but is simply a manifestation of the 3/4 day cycles. hence, one might believe a lockdown strategy has been successful after a sustained decrease of at least four days. spectral analysis [1, 5] of epidemics is not new, but most work has been carried out on epidemics observed over long time periods (seasons and years) using lengthy time series [6, 7, 8] . recent work [9] on covid-19 has applied popular autoregressive integrated moving average process [10, 1] models to a single prevalence time series with a sample size of n = 22. however, conclusions derived from such analyses on a single series with such small sample sizes [11] are questionable. for example, an autoregressive process of order one with parameter 0.9, normally considered to be a strong signal, is only distinguishable from white noise [12] approximately 20% of the time with sample size of n = 22; basic simulation studies show the large number of possible different models that can fit such short series apparently well. this indicates that it is virtually impossible to tie down the correct model with such a small sample size. phenomenological sub-epidemic models [13, 14] show much more promise and have been applied with some success to short-term forecasting of covid-19 cases in guangdong and zhejiang, china. these improve performance by using bootstrap methods on short case time series, but are still ultimately based on a parametric model of single series. our work is very different as it provides exceptionally accurate spectral estimates for a novel live epidemic that is still in its early days on short series, but reliably so by using recent bayesian spectral fusion techniques. [3] . the nonparametric nature of our analysis also permits us to split case time series at a boundary (e.g. lockdown or other intervention) and analyse the two halves separately, still with very short series in each. this is perhaps harder to do with classical parametric models and to maintain consistency between the two halves. on the other hand, our method relies on good quality case series from different regions, which is again not always the case for all epidemics. we transformed the number of new daily covid-19 cases by applying a signed log transform to the first differences of the new case time series (see methods). the transformed number of new daily cases for 16 countries are shown in figure 1 each showing a distorted noisy, but characteristic sinusoidal trace. the estimated log-spectrum for the uk transformed new daily cases is shown in figure 2 and for all other countries we analysed in the extended data figures. spectral estimates are commonly displayed on a logarithmic scale [16] . spectral peaks can be observed at wavelengths of 6.7, 3.2 and 2.3 days, respectively. although the peaks are visible, the credible intervals indicate that there is a fairly large degree of uncertainty, because this time series contains 52 observations. a frequentist analysis, e.g. using the spectrum function in r [16] , produces a similar result, but with even wider confidence bands. similar spectral analyses for each country indicate three similar spectral peaks, although not always as well-defined nor in precisely the same location. figure 3 shows an estimate that is the result of coherently fusing spectra from 18 countries, giving an an effective sample size of 916 days. here, the clear spectral peaks have narrow credible intervals, due to the large effective number of days afforded by using 18 countries together. the spectral peaks are located at wavelengths of 6.7, 4.1 and 2.7 days. the peak around 6.7 days is observed in the spectral plots for individual countries and we interpret it to be a weekly effect. such a weekly effect could be produced by reporting artefacts (e.g. paperwork being delayed until monday, or carried out differently at the weekend) or due to the behaviour differences of people at weekends. all countries analysed have a 5+2 working week/weekend pattern, although not necessarily the same days of the week (the actual days for a weekend are a phase effect, which does not effect the spectrum). clustering spectra and groups of countries with similar spectra we next clustered our 18 countries based on their spectrum, by calculating a dissimilarity between the spectra for each pair of countries, and then performing both a hierarchical cluster analysis and multidimensional scaling on the dissimilarity matrix. the scaling solution indicated that only two dimensions were required to encapsulate 72% of variation in the data. figure 4 shows the resultant twodimensional solution. attaching a meaning to the scaling axes in figure 4 is not easy. we hypothesise that axis 1 might indicate how badly a country has been perceived to have been affected by the virus with australia, new zealand and sweden less so and those on the left of the plot considerably more so. however, germany is the obvious anomaly to this interpretation as, currently, it has perhaps been perceived to have handled the crisis well so far. table 1 : spectral peaks for the three country groups in units of days. the peaks in the second and third rows have been arbitrarily labelled as peak a. and b. figure 5 show the spectral estimates for the three groups of countries identified in figure 4 , using the clustering techniques mentioned in methods. the peak frequencies for each of these groups is listed in table 1 , which shows differences between them. however, each group possesses a possible weekly peak and higherfrequency peaks labelled a., of around three to four days, and b., around 2.6 days. many countries experiencing the covid-19 pandemic have instituted a lockdown procedure to dramatically reduce virus transmission. at the time of writing, these countries have observed new daily covid-19 cases for between 43 and 54 days. we assume that, on average, it takes about seven days for the virus to incubate, for a person to seek attention and then be tested positive for the sars-cov-2 coronavirus. for each country, the number of days prior to and after the lockdown (19, 26) . for some of these countries the lockdown was applied over a period of two of three days and we took the median of these as the lockdown start date. the number of days before and after the lockdown are, in each case, too small to carry out anything other than the most simplistic time series to maintain statistical reliability. in particular, a spectral estimate in this situation would be subject to a high degree of uncertainty. however, figure 6 shows our coherently fused spectral estimates [3] across these countries before and after the lockdown period, making use of 192 effective days prior to lockdown and 196 days afterwards. the weekly peak is clearly visible in both estimates. the second and third peaks (labelled a. and b. in table 1 ) are visible pre-lockdown, but have all but disappeared post-lockdown. the spectrum is flat in the location where peak a. was previously, and spectral power declines considerably, relatively, where peak b. was located previously. this result is particularly interesting as it suggests that peaks a. and b. have been disrupted by the lockdown. the weekly effect seems relatively unchanged by the lockdown, indicating that perhaps it was strongly driven by non-epidemic effects, such as recording/paperwork or bureaucracy caused by weekends. the post-lockdown spectrum is higher overall than the pre-lockdown spectrum, this is due to the larger variation associated with the larger number of cases identified during the progress of the epidemic. our transformation suppresses this variation, but does not remove it entirely. we have had varied success in forecasting daily cases using a sum of two timemodulated cosine waves model, described in methods, and more research is required. we used the nelder-mead [17] optimisation routine built into r [16] , with starting frequencies of 0.31 and 0.44 taken from our uk spectral estimate plots, and built the model on the transformed cases up to april 11th. after optimisation, the fitted model resulted in modified frequencies ofω 1 = 0.34,ω 2 = 0.45, close to the starting frequencies (the other estimated parameters wereα 1 = 1.28,α 2 = 0.27,φ 1 = −0.102,φ 2 = −0.074,μ 1 = 1.3,μ 2 = −0.731,p 1 = 0.21,p 2 = 0.75). figure 7 shows transformed new daily uk cases, the model fit and forecasts. the model fit does not look too bad, many spectral peaks are being identified, but perhaps the amplitudes of them could be better matched. the untransformed forecasts for april 12th, 13th, 14th and 15th were 5250, 5200, 6373 and 6164, all with approximate 95% confidence interval of ±150. the actual number of cases for april 12th turned out to be 5288. in this case, the forecast was good. however, the two-step ahead forecast of 5200 was wrongthe true value turned out to be 4342 on april 13th. we also used several stochastic forecasting methods based on autoregressive integrated moving average modelling and exponential smoothing, but nothing that we tried was particularly successful. the series is difficult as its amplitude/variance is not constant and we suspect that frequencies are changing over time (as, e.g., the lockdown plot figure 6 indicated). however, rather than point forecasts, the general sinusoidal nature of the transformed cases suggests a further, perhaps more reasonable strategy. at this stage, the uk government and media are looking expectantly at the daily case numbers to try and detect a sustained downward trend in cases. excitement has been generated by a drop in cases two days in a row. this happened on april 5th with 5903 cases, followed by a drop to 3802 and then 3634 on april 6th and 7th and then, unfortunately, increasing to 5491 on the 8th. however, the general sinusoidal patten, with a wavelengths of about 2.7 and four days shows that we should only perhaps start believing that a downward turn is a downward trend after a sustained decrease of four days or more. however, caution needs to be applied here as there is no guarantee that the dynamics will remain unchanged. we analysed numbers of deaths using similar methods described here and found similar cycles. although we have not carried out a detailed analysis, if the number of deaths process can be approximated by a linear system [1, 10] with the numbers of cases as input, then similar cycles are to be expected. a time series with a fixed sampling rate and length has a minimum and maximum (nyquist) frequency that can be observed. [1, 10] although our spectral fusion methods [3] provide more accurate estimates of the spectrum in the normal range (equivalent to having a larger sample size), they can not provide information on frequencies outside of the normal range. to estimate lower frequencies, we would need a genuinely longer series and, for higher frequencies, we would require cases more frequently than once a day, which are arguably not really necessary for any practical purpose. our analyses assume approximate stationarity and linearity for the transformed series, which is unlikely to be exactly true in practice. for example, in the uk transformed case series in figure 7 , there are hints of the series oscillation speeding up over the last ten days. practically speaking, changes in the testing regime, recording practices, the lockdowns or other interventions will change the dynamics of either the pandemic itself or recording of it. ideally, it would be of interest to use methods for non-stationary time series [18, 19] , but the current series available to us are far too short for such analyses. all computations were executed in r [16] and packages that are mentioned specifically below. let y t , for t = 1, . . . , n c represent the number of new daily cases for n c days for country c. the spectral dynamics of the number of daily cases for different countries are all countries masked by the well-known and characteristic exponential increases (and decrease, for those countries that locked down and have now passed their peak). hence, we transform our number of daily cases series to reveal the spectral dynamics. after exploration [10] the following transform was used for all series l t = sgn(d t ) log(|d t |), where the sign function sgn(x) is +1, if x is positive or −1, if x is negative, and d t = y t − y t−1 for t = 2, . . . , n c . the transform is easily inverted, which is essential for forecasting the number of daily cases. we use the regspec [3, 20] bayesian spectral estimation method with a neutral white noise prior with prior variance of 1 and all default arguments, except for a smoothing parameter of 0.7, although the results are not sensitive to the latter. regspec straightforwardly enables the production of spectral estimates using multiple data sets, with each having different lengths and produces coherent credible intervals to properly ascertain the uncertainty inherent in the estimation process. regspec can also fuse spectra for multiple series recorded at different sampling rates, but we do not need to use this aspect of its functionality here as all our case time series are reported daily. however, if a country decided to release case numbers on some other sampling plan (e.g. every two days, or weekly) then regspec would be able to fuse the spectral estimates as described here. such a feature might be of use when dealing with reporting structures that are not equipped to provide daily reporting of cases or where weekly cases are thought to be more accurate. for example, this might apply to regions with fragile health or reporting systems or populations that are spread across widely dispersed geographical regions with poor communications. although the number of cases transformed time series show similar spectral behaviour, it is possible to observe closer similarities within certain subgroups of countries. we used unsupervised clustering and scaling techniques [21, 22] to depict the relationship between different countries and suggest a clustering for them. first, for each country we produced a spectral estimate using regspec as mentioned above, and then formed a dissimilarity for each pair of countries by computing the euclidean distance between their spectral values (using the dist function in r [16] ). classical multidimensional scaling was then used to produce an estimated configuration using the cmdscale function in r [16] . for clustering we use hierarchical cluster analysis on the dissimilarity matrix we computed. it is well-known that dendrograms are sensitive to the input dissimilarity matrix, so we used the clusterwise cluster stability assessment by resampling method to produce a stable clustering [15] . given the form of the transformed new daily cases we propose a model, m t , that is the sum of two time-modulated cosine waves, m t = m where i = 1, 2 indexes the two waves and t = 1, . . . , n c . initial values for forecast model fitting we used α i = 0.8, φ i = 0, µ i = 0.1, p i = 0.5, for i = 1, 2. for model evaluation we put more weight on getting later observations correct and use a residual weight vector w t = (t/n) 2 where t = 1, . . . , n and n is the number of cases. for short term forecasting, we fit m(t) to the transformed daily cases by weighted least-squares using standard r [16] optimisation functions and then extrapolate m(t), using recent weighted residuals to estimate the forecasting error. the number of daily covid-19 cases for countries can be found at the website of the european centre for disease prevention and control [2] . spectral analysis and time series european centre for disease prevention and control, covid-19 cases worldwide should we sample a time series more frequently? decision support via multirate spectrum estimation (with discussion) we don't know if coronavirus deaths will peak this week spectra analysis for physical applications travelling waves and spatial hierarchies in measles epidemics seasonality and the persistence and invasion of measles the dynamics of measles in sub-saharan africa application of the arima model on the covid-19 epidemic dataset the analysis of time series: an introduction forecasting: principles and practice white noise testing using wavelets a novel sub-epidemic modeling framework for short-term forecasting epidemic waves short-term forecasts of the covid-19 epidemic in guangdong and zhejiang flexible procedures for clustering, r package version 2.2-5 r: a language and environment for statistical computing (r foundation for statistical computing a simplex algorithm for function minimization locally stationary processes a test for second-order stationarity and approximate confidence intervals for localized autocovariances for locally stationary time series non-parametric bayesian spectrum estimation for multirate data an introduction to multivariate analysis the elements of statistical learning key: cord-000614-gl9cjmno authors: pang, xinghuo; yang, peng; li, shuang; zhang, li; tian, lili; li, yang; liu, bo; zhang, yi; liu, baiwei; huang, ruogang; li, xinyu; wang, quanyi title: pandemic (h1n1) 2009 among quarantined close contacts, beijing, people’s republic of china date: 2011-10-17 journal: emerg infect dis doi: 10.3201/eid1710.101344 sha: doc_id: 614 cord_uid: gl9cjmno we estimated the attack rate of pandemic (h1n1) 2009 and assessed risk factors for infection among close contacts quarantined in beijing, people’s republic of china. the first 613 confirmed cases detected between may 16 and september 15, 2009, were investigated; 7,099 close contacts were located and quarantined. the attack rate of confirmed infection in close contacts was 2.4% overall, ranging from 0.9% among aircraft passengers to >5% among household members. risk factors for infection among close contacts were younger age, being a household member of an index case-patient, exposure during the index case-patient’s symptomatic phase, and longer exposure. among close contacts with positive test results at the start of quarantine, 17.2% had subclinical infection. having contact with a household member and younger age were the major risk factors for acquiring pandemic (h1n1) 2009 influenza virus infection. one person in 6 with confirmed pandemic (h1n1) 2009 was asymptomatic. i n early april 2009, human cases of infection with a novel infl uenza virus of swine origin, pandemic (h1n1) 2009 virus, were identifi ed in the united states and mexico, and this virus spread rapidly across the world (1-3). on june 11, 2009 , the world health organization raised the pandemic level to 6, the highest level for pandemic alert (4) . estimating attack rates is a major task in characterizing pandemic (h1n1) 2009. some studies have reported attack rates of pandemic (h1n1) 2009 among household members and aircraft passengers (5) (6) (7) . these studies suggested that the transmissibility of pandemic (h1n1) 2009 virus was low. these studies were conducted in outbreak settings, and attack rates were calculated on the basis of clinical diseases that included infl uenza-like illness (ili) or acute respiratory illness (ari) of close contacts rather than confi rmed infection with pandemic (h1n1) 2009 virus. in addition, in these studies only symptomatic index and secondary cases were included. although most infections of pandemic (h1n1) 2009 infl uenza virus produce ili or ari symptoms (8) (9) (10) (11) (12) , subclinical infection can occur and can change the estimate of attack rate. in addition, the infectivity of asymptomatic case-patients has not been clearly defi ned (13) . because of the high rates of illness and death among the initial case-patients with pandemic (h1n1) 2009 (14) , the chinese government decided to prevent and contain the rapid spread of disease through tracing and quarantine of persons who had close contact with persons with confi rmed cases of pandemic (h1n1) 2009. beijing, the capital of the people's republic of china, took strict containment and control measures through october 2009. the beijing municipal government implemented border entry screening, ili screening in hospitals, health follow-up of travelers from overseas, and quarantine and testing of close contacts to identify new introduction of cases and local transmission. public health workers conducted epidemiologic investigation of all index case-patients (including those with subclinical infections) and traced and quarantined close contacts whose residence was within the jurisdiction of beijing. we estimated the attack rate of pandemic (h1n1) 2009 virus infection and assessed risk factors or correlates for infection among different types of close contacts, including household members and aircraft passengers. in 2009, under the guidance of the beijing center for disease prevention and control (beijing cdc), a network of 55 collaborating laboratories was established to perform reverse transcription pcr testing to confi rm cases of pandemic (h1n1) 2009 (15) . the confi rmed cases included symptomatic and asymptomatic cases, and these cases were detected mainly by border entry screening, ili screening in hospitals, health follow-up of travelers from overseas, and quarantine and testing of close contacts. once confi rmed, index case-patients were immediately quarantined in designated hospitals to receive treatment while in isolation. all the confi rmed cases were required by law to be reported to beijing and local cdcs. from may through october 2009, a detailed epidemiologic investigation was conducted for each confi rmed case of pandemic (h1n1) 2009 (including symptomatic and asymptomatic cases) by beijing and local cdcs within 6 hours after confi rmation of infection. patients with confi rmed cases were interviewed about demographic characteristics, course of illness, travel and contact history, and information about close contacts. patients with confi rmed cases were categorized as having imported cases (travelers) and locally acquired cases (no travel history) on the basis of where the infection was acquired. close contacts were defi ned as anyone who ever came within 2 meters of an index case-patient without the use of effective personal protective equipment (ppe) (including masks and gloves, with or without gowns or goggles) during the presumed infectious period. trained staff from local cdcs made the determinations on the basis of fi eld investigation. the relationships of close contacts to index case-patients were categorized as 1) spouses, 2) other household members, 3) nonrelated roommates, 4) contacts at workplace or school, 5) nonhousehold relatives, 6) passengers on the same fl ight, 7) friends, and 8) service persons met at public places. a close contact on an aircraft was defi ned as a passenger sitting within 3 rows in front and 3 rows behind the index case-patient. all close contacts were traced and quarantined for 7 days after the most recent exposure to the index casepatient. all index case-patients detected between may 16 (the fi rst case, the date of confi rmation) and september 15, 2009 (before widespread transmission in beijing), and their close contacts were included in this study. we excluded cluster or outbreak cases for which close contacts could not be determined clearly by epidemiologic investigation (the transmission chain was obscure). for each close contact, before quarantine, a pharyngeal swab specimen was collected for reverse transcription pcr testing, regardless of symptoms. a second pharyngeal swab specimen was collected for testing for pandemic (h1n1) 2009 virus if any of the following symptoms developed in a close contact during quarantine: axillary temperature >37.3°c, cough, sore throat, nasal congestion, or rhinorrhea. data were analyzed by using spss version 11.5 (spss inc., chicago, il, usa). median and range values were calculated for continuous variables, and percentages were calculated for categorical variables. differences in attack rates were compared between subgroups of close contacts by using the χ 2 test. for the signifi cant difference found in multiple subgroups, this test does not enable identifi cation of which multiple subgroups are signifi cantly different, only that across all the subgroups there are differences. the variables with p<0.10 in χ 2 test were included in multivariate analysis. multivariate unconditional logistic regression analysis was conducted to determine risk factors associated with infection in close contacts. backward logistic regression was conducted by removing variables with p>0.10. odds ratios (ors) and 95% confi dence intervals were calculated for potential risk factors of infection. the hosmer-lemeshow goodness-of-fi t test was used to assess the model fi t for logistic regression. all statistical tests were 2-sided, and signifi cance was defi ned as p<0.05. a total of 613 eligible index case-patients, detected from may 16 through september 15, 2009, were included in this study. through fi eld epidemiologic investigations, 7,099 close contacts were traced and quarantined in beijing. the median number of close contacts per index case per day was 7.0 persons (range 2.0-95.0 persons); the median number for an imported index case was 7.0 persons (range 1.7-95.0 persons) and for a locally acquired index case was 5.3 persons (range 1.0-25.0 persons). for the 601 symptomatic index case-patients, the median interval between illness onset and sample collection was 1.0 days (range −1.9 to 7.0 days). among close contacts with symptomatic infection, the median interval between illness onset and sample collection was 0.5 days. more than 85% of close contacts were quarantined within 72 hours after interview of the index case-patients. the median interval between fi rst exposure and quarantine was 3.4 days for the close contacts, and it was shorter, on average, for fl ight passenger contacts than nonpassenger contacts (1.7 days vs. 3.8 days). for symptomatic close contacts infected with pandemic (h1n1) 2009, the median of generation time (i.e., the time from illness onset in an index case to illness onset in a secondary case) were 2.4 days; it was shorter for fl ight passenger contacts than nonpassenger contacts (1.6 days vs. 2.5 days) ( table 1) . approximately 43% of the index case-patients were women; the median age was 20 years, and 38% likely contracted pandemic (h1n1) 2009 virus locally because they had not traveled recently. among the index casepatientss, 2% had subclinical infection. only 18% of index case-patients had close contacts with confi rmed pandemic (h1n1) 2009 (table 2) , and the total number of close contacts who were infected by the virus from 110 index case-patients was 167. fifty percent (3,514 of 7,032) of close contacts were women, and the median age was 27 years. approximately 12% of close contacts were household member of index case-patients (spouse or other household member), and aircraft passengers accounted for 44% of close contacts. approximately 61% of close contacts were exposed to symptomatic index case-patients during their symptomatic phase. about 70% were quarantined in a quarantine station ( table 2 ). the overall attack rate for infection among close contacts (positive test result) was 2.4% (167 of 7,099), indicating that 1 index case-patient transmitted infection to 0.27 close contacts (167 of 613) on average (reproduction number = 0.27). among those close contacts with a positive test result, 14.4% (24 of 167) had subclinical infection; among the close contacts with positive test results at the start of quarantine, 17.2% (20 of 116) had subclinical infection. attack rates did not differ by index case-patient's sex (p = 0.225). however, attack rates differed signifi cantly by index case-patient's age (p = 0.022), and the lower attack rate was found for older index case-patients. there was no signifi cant difference in attack rates between close contacts of patients with imported cases and those with locally acquired cases (p = 0.282). no infection was found in close contacts exposed to index case-patients with subclinical infection, and the attack rate observed in close contacts exposed to symptomatic index case-patients during their symptomatic phase was higher (p<0.001). almost identical attack rates were found among male and female close contacts (p = 0.808). however, attack rates were signifi cantly different among different age groups of close contacts (p<0.001), and the lowest attack rate was found for those >50 years of age. the attack rates were signifi cantly different across 8 contact types (p<0.001). the attack rate was 5.3% among spouses and 6.6% among other family members in the household, and was lower among other types of close contacts ( table 3 ).the attack rate among passengers on the same fl ight was low, 0.9% overall, and 1 index case-patient transmitted infection to 0.19 close contacts on a fl ight on average (28 of 147), and the attack rate was higher among the passengers with longer fl ight times (>12 hours, p = 0.001). the attack rate among close contacts of service persons at public places was 0.2%, and 1 index case-patient transmitted infection to 0.01 close contacts of service persons on average (1 of 113). nonpassenger close contacts with longer exposure duration (>12 hours), compared with those with shorter duration (>12 hours), recorded the higher attack rate (p<0.001) ( table 3) . by multivariate analysis, age and type of contact were the major predictors of infection ( (or 3.42; p = 0.002) and 0-19 years of age (or 7.76; p< 0.001) were at higher risk for infection. other signifi cant independent risk factors associated with infection included being a household member of a person with an index case (or 3.83; p<0.001), being exposed to index case-patients during their symptomatic phase (or 1.86; p = 0.003), and exposure duration >12 hours (or 1.83; p = 0.002). similar risk factors were observed among aircraft passengers. we estimated that pandemic (h1n1) 2009 virus was transmitted by 18% of index case-patients to their close contacts and that 2.4% (167 of 7,099) of close contacts we traced were infected. our data indicate that pandemic (h1n1) 2009 virus has low transmissibility in nonoutbreak settings. we found that 1 index case-patient transmitted infection to 0.27 close contacts on average, i.e., reproduction number = 0.27. this fi nding suggests that among those quarantined index case-patients, the number of persons with secondary cases who could be traced through rigorous fi eld investigation was small and far less than the number needed for the sustainable transmission of infectious disease in the population (reproduction number >1). however, the fact that the pandemic eventually spread in beijing indicates that contact and case tracing were far from complete, especially later in the summer and early fall of 2009. the strict control measures may have worked to some extent at the beginning but were outpaced by local transmission (16) ; the percentage of locally acquired infections ranged from <10% in june 2009 to >80% in september 2009 (data not shown). in this study, the median number of close contacts per index case-patient per day was 7.0 persons. although locating and quarantining these close contacts was done quickly, and stringent quarantine measures were used, which hindered implementation of control measures, the real number of close contacts was unknown and probably exceeded this number. many close contacts were persons met in public places, including public transportation, theaters or cinemas, and shopping malls, and it is nearly impossible to trace all of the contacts. in addition, some persons who had worn ppe during contact with index casepatients were excluded from close contacts management (i.e., they were not quarantined), but because wearing ppe might not protect (or fully protect) against infection, some persons excluded might have become infected. in addition, many persons with mild and asymptomatic cases cannot be detected, but they may transmit the virus. furthermore, the short generation time of pandemic (h1n1) 2009 shown in this study and in a previous study (13) could lead to the rapid accumulation of infection sources and close contacts. this rapid compounding could overwhelm response capacity and would have resulted in compromised effectiveness of containment measures. it should also be mentioned that we did not include persons with cluster or outbreak cases for whom close contacts could not be determined clearly by epidemiologic investigation to examine the basic feature of pandemic (h1n1) 2009 (e.g., generation time), and the reproduction number obtained from our data is an underestimate. attack rates of infection differed signifi cantly by contact type. among household members of index casepatients, the attack rate was the highest, as shown in the multivariate analysis after controlling for age and other factors. the most likely reason for this fi nding is that household members are more likely to have come into closer contact with index case-patients for a longer period with shorter distance and longer duration. another possible reason is that household members may have some certain linkage with index cases in genetic susceptibility or living habits that would cause higher predisposition in household members than in other close contacts. this fi nding is similar to fi ndings in other investigations of respiratory infectious disease (17) . close contacts on fl ights accounted for the highest proportion of all the close contacts, in part because of how the index cases were detected and the broad defi nition we used for close contacts. however, the attack rate was much lower than that for other close contacts; 1 index case infected only 0.19 close contacts on fl ights on average. this fi nding indicated that the possibility of transmission of pandemic (h1n1) 2009 virus on fl ights was low, and the yield of tracing and quarantining of close contacts on fl ights was limited. tracing contacts of service persons at public places was more diffi cult than tracing other categories of contacts, and the lowest attack rate (0.2%) was recorded in this category. despite extensive measures, on average, only 0.01 infected close contacts per index case-patient were identifi ed among service persons. tracing the contacts of service persons at public places seems far less cost-effective. criteria for close contacts on fl ights and those of service persons should be refi ned with respect to exposure duration and age of those exposed. exposure to index case-patients for >12 hours was a signifi cant independent risk factor for infection in fl ight passenger contacts. this fi nding suggests that limiting the time of contact with persons with ili on aircraft can reduce risk for transmission, and a long duration of exposure may be necessary for transmission to occur on aircraft. younger close contacts were at higher risk for infection than older ones. the possible reason was that younger persons had much closer contact with index case-patients than did older persons; another reason may be that younger persons were more susceptible to infection with pandemic (h1n1) 2009 virus (18) . this fi nding was consistent with fi ndings reported in other studies (5, 6) . no secondary cases were found among close contacts exposed to index case-patients with subclinical infection. the attack rate among close contacts who were exposed to symptomatic index case-patients during their symptomatic phase was much higher than that among those exposed to these case-patients before their illness onset. exposure to index case-patients during the symptomatic phase was a signifi cant independent risk factor for infection among close contacts. these fi ndings indicate that the infectivity of pandemic (h1n1) 2009 virus was higher after illness onset, and that the infectivity of symptomatic pandemic (h1n1) 2009 case-patients before illness onset was higher than that of persons with subclinical cases, although persons in each group were asymptomatic when in contact with other persons. in general, the earliest infectious time for pandemic (h1n1) 2009 was considered as 1 day before illness onset (19) . we found that index case-patients and infected close contacts shed pandemic (h1n1) 2009 virus <1 day before illness onset, which suggests that the infectious period of symptomatic persons with pandemic (h1n1) 2009 might be <1 day before illness onset. among close contacts with pandemic (h1n1) 2009, ≈14.4% were asymptomatic. it is noteworthy that specimens from some close contacts tested negative for pandemic (h1n1) 2009 virus before quarantine, but those persons could shed the virus during quarantine without symptoms. such infection could not be detected, and the proportion of subclinical infection was underestimated. therefore, we calculated the proportion of subclinical infection by cross-sectional analysis of the subclinical infection of close contacts before quarantine, and we found that ≈17% of case-patients with pandemic (h1n1) 2009 were asymptomatic. this study has several limitations. we could not fi nd all close contacts of persons with pandemic (h1n1) 2009 and did not know their infection status, so the infection parameters of pandemic (h1n1) 2009 that we found in this study might not be precise, especially for reproduction number, which may be underestimated to some extent. furthermore, we could not exclude the possibility that the infected close contacts had been infected from another unknown source before quarantine started, which might infl uence our conclusion to some extent. in summary, the attack rate among close contacts was low, even among household contacts. household member and younger age were the major risk factors for infection with pandemic (h1n1) 2009 virus among close contacts. approximately 17% of cases of pandemic (h1n1) 2009 were asymptomatic. table 2 were included in multivariate unconditional logistic regression analysis. hosmer-lemeshow goodness-of-fit test was used to assess the model fit for logistic regression. or, odd ratio; ci, confidence interval; na, not available, indicating not included in the final model. †one dependent variable (infection with pandemic [h1n1] 2009 virus) and 5 independent variables (age of index case-patient, type of exposure to index case-patients, age of close contacts, relationships to index case-patients, and exposure duration of close contacts) were included in multivariate analysis. one independent variable (age of index case-patient) was removed in the stepwise regression equation. the goodness-of-fit test suggested that the logistic regression model fitted well (p = 0.631). ‡one dependent variable (infection with pandemic [h1n1] 2009 virus) and 4 independent variables (age of index case-patient, type of exposure to index case-patient, age of close contacts, and exposure duration of close contacts) were included in multivariate analysis. two independent variables (age of index case-patient and type of exposure to index case-patient) were removed in the stepwise regression equation. the goodness-of-fit test suggested that the logistic regression model fitted well (p = 0.982). §one dependent variable (infection with pandemic [h1n1] 2009 virus) and 5 independent variables (age of index case-patient, type of exposure to index case-patient, age of close contacts, relationships to index case-patient, and exposure duration of close contacts) were included in multivariate analysis. two independent variables (age of index case-patient and exposure duration of close contacts) were removed in the stepwise regression equation. the goodness-of-fit test suggested the logistic regression model fitted well (p = 0.751). ¶exposed to symptomatic index case-patients before their illness onset or exposed to index case-patients who had subclinical infections. pneumonia and respiratory failure from swine-origin infl uenza a (h1n1) in mexico swine infl uenza a (h1n1) infection in two children-southern california pandemic potential of a strain of infl uenza a (h1n1): early fi ndings world health organization. world now at the start of 2009 infl uenza pandemic household transmission of 2009 infl uenza a (h1n1) virus after a school-based outbreak household transmission of 2009 pandemic infl uenza a (h1n1) virus in the united states transmission of pandemic a/h1n1 2009 infl uenza on passenger aircraft: retrospective cohort study novel swine-origin infl uenza a (h1n1) virus investigation team emergence of a novel swine-origin infl uenza a (h1n1) virus in humans clinical features of the initial cases of 2009 pandemic infl uenza a (h1n1) virus infection in china subclinical infection with the novel infl uenza a (h1n1) virus shedding and transmission of novel infl uenza virus a/ h1n1 infection in households-germany clinical and epidemiologic characteristics of 3 early cases of infl uenza a pandemic (h1n1) 2009 virus infection, people's republic of china european centre for disease prevention and control. ecdc risk assessment: pandemic h1n1 outbreak of swineorigin infl uenza a (h1n1) virus infection-mexico severe, critical and fatal cases of 2009 h1n1 infl uenza in china alternative epidemic of different types of infl uenza in 2009-2010 infl uenza season, china evaluation of control measures implemented in the severe acute respiratory syndrome outbreak in beijing incidence of 2009 pandemic infl uenza a h1n1 infection in england: a cross-sectional serological study interim guidance for emergency medical services (ems) systems and 9-1-1 public safety answering points (psaps) for management of patients with confi rmed or suspected swine origin infl uenza a (h1n1) infection we thank fujie xu for her suggestions and comments. all material published in emerging infectious diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. key: cord-245047-d81cf3ms authors: gupta, sourendu title: epidemic parameters for covid-19 in several regions of india date: 2020-05-18 journal: nan doi: nan sha: doc_id: 245047 cord_uid: d81cf3ms bayesian analysis of publicly available time series of cases and fatalities in different geographical regions of india during april 2020 is reported. it is found that the initial apparent rapid growthin infections could be partly due to confounding factors such as initial rapid ramp-up of disease surveillance. a brief discussion is given of the fallacies which arise if this possibility is neglected. the growth after april 10 is consistent with a time independent but region dependent exponential. from this, r0 is extracted using both known cases and fatalities. the two estimates are seen to agree in many cases; for these cfr is reported. it is seen that cfr and r0 increase together. some public health implications of this observation are discussed, including a target doubling interval if medical facilities are to remain adequate. sars-cov-2 is a virus which has newly entered the global human population [1] . as this host-parasite system evolves towards an equilibrium, its epidemiology has been studied extensively, but with some conflicting results [2, 3] . the true extent of its penetration into the population is as yet open to question [4] , since testing is fairly restricted in most countries. nor is the progression of the disease, covid-19, or its method of spread completely clear [5] . since the virus is already so widely established, it seems unlikely that it will be totally eliminated soon. so it is important to extract basic epidemiological parameters as cleanly as possible. india has managed to geographically contain the spread of the covid-19 epidemic with the nation-wide lock-down which started on 24 march, 2020. at the end of april the proportion of identified cases in india as a whole was a few tens per million, with 1-2 orders of magnitude more in hot spots. even if this were wrong by an order of magnitude, it would still mean that the epidemic remains at an early stage in india. this, combined with the lock-down, presents an opportunity to examine the growth of the epidemic in multiple isolated regions which implement essentially the same policy with regard to testing. this study examines the heterogeneity in the growth rate of the disease, in several ways. first, the doubling intervals, τ , of the cumulative number of identified cases, c(t), and the cumulative number of fatalities, d(t), is examined. from τ it is possible to extract the basic reproduction rate, r 0 , within epidemic models. marked heterogeneity are observed. after this the correlation between the case fatality ratio, cfr, and r 0 is studied. epidemic data, especially at the beginning is never clean. the public health system has to gear up for disease surveillance. the continuing recurrence of cholera epidemics [6] , the spread of dengue and chikungunya [7] , the successful surveillance and elimination of nipah [8] and zika [9] , show that india has a mixed record on epidemic surveillance. in addition to a possible lag between the beginning of the epidemic and its surveillance, there could be a problem of incomplete surveillance during the time the health service ramps up. any examination of data has to allow for the identification of confounding factors such as these. for the covid-19 surveillance data, there are further cautionary remarks. the icmr guidelines for testing [10] specify that only symptomatic cases should be tested using rrt-pcr. this part of the policy has been unchanged since the middle of march. depending on the fraction of cases which are symptomatic, this could miss the actual prevalence of the disease in the population. estimates of the fraction of asymptomatic infections range as high as 80% [11] , implying that, in this extreme case, the testing policy can never reveal more than 20% of the cases. the social stigma attached to covid-19 [12] also means that some fraction of infections may be cryptic. there are uncertainties in the statistics of fatalities also. it has been reported that in europe and the us the number of fatalities due to covid-19 may have been underestimated by a factor of 2-3. indian cities have fairly complete registries of deaths, so miscounting of covid-19 fatalities could come mainly from mistaken or incomplete reports of the cause of death. for larger regions, say districts and whole states, where most deaths happen at home and death certificates are not common [13] the errors in counting fatalities may be significantly larger, and hard to estimate at this time. one point about the quality test that is developed here is that absolute numbers are not as important for it as the check that fatalities and identified cases are independently tracing the same rate of growth of the epidemic. this is expected at the beginning of the epidemic, when all epidemic models become linear, and the growth of generic measures is driven by the maximum eigenvalue of the linearized models. however, in the extraction of the cfr, the absolute counts do matter. in spite of the uncertainties, the correlation of cfr and r 0 holds important lessons for public health in the inevitable later stages of this epidemic in india, and the middle and low income countries of the world. data has been extracted from official sources where possible. for ahmedabad city, the data is made publicly available by the municipal council of the city [14] . this well-organized site corrects data retrospectively for up to about 10 days. for chennai city, the data has been collected from daily tweets by the municipal council [15] . for indore city, the data was collected from daily bulletins of the chief medical and health officer and the collection is available for public use [16] . for mumbai city the data has been collated from the daily tweets by mcgm [17] into a publicly available site [18] . for pune district the data was collated from the daily tweets by the district authority [19] and the collection is publicly available [20] . for delhi and all other states, the data was taken from the publicly available collection at covid19india [21] . this site corrects data retrospectively for over a week. only data on the cumulative number of identified cases, c(t), and the cumulative number of known fatalities, d(t), are used in this analysis. for this work data collection stopped on may 1, 2020, and retrospective corrections made after this are not included. the unquantifiable parts of the errors in the counts of cases and fatalities due to covid-19 were discussed in the previous section, along with the reasons why their estimates need not be included in this analysis. however, there is another part of the errors in the daily counts of cases and fatalities which come from backlogs of tests or hospital records. these shuffle a fraction of the numbers from one day to another, and therefore cause errors in the daily counts. as long as the number of facilities keep pace with the growth of the epidemic, these errors remain proportional to the number of cases and fatalities. since the wait time for hospital beds for covid-19 cases has remained roughly constant during the period of study, this argument is expected to hold. in view of this, of 20% of the reported values of c(t) and d(t) are assigned as errors. the specific fraction, 20%, was chosen to in order to cover the long range fluctuations visible in the time series (for example in those visible on days 3 and 13 of figure 1 ). it has been seen that official reports and independent estimates of these number are generally within this range. at such an early stage in the infection, it is reasonable to assume exponential growth, i.e., doubling every τ days. within this assumption one can check how well the lock-down is working by letting the doubling interval become time dependent. the simplest function to try is a linear change in τ , i.e., and a similar set of three parameters for c(t). note that τ 0 has dimensions of time, whereas τ 1 is dimensionless. a fitting form with constant doubling interval was also used; this is denoted τ , dropping the subscript. the fitting procedure follows the methods of [23] , with gamma distributions used as prior probability distribution functions (pdfs) for τ 0 and c 0 . the additional parameter τ 1 is allowed to take positive and negative values, by letting the prior pdf to be a gaussian. for all these distributions, the widths are taken large enough that the posterior distribution is insensitive to the choice of priors. the appendix contains details of the relation between a time varying doubling interval and time variation of the basic reproductive rate r 0 . this requires choosing a model of the epidemic. using the seir model, and the median interval between the appearance of symptoms and the time of fatalities, t 2 = 17.8 days [22] , one has when a constant τ is used, one can set τ 1 = 0 in the above formulae and write r 0 and τ instead of r 0 0 and τ 0 . exactly the same procedure is followed for fits to the time series for d(t). estimates of the median values of the parameters, along with interquartile ranges (iqr) and 95% credible intervals (cri) are quoted for the doubling intervals as well as r 0 . the analysis of the time series for c(t) and d(t) lead quite naturally to the case fatality ratio, cfr. this is defined as the ratio cfr = d(t)/c(t). ( if c(t) is underestimated, then cfr is overestimated, and conversely, when d(t) is underestimated, then cfr is also underestimated. this was regulated using a bayesian estimator. since the outcome is binomial, the prior pdf used is a beta distribution with α = 1 and β = 2. these choices make the posterior distribution insensitive to doubling or halving the values of the priors. the posterior distribution is of the same form with α = 1 + d(t) and β = 2 + c(t) − d(t), with t taken to be the final day of the analysis. since c(t) and d(t) are both large, the following approximations for the median, µ, and standard deviation, σ, may be used: iii. results the time series of c(t) and d(t) is shown for the example of delhi in figure 1 . of the regions that we analysed, most cities show an initial rapid growth followed by a tempered growth. the exceptions are ahmedabad and chennai among cities, and the states of gujarat, kerala, and west bengal. note that day one is taken to be march 31, 2020, which is 7 days after the beginning of the national lock-down. since t 2 = 17.8 days, it might be expected that the growth rate of cases in the pre-lock-down period could manifest itself in that of fatalities until around day 11. in case of a successful lock-down, d(t) could then show an initial exponential growth, tempered after day 11. the initial data for fatalities in delhi, indore, mumbai and pune can indeed be described by an exponential. however, the doubling interval in pune turns out to be half of that in mumbai, although the average population density of mumbai is about 6 times larger than the average in pune city. the ansatz of eq. (1), i.e., a linearly varying doubling interval, was also examined for urban regions. the results are collected in table i . in most locations the initial doubling interval seems to be between half and day and two days. when converted to r 0 , one obtains extremely high values, far in excess of what has been quoted in the literature. certainly r 0 could vary from place to place, since it depends on infectivity of the virus as well as the social networks in each location, and the latter may change from one place to another. however, τ 0 for pune is one third that of mumbai, when mumbai has six times the average population density. the wrong dependence of the doubling time for fatalities on population density, together with the observation that c(t) shows a growth till the same date, supports the idea that there could be a more parsimonious explanation for this common period of growth. this is discussed in the next section. at the moment, any statistical evidence for a gradual slowing of the growth rate of the epidemic is hidden due to some confounding factors. in view of this, the analysis was continued with a constant doubling interval, τ , applying it to the period after day 10 or 11. for this part of the analysis data was from three states, namely gujarat, kerala, and west bengal, was also used. from figure 1 , one sees that this simpler model provides as good a description of the data as the model of eq. (1). furthermore, this yields more realistic values of r 0 implies that during the lock-down each of these places has seen a location dependent constant doubling interval. the values of τ , along with inferred values of r 0 , are collected in table ii . these are the primary results of this analysis. it was noted that the number of known cases, c(t), is definitely missing cases among those who have not been tested. this could include a possibly large, fraction of asymptomatic and non-critical or pre-symptomatic cases [24, 25] . however, india's disease surveillance mechanism has concentrated on identifying critical cases and contact tracing, which could be a good tracer of the growth of epidemics. if this reasoning is correct, then, during the early growth of the epidemic, one should be able to obtain reliable doubling intervals from the cumulative counts of test positives [23] . the results of this analysis are also given in table ii . the two independent estimates of r 0 agree well enough that a closer look reveals interesting patterns. the scatter plot in figure 2 of r 0 obtained in two different ways shows several interesting patterns. first, there seem to be two groups of outbreaks. most regions have r 0 below 3. among the regions that we studied, ahmedabad and gujarat were a separate group, which saw a faster epidemic growth, with r 0 above 3. finally there is kerala, a different outlier, whose doubling intervals are longer than t 2 , and therefore with very low values of r 0 . the case of kerala merits a separate remark. the cumulative number of fatalities reached 4 at the end of the period of study. with such low counts of fatalities the assumption of exponential growth cannot be well tested. the counts of total infections was larger, and supported the hypothesis of exponential growth over the period studied. second, one sees that most estimates lie close to the diagonal line. if the data was perfect, and the epidemic grew steadily, the estimates would lie exactly on this line. with this requirement we can separate the regions into two groups. one consisting of ahmedabad, chennai, delhi, gujarat, and west bengal are, within statistical uncertainties, on this line. the second group, with indore, kerala, mumbai, and pune, are not. this could indicate some issues with the data. on the other hand, if the data is as good as the other regions, then the fact that they are off the diagonal line should be understood. kerala, which is the only region which lies below the diagonal, is perhaps seeing a lower growth in new cases than fatalities, which could be indicative of a gradual slowing down of the epidemic. due to the lag by t 2 , fatalities would see the slowing down later. conversely, the regions which lie above the diagonal (namely indore, mumbai, and pune, and, possibly, chennai) could be seeing an increased growth in infections, not yet visible in fatalities because of the same time lag. whether these scenarios are true, or the data quality is not dependable, should be known to the health agencies now, and would become visible to the public later. when there is a statistically significant difference between the doubling interval determined by c(t) and d(t), then the ratio gives a time-dependent cfr. this is usually understood to be a transient phenomenon. in view of this, the analysis was restricted to ahmedabad, chennai, delhi, gujarat, kerala, and west bengal, i.e., the regions which lie on the diagonal line of figure 2 , and therefore are seeing a steady growth of identified cases as well as fatalities. the case fatality ratios for these regions are plotted against the r 0 inferred from d(t) in figure 3 . the most obvious trend is that for the group of three cities there is an overall trend towards smaller cfr with decreasing r 0 . this is also true of the two states. however, the cfr for states is displaced upwards from that for cities. both trends have strong implications for the public health outlook and will be discussed further in the next section. counts of known cases and fatalities of covid-19 from five cities (ahmedabad, chennai, delhi, indore, and mumbai), one district (pune), and three states (gujarat, kerala and west bengal) was investigated in this work. in two of the groups, there was one case each where the epidemic was not severe at the end of april (chennai among cities, and kerala among states). the others were known hot spots. kerala is special because the number of fatalities is too low for statistical tests to be meaningful. there are strong regional heterogeneity in the course of the epidemic, indicating the necessity of looking at its spread at extremely local scales in order to check and control it. the time series both of known cases and fatalities in four out of the six urban centers showed a rapid rise for about 18 days after the lock-down. this was followed by a much slower growth. since fatalities track cases with a delay of 17.8 days on the average, the early part of this data could track the growth in the time before the lock-down. however, it turns out that the data grows faster in less dense urban areas. moreover, this hypothesis is not tenable for the growth in the number of known cases. a possibility which resolves these difficulties is that this rapid rise of numbers in the early days tracks the rapid improvement of disease surveillance rather than the epidemic. the fact that the positive cases in kerala does not show such a rapid initial growth is consistent with reports that the state activated disease surveillance after the first infections came from abroad [26] . this could also be true of ahmedabad and gujarat, two other centers which show no such initial increase, since the state had passed through the surveillance challenge of zika virus in recent years [9] . due to this confounding factor, it is not possible to use the data until april 10 or 11 to make any statistically valid measurement of the growth of the epidemic before the lock-down. neglecting this leads to multiple fallacies, which i remark on next. the apparent slowing down of the growth in later stages may be falsely interpreted as a transition to polynomial growth. as shown in eq. (a5) and eq. (a7), this is equivalent to a time dependent doubling interval. it has been discussed in the previous subsection that this leads to highly unlikely properties of the covid-19 epidemic. the same apparent slowing down of the growth rate in india has also been interpreted within the homogeneous sir model with constant, time invariant, parameters [27] . in such a simple model the time dependence can only come from early evolution towards herd immunity. this gives rise to the unlikely conclusion that herd immunity will be reached for covid-19 while 99% of the population remains susceptible. a misrepresentation of data also arises when "instantaneous doubling intervals" or similar measures of exponential growth are constructed using c(t) for one day, or averaged over small windows of time [28] . this shows a spurious gradual slowing of growth during the first three weeks of the lock-down. in later weeks these estimates are also plagued by spurious effects which result when delayed reports are dumped into cumulative numbers on one day instead of being assigned to correct past dates. these appear as evidence of local spurts or slumps in growth. evidence of retroactive corrections from [14] shows that delays of as much as ten days may occur. when these artifacts are averaged over a moving window, this gives the mistaken appearance of peaks and troughs, and may put erroneous pressure to change policies. due to the reasons discussed in the previous subsections, the period after april 10 or 11 constitutes the base data for the main part of this analysis. as shown in figure 1 a constant growth rate in each locality during the the lockdown models the data as well as a growth rate which changes linearly with time. this is also the most parsimonious hypothesis about the growth of the epidemic. the observed doubling interval, and the derived quantity r 0 , fall into three groups (see table ii and figure 2 ). several geographical regions have r 0 less than 3. kerala has r 0 ≃ 1.7 (a doubling interval larger than t 2 , the interval from the emergence of symptoms to death). gujarat and ahmedabad have r 0 higher than 3. since this is the growth rate during the lock-down, population density effects are unlikely to be the major determinant of r 0 . it would be worthwhile to consider the role of individuals with extremely large number of contacts in this context, or a significant tail of the distribution with small number of contacts, but still above three. five regions pass the following data quality test-the value of r 0 obtained from the growth of fatalities and cases are equal. this does not mean that the number of cases is correctly counted. rather it indicates that the effort to find the cases requiring critical care, and tracing their contacts has successfully resulted in tracking a constant fraction of all infected persons. it may miss, for example, a large fraction of asymptomatic cases. for the five geographical regions which pass the quality test described in the previous section, a further study was performed. the dependence of the case fatality ratio, cfr (i.e., the ratio of the observed number of fatalities and cases) on r 0 was investigated. although the number of cases identified may be much smaller than the actual number of cases, the chance that cases are identified in these five regions are expected to be similar, since the rate of testing is about the same. a positive correlation between r 0 and cfr is observed. one possible reason for this is that with lower r 0 the number of critical cases grows slower, giving medical practitioners time to figure out good practices which prevent critical care patients from progressing to fatality. deeper studies of this factor, comparing case data from different regions, is called for in future. it is possible that this is one of the most positive, and least discussed, outcomes of the lock-down. another possibility may also be conjectured. careful maintenance of social distancing, necessary to reduce r 0 , results in evolutionary pressure on the virus. lock-down and similar methods force the virus to evolve in a direction which maximizes its ability to reproduce, which it can do if the disease becomes less critical or asymptomatic, and the chances of fatality decrease. it would be interesting to compare different regions across the globe for changes in the serial time and cfr. at the observed rate of growth, and with the current rate of testing, more than 0.5% of the population in hot spots will begin to test positive for infections in about a month. a constant rate of growth of infections means that the number of hospital beds will also grow at the same rate, for as long as the epidemic is growing. even if the rate is slowed down heavily, as it is already in delhi, mumbai, and chennai, the demand for hospital facilities will keep on growing, as long as the epidemic grows. this demand is already beginning to outstrip resources in the larger cities. the mean interval between the start of symptoms and discharge was estimated to be 24.7 days [22] . this means that unless the doubling interval is kept above 35 days (= 24.7/ ln 2), the demand on hospitals will keep rising. of the places we studied, only kerala has begun to approach this break-even point. cfr is currently small, partly because medical facilities have been able to cope with the rate of growth. if the number of cases exceeds the capacity of the medical system, cases which might have recovered will be harder to treat. inevitably in such cases cfr will climb. it is useful to note that in figure 3 the statewide figures for cfr are higher than those for cities. this is a reflection of the relative paucity of medical services outside cities, and is a pointer to what might happen when the number of infections rises beyond the sustainable capacity of hospitals. i thank rahul banerjee, prahlad harsha, d. indumathi, and r. shankar for sharing collated data on various cities. i thank jayasree subramanian for providing me with the reference [14] . in this form of the equation time is measured in units of the case resolution time. this equation assumes that the fraction of susceptible persons is close to unity, and the fraction of persons in any other compartment is very small. as argued before, this is a reasonable assumption to make. the cumulative number of infections is then found by integration. there is no closed form result for the general case. if only the linear term in the expansion of r 0 is retained, then the function erfi is defined through the integral it is possible to use an expansion for t ≪ t , which gives the form i(t) = i(0) 1 λ 0 e λ0t 1 + ǫ(1 − λ 0 t + λ 2 0 t 2 ) + o(ǫ 2 ) (a5) where the notation λ 0 = r 0 0 − 1, and ǫ = r 1 0 /(λ 2 0 t ) are introduced. the imaginary part vanishes exponentially. this is easy to match to the phenomenological form i(t) = i(0) 2 t/(τ0+τ1t/t ′ ) = i(0) 2 t/τ0 1 − ln 2τ 1 where an artificial expansion parameter t ′ is introduced. it is set to unity after expansion. matching these two expansions is accurate only when λ 0 t is large. then the phenomenological parametrization of eq. (a2) can be connected to the parameters of (non-autonomous) evolution equations for the epidemic. note that t and t ′ are both regularization scales, in the sense of a renormalization group, whose numerical value need not be specified. in order to change units of time to days, it is necessary to choose a model of the epidemic. if one uses the seir model, then the unit of time would be the median interval between the appearance of symptoms and the time of fatality or recovery, whichever is earlier. this quantity, t 2 = 17.8 days [22] . if one instead uses the sir model, then it is appropriate to choose the unit of time to be the median interval between the beginning of the infection and the earlier of the time of fatality or recovery. this is t 1 + t 2 , where t 1 is the median pre-symptomatic period, t 1 = 5.1 days. here the conversion is made within the seir scheme. this gives when a constant τ is used, one can set τ 1 = 0 in the above formulae and write r 0 and τ instead of r 0 0 and τ 0 . evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic changes in contact patterns shape the dynamics of the covid-19 outbreak in china effect of non-pharmaceutical interventions to contain covid-19 in china first antibody surveys draw fire for quality, bias coronavirus disease 2019 (covid-19): a literature review identification of burden hotspots and risk factors for cholera in india: an observational study emergencies preparedness, response who, zika virus infection -india, emergencies preparedness, response strategy for covid19 testing in india global covid-19 case fatality rates ministry of health and family welfare, government of india, addressing social stigma associated with covid-19 undated advisory nationwide mortality studies to quantify causes of death: relevant lessons form india's million death study amdavad municipal corporation, covid-19 website greater chennai corporation, official twitter page collection of press releases by praveen jadiya, chief medical and health officer municipal commission of greater mumbai, health department, official handle district information office, pune, official twitter account covid-19 india api estimates of the severity of coronavirus disease 2019: a model based analysis inferring epidemic parameters for covid-19 from fatality counts in mumbai estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) coronavirus: surveillance is the key, kerala shows the way singapore univ. of tech. and design, data driven innovation lab, predictive monitoring of covid-19 coronavirus (covid-19) cases our world in data in this appendix the unit of time will be taken to be the inverse of the mean rate of fatality of the infected. in these units, r 0 is the average number of new infections caused by an infected person. r 0 depends on the infectivity of the virus, as well as an average degree of the contact network. as a result, it may be affect by public health policies, such as a lock down. say a policy measure has a time scale is t . due to this, r 0 may become time-dependent, and one may write a taylor series expansionone may introduce this into a typical epidemic model equation, to obtain di dt = (r 0 − 1)i, which gives log i(t) i(0) = t r 0 0 − 1 + key: cord-018792-oqwbmyft authors: ammon, andrea; sasse, julia; riedmann, klaus title: early disease management strategies in case of a smallpox outbreak date: 2007 journal: poxviruses doi: 10.1007/978-3-7643-7557-7_20 sha: doc_id: 18792 cord_uid: oqwbmyft as a consequence of the threat of smallpox being potentially used as a means of bioterrorism, many countries have developed preparedness plans for smallpox in the past few years. this chapter summarizes some of the most important issues for the management of smallpox. usually, the strategy for the management of clinical cases of poxviruses includes the early detection of cases, rapid laboratory diagnosis, an assessment of the risk of further spread and containment measures. for the early detection, different systems are being tested to identify suspected cases before a diagnosis is confirmed (e.g., syndromic surveillance). also it is necessary to provide special training on the disease pattern, including differential diagnosis, to clinicians and practitioners. if a suspected case has been identified, rapid diagnostic tests are required. in addition to the national and international notifications based on given case definitions, certain measures are necessary to allow an initial risk assessment of the epidemic development. for a rapid risk assessment, the investigations should follow the algorithms of epidemiological outbreak investigation such as the tracing and identification of exposed contacts and the sources of infection. further decisions have to be taken on the basis of a continuous risk assessment. countermeasures can be divided into medical and non-medical ones. the choice of an adequate vaccination strategy as a medical countermeasure for the case of a re-emergence of smallpox very much depends on the epidemic scenario, and the general availability and quality of a vaccine. logistic aspects of the vaccination strategies have to be considered in preparedness planning (e.g., resources necessary for the implementation of mass vaccinations), and also the prioritization of groups to be vaccinated. in addition non-medical measures to prevent the spread of infection, such as the isolation of cases and quarantining of exposed persons (e.g., contact persons of confirmed cases) have to be foreseen. the effectiveness of other measures like prohibition of mass gatherings or closure of institutions is often assessed in the light of historical events. however, they have to be considered within today’s ethical and societal context, taking into account, in particular, the increased number of people who are immunocompromised. since our knowledge of how the virus would behave today is limited to extrapolations from historical data and is therefore imperfect, these measures are still under discussion. all relevant groups should be involved in exercises to assure the effective operation of the plan mainly regarding communication and cooperation. after the eradication of smallpox, it was possible to cease the most successful strategy against smallpox, namely vaccination. apart from rare events like the outbreaks of monkeypox in the democratic republic of congo or in the usa [1, 2] , there has been no need to think about the management of this disease anymore. however, the threat of smallpox being used as a means of bioterrorism has forced reconsideration of the need for smallpox vaccinations and other measures to manage potential cases or outbreaks of smallpox. in the past few years, many countries have developed preparedness plans for smallpox. in the following chapter we have tried to summarize some of the most important issues for the management of smallpox. a full description of all the necessary parts of the preparedness plans would go beyond the space available here. the strategy for the management of clinical cases of poxviruses (occurring sporadically or in outbreaks) usually includes the early detection of cases, rapid laboratory diagnosis, an assessment of the risk of further spread and containment measures. early detection of a first smallpox case will be crucial for a successful management of any new outbreak. the earlier anti-epidemic countermeasures are initiated, the more likely the epidemic can be controlled or prevented in time and casualties can be limited. conventional surveillance systems like epidemiological surveillance of a well-defined set of clinically suspected diseases or laboratory confirmed agents are important to monitor and control the occurrence of infectious diseases. yet, these systems usually detect outbreaks or unusual epidemic developments only with a certain time delay. therefore, planning considerations include concepts that identify an attack as early as possible [3] . among such systems are for example strategies to monitor the number of emergency department visits, over-the-counter medication sales or school absenteeism. also, environmental monitoring systems like air samplers, which permanently test the air for threat agents to detect a biological agent before it causes symptoms, have been suggested. since they only cover selected areas and have to be analyzed against a background noise, they do not necessarily guarantee a timely recognition of a biological threat [3] . after 11th september 2001, various models of syndromic surveillance have been established and tested in the united states for different syndromes (e.g., [4] ), but they also still need to prove their value in detecting a bioterrorist attack in a timely manner. most likely a deliberate release of smallpox would not be detected unless one or more human cases with clinical symptoms of the disease occurred. the early clinical detection of a smallpox case requires familiarity with the disease pattern. the number of the actually practicing physicians who have clinical experience with smallpox patients is decreasing, and it is therefore necessary to provide special training on the disease pattern, including differential diagnosis to clinicians and practitioners. the emergence of highly contagious diseases with high mortality and morbidity rates pose an immediate threat to public health and ask for a real time detection of the onset. as a separate chapter in this book describes poxvirus diagnostics, we will not go into specific diagnostic techniques. a very important issue is the necessity to confirm any suspicion of smallpox as fast as possible to avoid false alarms with far-reaching consequences. to ensure the safety of staff involved in taking samples and performing the diagnostics, good cooperation and agreed procedures between health authorities, clinicians and laboratory staff are required. electron microscopy and nucleic acid detection are the fastest methods and can give results within 24 h. for culturing the virus, biosafety level 4 facilities are required. an initial suspected smallpox case triggers various notifications according to the requirements of national and international health legislation and regulations. furthermore, if a deliberate release of the virus seems possible, 1 an actual threat to the affected state has to be presumed. in this case, disaster management and law enforcement agencies will assist the responsible health authorities to guarantee a comprehensive management in case of a confirmation and the likely spread of the disease. epidemiological and criminal investigation should be coordinated. in addition to the national and international notifications based on given case definitions, certain measures are necessary to allow an initial risk assessment of the epidemic development. these measures should follow the algorithms of epidemiological outbreak investigation, such as the tracing and identification of exposed contacts and the sources of infection. further decisions have to be taken on the basis of a continuous risk assessment. immediate anti-epidemic measures are of considerable importance. a permanent monitoring of the epidemic is necessary to guarantee that the effectiveness of the measures taken can be accurately evaluated, which in turn can lead to new measures or to a modification of the actual strategy. the following target groups for intervention measures can be distinguished: smallpox patients must be transferred immediately to a hospital with an isolation unit for further treatment. if no adequate infrastructure is available, isolation standards should be followed as well as possible (for requirements for isolation and isolation facilities see tab. 1). most important is the vaccination of the contact persons as soon as possible within the first 4 days after exposure and their isolation and observation either at home or in hospital. contraindications, e.g., history of severe eczema or immunodeficiency have to be weighed against the risk of disease. the treatment of complications resulting from vaccination must be also taken into account. even after a deliberate release, it is rather unlikely that a major epidemic or pandemic will occur if the appropriate countermeasures are taken in time. in the event of a smallpox outbreak the population can be protected by the prompt implementation of a vaccination campaign adapted to the epidemic realities. due to the historical experience, a second eradication of the smallpox disease is possible on the basis of the known eradication measures. the bigger challenge will be the identification and elimination of the sources of the intentional release. furthermore, the spread of a smallpox epidemic can be counteracted by limiting access to public facilities and events and by restricting freedom of movement. in addition, recommending appropriate protective measures and risk avoidance behavior to the population will be helpful. it is most important that all the measures taken are communicated to the public according to best practice of a consistent risk communication. the general public has to be given consistent information adapted to target groups and the situation via the available media. information of general relevance can be broadcast nationwide by television, for example, whereas information of regional or local relevance can be transmitted via other media (radio, local newspapers, cars with loudspeakers, leaflets, etc.). the information to be disseminated will include recommendations for protective measures as well as the announcement of restrictions on entry to events and facilities. the protection of the non-infected population will necessitate quarantine measures for suspect cases. as viruses do not recognize national borders, international cooperation is also of decisive importance. this may include technical and personnel support as well as the exchange and coordination of information but also coordinated action. in the revised international health regulations adopted by the world health assembly in 2005, smallpox is one of the four diseases (the other three are poliomyelitis due to wild-type poliovirus; human influenza caused by a new subtype; severe acute respiratory syndrome, sars) for which just a single case case is considered unusual or unexpected with potentially serious public health impact, and thus must be notified (http://www.who.int/csr/ ihr/wha58_3-en.pdf, accessed 6th may 2006). who member states have 5 years to implement the necessary systems for surveillance and response including national focal points, which have to be accessible at all times for communication with the who focal points. the choice of an adequate vaccination strategy for the case of a re-emergence of smallpox in a country very much depends on the epidemic scenario one has in mind and the general availability and quality of a vaccine. at the same time, logistic aspects of the vaccination strategies have to be considered in preparedness planning, i.e., the facility and personnel resources necessary for the implementation of mass vaccinations have to be determined and identified. with the exception of the very unlikely situations of an accidental release or a natural re-emergence [caused, for example, by mutants of orthopoxviruses (camel-or monkeypox)], the only realistic scenario for a re-emergence of smallpox is a deliberate release of the agent, which does not necessarily have to follow historic patterns of epidemic spread. simultaneous and multilocal outbreaks are possible and have to be included as possible scenarios for a comprehensive preparedness planning. predictive modeling of the epidemic spread has to rely entirely on historic data and is of limited value. the availability and quality of a vaccine has the most significant influence on the strategy, as there is no evidence of an effective therapy with antiviral drugs against a smallpox infection in humans. the chosen strategy will be determined by the particular epidemiological situation and consideration of the threat of further releases and the risk of secondary infections compared with the well-known adverse effects of the currently available vaccines. unlike during a natural outbreak, the threat of additional intentional releases has to be considered for a vaccination policy. various models have been developed to assist in identifying the best use of the available vaccines (e.g., [5] [6] [7] [8] ), as well as other control measures like case isolation and contact tracing or combinations thereof [9, 10] . since all these models have different assumptions for important parameters (like r 0 ), the conclusions also vary. following historical data from the last natural, in this case imported, smallpox cases in europe in the decades before and during the eradication, the first step will be -after the immediate isolation measures have been initiated -the vaccination of contacts and simultaneous ring vaccinations. there are efforts to predict the best anti-epidemic measures on the base of mathematic modeling [7, 9, [11] [12] [13] [14] [15] . such models are fitted in such a way that they can reproduce historical outbreaks very well and try to predict the effects of different anti-epidemic measures on the basis of historical data. the quality and predictive value are limited and depend very much on the inclusion of a sufficient number of necessary and correct parameters. a slight change in a parameter can lead to exaggerated effects that do not follow the common sense experience. a lot of the decisive factors can only be roughly estimated, like transmission rate, population immunity or the effectiveness of a post-exposure vaccination. furthermore, as the re-emergence of smallpox is most likely to result from a deliberate release and multiple geographically unlinked outbreaks may be possible, this historically based vaccination strategy might seem idealistic. public and political pressure and security considerations may quickly lead to the ultimate step, the mandatory vaccination of the entire population. nevertheless, this should be done after a careful risk-benefit-calculation considering the serious adverse effects of the available vaccines. vaccination priorities: first responders, other priority groups no matter which strategy is chosen the availability of vaccine is a key issue. most industrialized countries have acquired a certain stockpile of first or second generation vaccine. the sizes of the stockpiles vary from country to country. some countries have sufficient stockpiles to cover the whole population, some do not. therefore, priority population groups have to be identified for vaccination -in accordance with epidemiological, political, ethical and societal necessities and based on a public consensus. as long as there are no smallpox cases worldwide, obligatory prophylactic vaccinations especially of entire populations are not necessary. the re-emergence of smallpox has a limited likelihood, whereas the certainty of serious adverse effects due to vaccination is a proven fact. nevertheless, it can be necessary if there is an increased likelihood of occupational expo-sure. 2 prophylactic vaccination may seem useful for the staff of special isolation units, which are most likely to treat the first smallpox cases or of those laboratories designated for confirmatory diagnostics. in this phase also members of infectious disease task forces (interdisciplinary teams on any administrative level for the initial risk assessment and subsequent investigations) may be offered vaccination on a voluntary basis. as soon as a first smallpox case is confirmed worldwide, and a real threat and exposure seem more likely, the offer of voluntary vaccination to all professional groups who are required to keep the necessary public services running during a smallpox epidemic has to be considered. these groups include mainly medical staff, fire brigades and disaster relief organizations, red cross etc., but also people working in critical infrastructures (power and water supply, public transportation and communication) or for public security and order or on the administration or political level, i.e. those population groups who are relevant for the maintenance of public life. once a smallpox case is confirmed, vaccination strategies should focus on the necessities of an anti-epidemic management. first of all the population being affected or at risk must be vaccinated. if the epidemic spread cannot be controlled, mandatory mass vaccinations will be necessary. smallpox can be spread by droplets and by direct or indirect contact with the pustules on the skin. this assumes that all primary contact persons of a confirmed smallpox case (see tab. 2) may be infected and must be identified as soon as possible. the risk of infection for persons with an extended contact time or a close contact distance is much higher than for persons with a short contact time. according to historical data, the highest risk of infection exists for household members or hospital contacts. the european outbreaks between 1950 and 1971 showed that 55% of the infected persons contracted smallpox at a hospital, 20% in the family, 14% at their working place or school and 3% of the infected persons were working in a laundry, while 8% were unidentified contacts. none of the 945 smallpox cases in europe since the second world war contracted it on an airplane, a train or a bus [16] . yet, under special conditions, an airborne transmission may be possible. in a hospital in meschede, germany, patients and nurses from the two floors above the floor where a smallpox patient was treated were infected by air circulation [17, 18] . based on publications on smallpox transmissions, table 2 describes the risks of infection. it might be impossible to control an outbreak of smallpox using only vaccination, therefore isolation of cases and monitoring of the contacts may be necessary in addition [9, 19] . quarantine in an isolation ward for all persons who were exposed seems to be the safest way, but it has some limitations, like the quantity of qualified isolation wards, the supply of the population with food, drinking water etc. and the cooperation of the population. therefore, it will be helpful to adjust the anti-epidemic measures to the likelihood of developing the disease (tab. 1) [20] . the isolation concept should be adapted to the epidemic situation, the requirements on effective isolation and the expected number of contact persons. the personnel in all hospitals/facilities must be vaccinated and trained, personal protective equipment (including gloves, masks, goggles, gowns) and means to follow the hygiene measures must be available. if prihigh risk -persons who are living in the same household with the patient and persons with a similar risk of infection (members of the family and household contacts, etc.) -persons who have had "face-to-face-contact" with a sick person, which includes all persons, who have been so close to the patient that they could be infected by droplets, or who have touched the efflorescence of the skin [e.g., friends or neighbors who have taken care of the patient, physicians who have been consulted before the hospital, hospital staff (medical doctors, nurses, cleaning staff), persons in a public traffic system with direct contact, i.e., less than ca. 2 m to the infectious case of smallpox, etc.] -persons who have been longer in the same (confined) room with a patient (e.g., work colleagues, transport staff of the ambulance, etc.) -persons who have direct contact with the dead body of a smallpox patient (e.g., undertaker, pathologist, priest, etc.) -persons who have worked with infectious samples of a smallpox patient without appropriate protection -persons who have touched scabs of a smallpox patients without appropriate protection -persons who have had direct, non-protected contact with the personal clothes, bed linen or other personal belongings, materials that a smallpox patient wore or used after the onset of fever medium risk -persons who are in the same building as a smallpox case, if this building has a ventilation system, air conditioning or comparable installation systems that circulate the air between different rooms in the building -persons who have traveled in the same compartment of a public transportation system or airplane with a ventilation system, air conditioning or comparable installation systems to circulate the air low risk -persons with a short and/or not close contact to an infectious smallpox case (e.g., a short stay in the same room, or a longer stay in the same building without ventilation system, air conditioning or comparable installation systems to circulate the air; sharing the same public transportation system without ventilation system, air conditioning or comparable installation to circulate the air; distance to the index case > 2 m) -medical staff, if they have used appropriate personal protection equipment mary contacts develop fever and other typical symptoms of smallpox, their transfer to a hospital with isolation ward is immediately necessary. for contact persons with a low risk of infection and a timely, successful vaccination, segregation at home seems to be appropriate as long as they have not developed fever, all household contacts have been vaccinated and the local health authority has the capacity to observe them daily. nevertheless, it must be kept in mind that a vaccination, even when administered in time, does not yield 100% protection. according to historical data, the risk of infection for vaccinated household contacts of a smallpox patient in the past was 3.7% [21] , in comparison to 65% of unvaccinated household contacts. these data did not give any information about when the contact persons had had their last vaccination. vaccination should also be offered to secondary contact persons. they must be registered because they will become primary contacts themselves if the originally primary contact develops the disease. since transmission of smallpox is favored by close distance between persons, so-called "social distancing" measures are considered as further intervention measures to stop the spread. whereas the isolation of cases or segregation of exposed persons (contacts) is not under debate, the effectiveness of other measures like prohibition of mass gatherings, closure of institutions or even curfews are often assessed in the light of historical events. however, they should be considered within today's ethical and societal context, taking into account differences in the society, in travel behavior, and the increased recognition of contraindications to vaccination [10] . also, the number of people who are immunocompromised (due to hiv, chemotherapy, transplantations etc.) has increased [10] . these measures are still under discussion, since we have limited knowledge of how the virus would behave today. according to the vaccination strategy described above, the majority of vaccinations would be carried out in the case of the real event. therefore, elaborate preparations have to be implemented in the pre-event phase. smallpox vaccine and bifurcated needles have to be procured and stockpiled. some governments have a national stockpile of smallpox vaccines, but not all of them have a stockpile covering the need of their entire population. therefore, multi-lateral support in the case of an event has to be assured in time. within the european union, a task force on bioterrorism was set up in may 2002 with the main objective of implementing the health security program [22] . the world health organization (who) has to convince some states to contribute to an international stockpile at who level. for national stockpiles, the logistics for storage, transport and distribution have to be determined in advance as well. to allow immediate mass vaccinations, the required infrastructure, such as facilities or personnel, has to be identified and the latter informed and trained in time. the entire process should be tested and practiced in simulation exercises. when choosing vaccination facilities important aspects have to be considered to enable the vaccination of a large number of people in a very short time, such as: -number and size of vaccination facilities according to population density -transport connections -easy access, also for handicapped people -water and energy supply -toilets -possibility of separate treatment of suspected cases -availability of rooms for personnel, first aid, treatment -phone -furniture material for documentation of the vaccinations and checking of contraindications (questionnaires, vaccination list/card) as well as information for the public has to be produced in advance and distributed to the authorities. they take care of the implementation of preparedness measures on the regional and local level. other tasks have to be achieved or initiated in the pre-event phase as well: vaccination of the vaccinators, training of the necessary staff and provision of the material needed at the vaccination facilities. a survey of over 14 million vaccinations in the usa in 1968 showed that per million vaccinations there were 75 serious adverse effects, including 1 death [23] . some of the known adverse effects that may arise from smallpox vaccination are post-vaccination encephalitis, progressive vaccinia, eczema vaccinatum or generalized vaccinia. therefore, the production of modern and more compliant vaccines is under consideration. a way to minimize the adverse events of smallpox vaccination might be the use of modified vaccinia virus ankara (mva), which was developed in the 1970s by more than 500 passages in chicken embryo fibroblasts [24] . however, smallpox had been eradicated before the efficiency of the protective effect of mva could be tested. experiments with animals indi-cate that there may be fewer complications after vaccination with mva [25] [26] [27] , and show also that mva provokes a high antibody titer and a high concentration of ifn--positive cells. some data show that mva-vaccinated animals are protected against smallpox infection [26, 28] , but other results allow the interpretation that a mva-vaccination alone can not guarantee a full protection against infection [25] . mva might be a good candidate for a pre-immunization [25] or for persons with strong contraindications [26, 29] . other replication-deficient vacv strains have also been developed for immunization [14, [30] [31] [32] . some mva strains currently under development require a higher virus titer as they do not replicate in the human body. vacv strains have the potential to inducing post vaccination encephalitis. derived from historical data with 1-2 cases per million, the vaccination of the entire population of a country like germany would lead to 80-160 cases of severest adverse effects. finally, a lot of research is being performed to develop new vaccines. experiments on a dna basis are very promising, even if these vaccines do not fully protect from infection yet [33, 34] . all the vaccines under development are still in the pre-clinical state. usually, vaccination strategies are chosen on the basis of scientific evidence and national health legislation. for the special case of smallpox, the only vaccine which has proven its efficiency decades ago is known to produce serious side effects. therefore, legal regulations for the financial compensation of vaccination damages have to be agreed upon and guaranteed before the implementation of vaccinations, no matter if they are being recommended for occupational safety reasons in the pre-event phase or as antiepidemic measure in the case of an event. more than 20 years after the eradication of smallpox only very few health professionals have practical experience with the management of this disease. therefore, all relevant professions involved in the management of a smallpox outbreak or epidemic have to be trained on the disease pattern and its specific consequences on their professional tasks. training must include the professional implementation of sampling techniques as well as safe transport, which have to be arranged in advance to avoid any unnecessary delay or hazard from improper handling or packaging. the laboratories selected for smallpox diagnostics have to guarantee that this can be done both rapidly and with assured quality. these labora-tories have to immediately report a suspected or confirmed 3 laboratory diagnosis to the appropriate authorities. public health officers, clinicians and practitioners for example have to update their knowledge on the clinical picture to guarantee an early recognition of the disease and also get familiar with the treatment and therapy of smallpox cases. laboratory personnel have to be trained in the diagnostics of smallpox on the basis of the standard operating procedures. the validity of the diagnosis is improved by regular participation in a quality assurance system. in general, if preparedness plans exist, they have to be evaluated among all the relevant groups by exercises to assure the effective operation of the plan mainly in the field of communication and cooperation. public health services might test the implementation of mass vaccinations or the reporting systems for a smallpox alert; clinicians might check the clinics' preparedness plans for cases of highly contagious diseases, ambulance services might train for the transport of highly contagious patients and all together they might check the interaction between the relevant actors aiming at a harmonization of the preparedness planning. outbreak of human monkeypox update: multistate outbreak of monkeypox -illinois advances in 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without immune deficiencies against pathogenic vaccinia virus challenge modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses modified vaccinia ankara; potential as an alternative smallpox vaccine immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus ankara induction of potent humoral and cell-mediated immune responses by attenuated vaccinia virus vectors with deleted serpin genes genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia lc16m8 smallpox vaccines: looking beyond the next generation smallpox dna vaccine protects nonhuman primates against lethal monkeypox key: cord-244687-xmry4xj4 authors: hsieh, chung-han title: on control of epidemics with application to covid-19 date: 2020-11-02 journal: nan doi: nan sha: doc_id: 244687 cord_uid: xmry4xj4 at the time of writing, the ongoing covid-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), had already resulted in more than thirty-two million cases infected and more than one million deaths worldwide. given the fact that the pandemic is still threatening health and safety, it is in the urgency to understand the covid-19 contagion process and know how it might be controlled. with this motivation in mind, in this paper, we consider a version of a stochastic discrete-time susceptible-infected-recovered-death~(sird)-based epidemiological model with two uncertainties: the uncertain rate of infected cases which are undetected or asymptomatic, and the uncertain effectiveness rate of control. our aim is to study the effect of an epidemic control policy on the uncertain model in a control-theoretic framework. we begin by providing the closed-form solutions of states in the modified sird-based model such as infected cases, susceptible cases, recovered cases, and deceased cases. then, the corresponding expected states and the technical lower and upper bounds for those states are provided as well. subsequently, we consider two epidemic control problems to be addressed: one is almost sure epidemic control problem and the other average epidemic control problem. having defined the two problems, our main results are a set of sufficient conditions on a class of linear control policy which assures that the epidemic is"well-controlled"; i.e., both of the infected cases and deceased cases are upper bounded uniformly and the number of infected cases converges to zero asymptotically. our numerical studies, using the historical covid-19 contagion data in the united states, suggest that our appealingly simple model and control framework can provide a reasonable epidemic control performance compared to the ongoing pandemic situation. at the time of writing, according to the world health organization, the ongoing covid-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), had already resulted in more than thirty-two million cases infected and more than one million deaths worldwide; see [19] , and even worse, the pandemic seems to be "showing no clear signs of slowing down" in many countries. to this end, governments are persistently striving to control and slow down the spread of covid-19; e.g., minimizing the contact rate by consulting non-pharmaceutical intervention mechanisms such as lock-downs and social distancing or relying on some pharmaceutical interventions such as enhancing medical treatments, and developing possible cure or remedies. with this motivation in mind, the focal point for this paper, as a preliminary work, is to understand how the disease spreads and how it would be possibly controlled. the remainder of the paper is organized as follows: in section 2 we describe the preliminaries involving the details of our modified sird-based epidemiological model. then two problem formulations for epidemic control: almost sure epidemic control problem and average epidemic control problem are stated. subsequently, in sections 3, detailed analyses on our epidemiological model are provided. in sections 4 and 5, using a linear epidemic control policy, we provide sufficient conditions on feedback gain so that the epidemic can be properly controlled in either expected value sense or almost sure sense. next, in section 6, we describe a simple model parameter estimation approach and illustrate its use via numerical examples involving historical covid-19 data from the united states, the country among those with a relatively high confirmed cases in the middle of 2020. the control performance is also discussed. finally, in section 7, we provide some concluding remarks and promising directions for future research. in the sequel, we take k to be the index indicating the day number and u(k) ≥ 0 to be the corresponding abstract epidemic control policy implemented by governments. 1 we view that the large values of u(k) correspond to enhanced medical treatments or stringent non-pharmaceutical interventions such as mandating social distancing with wearing the masks. the smaller values of u(k) might correspond to diminished medical treatments or relaxation of the rules. in the analysis to follow, we assume that the epidemic control policy u(k) is causal ; i.e., it only depends on the past and current information that is available at hand-not the future. for k = 0, 1, . . . , let s(k) be the number of susceptible cases at the kth day, i(k) be the infected cases at the kth day and r(k) be the recovered cases at the kth day, and d(k) be the deceased cases at the kth day. take n (k) be the underlying human population at the kth day satisfying n (k) = s(k)+i(k)+r(k). as mentioned in section 1, many infected individuals may go undetected or asymptomatic [16] and the effectiveness of a control policy u(k) may be uncertain at the time, we introduce two uncertainty quantities: the uncertain rate of infected cases which are undetected or asymptomatic, call it δ(k), and the uncertain effectiveness rate of control, call it v(k), in our model to follow. now, with initial values s(0) := n (0) := s 0 , i(0) := i 0 > 0, and r(0) = d(0) = 0, we consider a discrete-time stochastic epidemiological model with uncertainties described by where d i (k) is the death rate for infected cases satisfying d i (k) ∈ [0, d max ] with d max < 1 for all k. we assume that the sequence d i (k) are independent and identically distributed (i.i.d.) random variables. in addition, we assume that both of δ(k) and v(k) are i.i.d. random variables with arbitrary distribution but known bounds 0 ≤ δ(k) ≤ δ max and 0 < v min ≤ v(k) ≤ v max ≤ 1, respectively. 2 if v(k) = 1, it corresponds to the case where the control policy is extremely effective. on the other hand, if v(k) ≈ 0, it corresponds to an extremely ineffective case; i.e., the people are not disciplined and may be unwilling to follow the medical advise for masks or social distancing; see also a discussion in section 3.5. in the sequel, we assume further that i 0 δ max < s 0 ; and v(k), δ(k), and d i (k) are mutually independent. for the sake of notational convenience, we also take the shorthand notations δ : remark on the epidemiology model. as a preliminary work, while we allow uncertainties on both infected cases i(k) and control u(k), our epidemiological model 1 assumes that u(·) can interact with the infected cases instantaneously without any time delay. the action with delay is left for future work; see also section 7. it is also worth mentioning that, if one considers a linear control policy u(k) = ki(k) with constant k ≥ 0, then a basic reproduction ratio of sorts, call it r 0 (·), can be obtained as follows: for k ≥ 0, where β(k) := δ(k)(s(k) + i(k)))/s(k) is the infectious rate and γ(k) := v(k)k is the recovery rate. if r 0 (·) > 1, the number of infected cases is expected to increase; if r 0 (·) < 1, then the infected cases decrease. finally, we should mention that, in terms of the terminology in systems and control theory, the epidemiological dynamics described by equation (1) is indeed an uncertain linear time-varying system. this observation is useful in the following sections to follow. in this subsection, we introduce the first epidemic control problem, which we call the almost sure epidemic control problem. that is, given the modified sird model 1, we seek a control policy u(·) which assures the following conditions hold: (i) the ratio of infected cases converge to zero with probability one; i.e., remarks. the results related to this almost sure epidemic control problem is discussed in section 4 when a linear control policy of the form u(k) = ki(k) with pure gain k ≥ 0 for all k ≥ 0 is applied. while almost sure epidemic control policy, if exists, can be a good candidate to mitigate the pandemic. however, in practice, some potential issues remain. for example, in some cases, the cost for implementing an almost sure epidemic control policy may be still too expensive in practice; see remark 4.1.2 in section 4. to hedge this issue, we now introduce our second epidemic control problem which we call the average epidemic control problem aimed at controlling the "expected" infected cases and "expected" deceased cases. to address the issue raised in previous subsection, we consider the second epidemic control problem which we call the average epidemic control problem aimed at controlling the "expected" infected cases and "expected" deceased cases. rigorously speaking, given the modified sird model 1, we seek a control policy u(·) which assures the following conditions hold: remark. similar to the previous remark, the results related to this average epidemic control problem is discussed in section 5 when a linear control policy of the form u(k) = ki(k) with k ≥ 0 for all k ≥ 0 is applied. in this section, to understand the contagion process and the evolution of the epidemic, we assume that there exists a control policy u(·) which assures that infected cases i(k), susceptible cases s(k), recovered cases r(k) and deceased cases d(k) are all nonnegative for all k with probability one. 3 the analytic expression for infected cases, susceptible cases, recovered cases, and deceased cases are provided. the results obtained in this section are useful for the subsequent sections to follow. in the sequel, we use a shorthand notation for integer k ≥ k 0 ≥ 0. we mention here that φ(k, k 0 ) is a state transition function for infected cases from day k 0 to k. the reader is referred to [7] for detailed discussion on this topic. we may sometimes write φ i (·, ·) instead of φ(·, ·) to emphasize that the function is related to the infected cases. the following lemma characterizes some useful properties of the function. proof. to prove part (i), we let f (k) := 1 + δ(k) − d i (k). since δ(k) ∈ [0, δ max ] and d i (k) ≥ 0 for all k ≥ k 0 ≥ 0, we have 1 − d max ≤ f (k) ≤ 1 + δ max for all k with probability one. note that the upper and lower bounds are nonnegative. hence, a straightforward multiplication leads to the desired inequality and the proof of part (i) is complete. to prove part (ii), with the aid of the fact that δ(k) and d i (k) are i.i.d. in k and are mutually independent, we obtain having proved lemma 3.1, we are now ready to characterize the infected cases i(k). given i 0 > 0, the infected cases at the kth day is described by for k ≥ 1. moreover, the expected infected cases satisfy proof. since the infected cases dynamics (1) is a linear time-varying system, the solution i(k) and its corresponding proof are well-established; hence we omitted here and refer the reader to any standard textbook in system theory; e.g., see [7] . to complete the proof, we must show that the expected infected cases can be characterized by the form described in the lemma. using lemma 3.1 and the fact that v(k) ≥ v min and are i.i.d., we observe that note that u(i), as assumed in section 2, is causal; i.e., it only depends on the information available up to the ith day. in addition, according to equation (2) , the state transition function φ i (k, i + 1) = as seen later in the next section, if one adopts the linear feedback policy of the form u(k) = ki(k), then the solution i(k) described in lemma 3.2 can be greatly simplified. the following lemma gives a closed-form expression for the susceptible cases s(k). with s(0) = s 0 > 0, the number of susceptible cases at the kth day is given by using lemma 3.2, we obtain in this subsection, we characterize the solution of recovered cases and some of its useful properties. with r(0) = 0, the recovered cases at the kth day is given by for k ≥ 1. proof. while the proof is simple, for the sake of completion, we provide a full proof here. we first recall that r(0) = 0 by assumption on initial condition. to complete the proof, we proceed a proof by induction. begin by noting that for k = 1, the recursion on r(k) tells us that to complete the proof, we now show the desired expected recovered cases and its bounds. for which is desired. it is readily seen that r(k) is increasing in k since u, v are nonnegative. the number of deceased cases and its expectation are provided in this subsection. for k ≥ 1. moreover, the expected number of death is bounded by proof. with d(0) = 0, we begin by recalling that the deaths dynamics (1) is given by d(k + 1) = d(k) + d i (k)i(k) for k ≥ 0. an almost identical proof as seen in lemma 3.2 leads to to complete the proof, we take expectation on equation 5 and, with the linearity of expectation and the fact 0 ≤ d i (k) ≤ d max , the desired inequality follows immediately. if there are low or no medical supports, i.e., u(k) ≈ 0, or the medical treatments are lacking or extremely ineffective, due to the reasons such as the people are not disciplined or unwilling to follow the medical advise to wearing masks or social distancing; i.e., v(k) ≈ 0, then it is readily verified that the susceptible cases, infected cases, recovered cases, and deceased cases become consistent with the intuition, the equation (6) above tells us that the recovered cases r(k) are close to zero approximately. on the other hand, the infected cases i(k) and the deceased cases d(k) are both increasing monotonically; i.e., an "outbreak" is seen. to mitigate the pandemic, in this paper, we consider a class of linear epidemic control policy of the form u(k) := ki(k) for k ≥ 0 where k ≥ 0 is the feedback gain which represents the degree of the epidemic control effort on providing medical treatment or mandating the non-pharmaceutical interventions. in the sequel, we sometimes call the linear epidemic control policy above as linear feedback policy or just linear policy for the sake of brevity. as mentioned in section 2, our objective of this section is to address the almost sure epidemic control problem using the linear control policy. we begin with discussing the control of infected cases in the almost sure sense. the following lemma is useful for deriving one of our main results: theorem 4.2. . then the infected cases dynamics becomes therefore, the product of f (i) is also nonnegative which implies that i(k) ≥ 0 for all k with probability one. to complete the proof of part (i), we note that for any k ∈ [0, 1−dmax vmax ], the infected cases satisfy to prove part (ii), an almost identical proof as seen in part (i) applies. that is, if k < (1 − d max )/v max , then i(k) > 0 for all k with probability one. according to part (ii) of the lemma above, we see that if k < (1 − d max )/v max , then the infected cases i(k) is strictly positive for all k with probability one. said another way, the infected cases can not be eradicated at any time. however, as seen in the next theorem, the ratio of the infected cases can go down to zero asymptotically for some k. then we have (i) lim k→∞ i(k)/i 0 = 0 with probability one. (ii) the controlled infected cases are upper bounded uniformly; i.e., i(k) < i 0 for all k with probability one. if k = δmax vmin , then i(k) ≤ i 0 . proof. to prove (i), we observe that as defined in the proof of lemma 4.1. with the assumed inequalities and feedback gain δmax vmin < k < 1−dmax vmax , it is readily verified that 0 < f (i) < 1 for all i = 0, 1, . . . , k − 1. to show the limit of the ratio i(k)/i 0 is zero with probability one, we write we also note that the logarithmic function above is well-defined for k within the assumed range. hence, now, since f (i) are i.i.d., by the strong law of large number (slln); e.g., see [10] , we have as k → ∞ with probability one. now, using the fact that the logarithmic function is strictly concave, jensen's inequality yields where the last inequality hold by the monotonicity of logarithmic function. hence, log(1+δ−kv) ≤ 0 for any k ≥ δ max /v min ≥ δ/v. therefore, e[log f (0)] < 0, which implies that lim k→∞ i(k)/i 0 → 0 and the proof for part (i) is complete. to prove (ii), we must show that the i(k) is upper bounded by the initial infected cases i 0 . as seen in the proof of part (i) of lemma 4.1, we have, for all k, where the last inequality holds by using the assumed fact that k ≥ δmax vmin . in practice, since the medical resource is limited, it is natural to put additional constraints on the feedback gain such as k ∈ [0, l] with l ≥ 1 being the constant which corresponds to the maximum allowable medical resources to be used. hence, other than the sufficient condition 7, for any δ max < vmin(1−dmax) vmax , one might require k to satisfy however, one should note that the set k might be an empty set. to see this, we consider l = 1, v min = v max = 0.01 and δ max = 0.1 and d max = 0.01. then it is readily verified that the condition δ max < vmin(1−dmax) vmax but k = (10, 99)∩[0, 1] = ∅. if k = ∅, it tells us that the infected cases does not converge to zero asymptotically and the epidemic is uncontrollable. if this is the case, one should put whatever they can to suppress the disease; e.g., by putting k := l for possible l ≥ 1. on the other hand, given the fact that the higher feedback gain would cause a higher consumption on medical/economical resources, one should choose the lowest feedback gain which is nonzero. hence, an immediate "sub-optimal" choice would simply be as follows: if δ max ≤ v min we take k * := δ max /v min with the aim that i(k) ≤ i 0 can be guaranteed at least. on the other hand, if δ max > v min , we take k * := l. or, we can write it in a more compact way as follows: provided that δ max < vmin(1−dmax) vmax where 1 {δmax≤lvmin} and 1 {δmax>lvmin} are the indicator function; see [11] for a detailed discussion on this topic. as seen later in section 6, the k * above will be adopted to test the epidemic control performance using historical data. in this subsection, the recovered cases r(k), susceptible cases s(k), and deceased cases d(k) under linear control policy are discussed. the analysis of recovered cases is simple. we begin by recalling lemma 3.4 and write r(k) = k−1 i=0 v(i)u(i). now, by taking linear control policy u(k) = ki(k) with gain 0 ≤ k ≤ (1 − d max )/v max , we have r(k) ≥ 0 for all k with probability one and where the last inequality holds by using lemma 4.1. for k = 1, 2, . . . , ⌊s 0 /(i 0 δ max )⌋ with probability one where ⌊z⌋ is the floor function satisfying ⌊z⌋ := max{n ∈ z : n ≤ z}. proof. similar to the proof of lemma 3.3, recalling that s(k+1) = s(k)−δ(k)i(k), a straightforward inductive calculation leads to using lemma 4.1, the desired form of solution follows immediately. to complete the proof, we note that thus, via a lengthy but straightforward calculations, it is readily verified that s(k) ≥ 0 for k ≤ ⌊s 0 /(i 0 δ max )⌋ with probability one. remark. while the lemma above tells us that s(k) ≥ 0 holds up to k ≤ ⌊s 0 /(i 0 δ max )⌋, we should note that the initial cases s 0 = n (0) are often far larger than the denominator i 0 δ max . hence, without loss of generality, in the sequel, we deemed that s(k) ≥ 0 for sufficiently large k. the next lemma indicates that the deceased cases under the linear control policy. for all k ≥ 1 with probability one. proof. we begin by recalling that d(k) = k−1 i=0 d i (i)i(i) for all k ≥ 1. hence, using lemma 4.1, we have note here that the assumption k ≤ (1 − d max )/v max assures i(k) ≥ 0 for all k with probability one; hence the inequality above is well-defined. to complete the proof, with the aid of sum of geometric series, we have which completes the proof. with the aids of theorem 4.2 and lemma 4.4, we see that if we take linear feedback policy with constant gain; i.e., u(k) = ki(k) and assuming that δ max < v min ( such that infected cases i(k) ≤ m d for all k with probability one. therefore, the almost sure epidemic control problem is solved. while the almost sure epidemic control policy, if exists, can be a good candidate to mitigate the pandemic, some potential issues remain in practice. that is, recalling remark 4.1.2 in section 4, it tells us that, in some cases, the almost sure epidemic control policy, while exist, may not be possible to implement. to address this, as mentioned in section 2, we now move to our second epidemic control problem which we call the average epidemic control problem. in this setting, the aim now becomes to control the "expected" infected cases and "expected" deceased cases in this section, we provide our results on control of epidemics in the sense of expected value. we begin with discussing the control of expected infected cases. the lemma below provides an useful analytical expression for the expected number of infected cases. proof. the proof is straightforward. we begin by noting that, for any linear policy u(k) = ki(k) with gain k ∈ [0, (1 − d max )/v max ], i(k) ≥ 0 for all k with probability one and i(k) = k−1 i=0 (1 + δ(i)−d i (i)−kv(i))i 0 . since δ(k), d i (k) and v(k) are i.i.d in k and are mutually independent, taking the expected value on the i(k) above yields and the proof is complete. with the aid of the lemma above, we are now ready to provide our second main result. proof. the idea of the proof is similar to the one used in theorem 4.2. however, for the sake of completeness, we provide our full proof here. we begin by assuming that δ < (1−dmax)v vmax and δ−di v /v max , it implies that i(k) > 0 for all k with probability one. now, according to lemma 5.1, we have where note that hence, log(1 + δ − d i − kv) < 0 for any k > (δ − d i )/v and the logarithmic function is welldefined for k within the assumed range. therefore, we have log e[f (0)] < 0, which implies that lim k→∞ e[i(k)]/i 0 → 0 and the proof for part (i) is complete. to prove part (ii), we fix k and simply note that, with the assumed assumptions on δ and k and the fact that d i (·) ≥ 0, it is readily verified that e[i(k)] ≤ i 0 , which completes the proof of part (ii). to prove part (iii), take k = (δ − d i )/v and substitute it back into the equation 12, we obtain e[i(k)] = i 0 for all k and the proof of part (iii) is complete. similar to remark 4.1.2, to accommodate the practical considerations, we fix l ≥ 1 and require due to the limited available medical resources, it is reasonable to choose a "sub-optimal" k in the sense of minimizing the potential use of medical resources. for example, we let 1 {δ≤lv} and 1 {δ>lv} be the indicator functions and consider provided that δ < v(1−dmax) vmax to be our choice of feedback gain for controlling of epidemics in the sense of expected value. note that k * > δ−di v for d i > 0; hence, the theorem above applies. this usage of k * will be also seen later in section 6. where the last inequality holds by using lemma 5.1. the next lemma states the expected susceptible cases. we begin by recalling that lemma 4.3 tells us that s(k) ≥ 0 for stage k = 1, 2, . . . , ⌊s 0 /(δ max i 0 )⌋. now using the facts that δ(k) are i.i.d. in k, and δ, d i and v are mutually independent, we have using the geometric series, we conclude which completes the proof. for k ≥ 1, any linear feedback control policy of the form u(k) = ki(k) with 0 ≤ k ≤ (1 − d max )/v max yields the expected deceased cases proof. the proof is similar to lemma 4.4. using lemma 4.1 and the fact that v(k), d i (k), and δ(k) are independent, we have it is easy to verify that d i (i) and (1 + δ(j) − d i (j) − kv(j)) are independent for all j = 0, . . . , i − 1; hence, we have using the geometric series, the equality above reduces to which is desired. for u(k) = ki(k), one can readily verify that with the aids of theorem 5. such that expected deceased cases e[d(k)] ≤ c d for all k. therefore, the average epidemic control problem is solved. in the next section to follow, we provide an illustrative example using historical covid-19 data to demonstrate our epidemic control performance. we now illustrate the application of our control methodology on the epidemiological model using historical data for year 2020 available in https://ourworldindata.org/coronavirus, which contain the number of daily confirmed cases, denoted by c(k), and daily reported deaths, denoted by d(k) for k = 0, 1, . . . , n − 1 for some fixed integer n . to study the epidemic control performance, there are various way to estimate the uncertain parameters; e.g., one can consult "standard" approach such as minimizing the least-square estimation error to obtain the "optimal" parameters δ, d i and v; e.g., see [5, 6, 9] . however, for the sake of simplicity, we now provide a simple "mean-replacing" approach to estimate the uncertain rate of infected cases δ(·) and the death rate d i via the available data of confirmed cases and the number of reported deaths. that is, we begin by recalling that the deceased cases satisfy d(k + 1) = d(k) + d i (k)i(k). for k = 0, 1, . . . , n − 1, given the confirmed (infected) cases c(k) and reported deaths d(k) and taking c(k) := i(k) and d(k) := d(k), we obtain then, the estimate of death rate d i (k), call it d i , is defined by 4 having found d i , we can now estimate the remaining two uncertain quantities δ(k) and v(k). to this end, assume that the adopted epidemic control policy is linear of the form u(k) = ki(k) with k := 1; i.e., we ideally assumed that government had put all available resources to control the pandemic. now, setting c(k) := i(k), d i := d i (k), and replacing v(k) by its mean v, we obtain with the aid of equation (16), the estimate of δ(k), call it δ, is given by having obtained the estimates δ and d i , we then ready to apply the epidemiological model and use it to compare it with the available data; see next subsections to follow. using the data from the start of the epidemy, over a horizon starting from march 1, 2020 to september 8, 2020, we obtain the estimates δ with δ max := δ ≈ 0.5135 and d i := d max ≈ 0.0449. in addition, we take l = 2 and assume that the effectiveness rate of control v(k) follows a uniform distribution with v min = 0.1 and v max = 0.2; i.e., 10% to 20% effectiveness rate on control. observe that hence, theorem 4.2 does not apply. however, we can still choose a suboptimal feedback gain discussed in section 4.1.2; i.e., this means that, if the infected cases can not be well-controlled in almost sure sense, governments should put whatever they can to suppress it; here, we see a 100 · l% feedback gain is applied. the epidemic control performance is shown in figure 1 where the black solid lines depict the confirmed cases c(k) and reported deaths d(k) from historical covid-19 data in the united states. the other thinner lines with various colors depict the epidemic control performance, in terms of c(k) and d(k), under the linear policy u(k) = kc(k) with k := l. interestingly, the figure also tells us that if l = 2 is possible, it may yield, in average, a lower confirmed cases. with the aid of equations 15 and 17, we obtain the estimates d i ≈ 0.002 and δ := δ ≈ 0.215. similar to the previous example, we again assume that v(k) follows a uniform distribution with v min = 0.1 and v max = 0.2; hence the average v = 0.15. it is readily verified that our estimates satisfy hence, theorem 5.2 applies if we take the control policy above tells us that the government may need to bring in extra medical resources to achieve k > 1. the corresponding epidemic control performance is shown in figure 2 where the confirmed cases is on the top panel and the deaths is on the bottom panel. in the figure, we see a downward trend occurs on the confirmed cases and the saturated deaths as time increases. the figure 3 shows, with y-axis in log-scale, the epidemic control performance comparison where the black solid lines are the reported confirmed cases (top) and reported deaths (bottom). this preliminary work has been done in the urgency of the ongoing covid-19 pandemic, with the mind of providing a simple yet explainable epidemiological model with a rigorous study on the effectiveness of a linear epidemic control policy class. consistent with the existing literature on epidemic modeling and control, this paper considers a modified stochastic sird-based model. we analyzed the model and considered two epidemy control problems: one is the almost sure epidemic control problem and the other is the average epidemic control problem. then, for both of two problems, with linear control policy, we show sufficient conditions on feedback gain so that the epidemy is deemed to be "well-controlled" in the sense that infected cases goes down to zero asymptotically, and both infected cases and deceased cases are upper bounded uniformly. subsequently, we provide a simple data-driven parameter estimations and show some promising numerical results using historical covid-19 data in the united states. based on our work to date, two important directions immediately present themselves for future work which described in the next subsections to follow. it is possible to extend our analysis to involve multi-population epidemics. to illustrate this, below we consider only the infected cases dynamics. fix m populations, then for each population i = 1, . . . , m, we write with limited medical resource for some u max > 0 and the overall infected cases are it is also possible to consider the case where the control policy is with delay effect; i.e., u(k −d) with delay timed; see also [13] for a discussion on handling the time delay considerations in a class of positive finance systems. another possible research direction would be to take the economic budget into play. as seen in remark 4.1.2, the idea of minimizing the expenditure induced by implementing control policy may lead to a version of "optimal" choice for feedback gain k. in particular, one can even consider the budget dynamics and carry out the optimization. specifically, let b(k) be the available medical budget at the kth day which satisfies b(k + 1) = b(k) + p(k)u(k) where p(k) is the price to pay per control policy at each stage, which can be modeled as a random variables with finite support. then, it is readily verified that for k ≥ 1. from here, there are many possible directions to pursue. for example, one can consider a optimization problem which minimizes the expected budget cost and infected cases; i.e., one might seek to find a sequence of k which solve inf k e[βb(k) + γi(k)] for some constants β and γ. data-driven methods to monitor, model, forecast and control covid-19 pandemic: leveraging data science, epidemiology and control theory some discrete-time si, sir, and sis epidemic models, mathematical biosciences infectious diseases of humans: dynamics and control the mathematical theory of infectious diseases and its applications a data-driven control-theoretic paradigm for pandemic mitigation with application to covid-19 a modified sir model for the covid-19 contagion in italy linear system theory and design a time-dependent sir model for covid-19 with undetectable infected persons fitting dynamic models to epidemic outbreaks with quantified uncertainty: a primer for parameter uncertainty, identifiability, and forecasts probability: theory and examples probability and random processes for electrical and computer engineers the mathematics of infectious diseases, siam review on positive solutions of a delay equation arising when trading in financial markets containing papers of a mathematical and physical character robust and optimal predictive control of the covid-19 outbreak substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) how control theory can help us control covid-19 world helth organization, weekly epidemiological and operational updates key: cord-002426-5e1xn7kj authors: falcón-lezama, jorge abelardo; santos-luna, rené; román-pérez, susana; martínez-vega, ruth aralí; herrera-valdez, marco arieli; kuri-morales, ángel fernando; adams, ben; kuri-morales, pablo antonio; lópez-cervantes, malaquías; ramos-castañeda, josé title: analysis of spatial mobility in subjects from a dengue endemic urban locality in morelos state, mexico date: 2017-02-22 journal: plos one doi: 10.1371/journal.pone.0172313 sha: doc_id: 2426 cord_uid: 5e1xn7kj introduction: mathematical models and field data suggest that human mobility is an important driver for dengue virus transmission. nonetheless little is known on this matter due the lack of instruments for precise mobility quantification and study design difficulties. materials and methods: we carried out a cohort-nested, case-control study with 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) with the goal of describing human mobility patterns of recently dengue virus-infected subjects, and comparing them with those of non-infected subjects living in an urban endemic locality. mobility was quantified using a gps-data logger registering waypoints at 60-second intervals for a minimum of 15 natural days. results: although absolute displacement was highly biased towards the intradomestic and peridomestic areas, occasional displacements exceeding a 100-km radius from the center of the studied locality were recorded for all three study groups and individual displacements were recorded traveling across six states from central mexico. additionally, cases had a larger number of visits out of the municipality´s administrative limits when compared to intradomestic controls (cases: 10.4 versus intradomestic controls: 2.9, p = 0.0282). we were able to identify extradomestic places within and out of the locality that were independently visited by apparently non-related infected subjects, consistent with houses, working and leisure places. conclusions: results of this study show that human mobility in a small urban setting exceeded that considered by local health authority’s administrative limits, and was different between recently infected and non-infected subjects living in the same household. these observations provide important insights about the role that human mobility may have in dengue virus transmission and persistence across endemic geographic areas that need to be taken into account when planning preventive and control measures. finally, these results are a valuable reference when setting the parameters for future mathematical modeling studies. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 dengue fever (df) is the most important arthropod-borne viral disease in the world. it is caused by infection with any of the four dengue virus (denv) serotypes. nearly half of the human population inhabits areas with denv transmission. in mexico dengue incidence and severe cases have been increasing in the last decade. to date, there is no specific treatment or vaccine for df and vector control stands as the cornerstone for df prevention [1, 2] . df is an important public health problem, especially in urban areas [3] , where it usually presents in large outbreak. the costs of treatment and management during a df outbreak are a serious burden for health systems, especially when there is a risk for saturation of health facilities [4] . the actors that are necessary for denv transmission are fairly well understood. nonetheless, some of the dynamical features about these actors still need to be elucidated in order to understand how they impact on transmission. human mobility has been studied in relation to other infectious diseases, where its role as an important driver for disease transmission has been proven [5, 6] . mathematical models have suggested that local scale human mobility may play a role in denv transmission, outbreak persistence, and control efficiency [7, 8] . however, little information is available on detailed human mobility patterns in geographic areas where df is endemic or on confirmed cases during an outbreak. recently, gps-based technologies have been tested and shown to be a reliable and acceptable tool for quantifying human mobility [9, 10] . human mobility has been described in relatively recent reports by using indirect measures [11, 12] . for these reasons, we studied the micro and macromobility of dengue virus-infected subjects in an endemic locality. here we present the results of a cohort-nested case-control study on a dengue endemic urban locality in mexico. the protocol of the project was reviewed and approved by the comité de etica y de prevención de conflictos de interes (institutional review board) ci 1046 no 1160 and departamento de investigación (external review) servicios de salud de morelos, mexico dei/cei/0281/2012. cohort-nested, case-control study. sample: 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) with age older than 12, and residents in axochiapan, morelos state, méxico, were selected from the cohort "peridomestic infection as determinant for dengue virus transmission" [13] . they were assigned into three study groups: a. cases were individuals with laboratory evidence of recent, symptomatic or asymptomatic, denv infection, and identified as the only persons infected within their households during the study. b. intradomestic controls, were individuals with a negative serological result for recent denv infection, living in the same household with a case; c. population controls were individuals with a negative serological result for recent denv infection, randomly selected from the same locality. all controls tested negative for denv during the same period and using the same validated techniques than cases (igm or igg capture elisa) as reported in the cohort study [13] . partici-pant´s selection was performed as follows: cases were approached first, if accepted participation an intradomestic control was randomly assigned from the pool of subjects living in the same house that had both baseline and final negative elisa results. for each pair of in-house participants, a randomly selected population control was then assigned (fig 2) . given the limited number of available gps loggers and the three-month time frame for the follow-up, the recruitment was limited to a maximum of 50 cases with their respective intradomestic and population controls. between may and september, 2012, with prior signed informed consent, all participants were provided with a portable gps (gps data-logger, transystems mod. 747 a+), programmed for recording its position at 60-second intervals (variables date, time, latitude, longitude, altitude and speed), during 24 hours a day, for a minimum period of 15 days. participants were instructed to carry their gps at all times whenever they left their homes and to recharge the equipment's battery daily during their in-home resting times. this follow up was performed in cases identified during the immediate previous season, on average one year after diagnosis confirmation, in order to match the activities performed during the high transmission season, and under the assumption that their mobility patterns remained unchanged after disease, and constant through time. a web-based interface was developed to import the text files from gps equipment to a main database. variables were homogenized, waypoints in the initial and final days of each individual gps track (comprising incomplete days) were eliminated in order to standardize the period of time to be analyzed starting at 00:00:00 hours on the second day of follow up and ending at 23:59:59 on the last to final day of follow up. data were converted into a feature dataset and projected from the geographic coordinate system to a lambert coordinate system (from hexadecimal to metric units) with the purpose of performing arithmetic operations for distance calculations. origin or routinely residence sites were identified by means of an iterative algorithm (mean center) employing waypoints from 00:00:00 to 04:59:59 hours, monday to friday. routine residence coordinates were added to the database. distance to home variable (dhome) was calculated for each extradomestic waypoint using sql applying the following formula: where; x 1 = xhome (x coordinate from home), x 2 = xccl (x coordinate from each waypoint), y 1 = yhome (y coordinate from home) y, y 2 = yccl (y coordinate from each waypoint). waypoints within the peridomestic area (dhome < 50m) were identified. distance, speed, altitude and time differentials were created: where: xccl 1 = (x coordinate from previous waypoint), xccl 2 = (x coordinate from current waypoint), yccl 1 = (y coordinate from previous waypoint) and, yccl 2 = (y coordinate from current point) displacement and spatial permanency variables for each subject with complete data were generated. visit sites were defined as those areas out of the individual's home with a 50 m radius in which each participant remained static for a period enough to allow a potential effective interaction with local vectors. these sites were identified by generating an algorithm through which visit clusters were formed using the following criteria: stops lasting 5 minutes or longer, a distance from home of 50 m or farther, distance of the current waypoint from previous waypoint < 50 m, and speed for the current waypoint of 2 km / h or less. for each cluster (visit site) a centroid was calculated. common visit sites for cases were identified as hexagonal cells with 50 m radius [14] which were visited by at least two different cases at a given time, and where the proportion of different visiting cases was at least two thirds of the total visiting population for that cell. common visit sites for controls were identified as hexagonal cells with 50 m radius which were visited by at least two members from each control population and not visited by any of the cases. the geographic universe in the study was divided in five areas (1.-inside the house, 2.-out of the house but in the locality, 3.-out of the locality but in the municipality, 4.-out of the municipality but in the state, 5.-out of the state), limited by four buffers. a circular buffer with 50 m radius around each participant's home limited the first area and three additional polygonal buffers were drawn according to the administrative limits for the locality, municipality and state. arcgis arcinfo 10 was used for processing and analyzing spatial data, sql server was used to create a geodatabase, arc sde 10 was used as interpreter between entre sql and arcgis. statistical analyses were performed using stata 12. fifty randomly selected cases were asked to participate in the study from which 42 (84%) accepted participation. all approached controls agreed to participate. in total 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) were recruited. our drop-out rate was lower than 1% (1/126) since one participant (intradomestic control) did not finish the follow-up due to the loss of the assigned gps logger. table 1 describes the main characteristics of the subjects in each group. no statistically significant differences were observed in most of variables except in age, since cases were significantly younger than the intradomestic or population controls (cases mean: 29.2, sd: 17.7; intradomestic controls mean: 35.9 sd: 13.5; population controls mean: 37.4 sd: 16.6. p = 0.0315). of 126 participants, 125 (99.2%) participants completed their follow up since one gps used by an intradomestic control went missing. the final database contains 3,064,887 waypoints from these 125 participants, and all participants were followed by a mean of 15.9 continuous days. as for the number of days of follow-up for each group no differences were recorded. as expected, most of the waypoints in the population fell within the intradomestic area (< 50 m radius from home centroid). as distance from home (absolute displacement) increased, we observed a marked decrease in the proportion of waypoints. all three groups presented a small peak when the distance reached the 100 m radius. from this point the proportion of waypoints quickly decayed (fig 3) . no differences were noticed for absolute displacement among the groups. the hourly distribution of recorded waypoints out of the participant's homes is shown in fig 4. as expected, participants usually left their homes early in the morning and returned by the end of the day. although we recorded waypoints out of the participants' homes in every hour of the day, the period comprised between 08:00 pm and 12:00 pm registered the peak in the number of waypoints recorded out of the homes, and this number decreased steadily as the day progresses. the pattern during weekdays ( fig 4a) suggests that cases leave their homes and return to them slightly earlier than control groups. as for the weekends (fig 4b) , both control groups show a similar pattern to that observed for weekdays, nonetheless, cases seem to remain in their homes more often and return earlier. table 2 shows values for different mobility variables. no significant differences were recorded among groups for the following variables: mean distance from home at all times, maximum recorded distance at any given time, and mean time spent in each geographic area at any speed or at static speed. nonetheless, when comparing the number of visits per geographic area, the cases had fewer recorded visits in the area out of the locality but in the municipality (3.1 vs 18.7, p = 0.0428), and more visits in the area out of the municipality (10.4 vs 2.9, p = 0.0282), both compared to intradomestic controls. these differences were statistically significant. consistent with this behavior, although non-statistically significant, cases visited more states, municipalities, and regions with high dengue incidence through their follow up, in comparison to both control groups. we next examined the proportion of waypoints recorded by the comparison groups in each area stratified by age (fig 5) . there is a notorious difference in the proportion of waypoints among the cases, observing an increase of nearly 8 percentile points in the intradomestic waypoints, recording the highest frequency in cases under age 25 (fig 5a. however the difference not statistically significant (cases < 25: median 90.2%, interquartile-range 75.5-95.5; cases ! 25: 80% iqr 66-86.4; p = 0.079). we also observed a difference in the area out of the municipality but in the state, where the group of cases aged 25 and older spent the highest proportion of time (age < 25: 0% iqr 0-1%; age ! 25: 1.1% iqr 0-4.6; p = 0.0233). there was no significant difference between cases and population controls, regardless of age. when comparing cases versus intradomestic controls, we observed a statistically significant difference in time spent in area out of the municipality but in the state (cases: 1.1% iqr 0-4.6; ic: 0% iqr: 0-0.6; p = 0.009). we found differences in mobility patterns when analyzing data by gender ( fig 5b) . women had a higher proportion of waypoints within the intradomestic area than men. these differences were statistically significant for intradomestic area (male: 77.4% iqr: 65.8-86.9; female: 89.4% iqr 80.5-93.5; p = 0.002), out of their homes but in the locality (male: 15.5% iqr 5.3-25; female: 7.2% iqr 4.6-16.5; p = 0.0063) and the area out of the locality but in the municipality (male: 1.1% iqr: 0.1-4.9; female: 0.1% iqr: 0-0.4; p<0.0001). nonetheless, linear mean (p = 0.061) and maximum (p = 0.1468) distances were not. a) cases vs. intradomestic controls. a conditional logistic regression analysis was performed, including variables identified in the bivariate analysis as having a p value < 0.20, and by data mining techniques using all variables as reported previously [15] . for bivariate analysis variables were age (continuous and dichotomic [under 25 or 25 and older]) gender, occupation (intra or extradomestic), education (dichotomic), and the proportion of time spent in each geographic area. for the data mining we considered the whole data base. the final model included age (or: 0.015 ic95% 0.0005-0.488; p = 0.018) and the area out of the municipality but in the state (or 2.61 ic 95% 1. 16-5.88 ; p = 0.021). we observed a protective effect in the 25 and older group, and a risk effect when the proportion of time spent in the area out of the municipality but in the state is increased. b) cases vs. population controls. a multiple logistic regression analysis was performed including variables identified with p value < 0.20 in the bivariate analysis and data mining techniques using all variables as reported previously [15] . for bivariate analysis, only the variables age (continuous and dichotomic [under 25 or 25 and older]), gender, occupation (intra and extradomestic), education (dichotomic), proportion of time spent in each area and linear distance were taken into consideration. for the data mining we considered the whole database. the final model included: occupation (or 3.02 ic 95% 1.02-8.88; p = 0.045), proportion of time in the area out of the municipality but in the state (1.42 ic 95% 1.02-1.98; p = 0.035), proportion of time in the area out of the locality but in the municipality (or 0.74 ic 95% 0.57-0.96; p = 0.023), and age (or 0.26 ic 95% 0.09-0.78, p = 0.017). next we analyzed the geographic distribution of the recorded waypoints for each group, both locally and regionally (fig 6) . all three groups recorded waypoints exceeding a 100 km radius from their homes (fig 6a, 6b and 6c ). as expected, most of recorded waypoints were located within the locality. nonetheless, all three groups recorded waypoints exceeding the locality, municipality and state limits. these trajectories were headed mainly to the east, north and west of the locality, and were consistent with the location of the main cities in the area, including cuernavaca (population 338,650) and cuautla (population 154,358), the capital city and the second most important city in the state of morelos, respectively. the geographic distribution of the visits performed by cases and df cumulative incidence for the central mexico region, during year 2012, is shown in fig 7. as seen, this group performed visits to locations with and without df transmission. the number of states, municipalities and regions with high dengue incidence is shown in table 2 . within the locality of axochiapan the most visited areas were identified by dividing the locality in 50 m-radius hexagonal cells (fig 8a) . the most visited cells were those located in the locality's central area, which correspond to the location of the main market, road junctions and main administrative and / or service offices. the location of the cells considered as common visit sites for cases are shown in fig 8b. unlike in fig 8a, the geographical distribution of these 15 cells tends to be peripheral with respect to the locality. using google earth™ we identified the geographic features of each of the 15 cells that was classified as a common visit site for cases. fourteen out of fifteen cells were geographically located within the locality of axochiapan, morelos, and the last one was in the central area of a neighboring small locality (town of tzicatlán) in the state of puebla. as for their typology, xix out of 15 cells clearly corresponded to residential areas (including that in the neighboring state), one cell was a residential area adjacent to a local large business, four cells included small processing plants, a warehouse and a local business, three peripheral cells were crop fields and one cell was clearly a soccer field. previous works have used gps tools for measuring exposure to infectious diseases [16] [17] [18] . in df, recent works in the endemic area of iquitos, peru, have elegantly described human population mobility [19, 9] . however, few data are available for infected cases so far. our work builds upon our knowledge of the role played by mobility in denv transmission documenting spatial mobility of subjects from an endemic region that had, or had not been recently infected by denv. our data show that the people from axochiapan stay within their houses or surrounding areas most of the time. this is consistent with previous observations in iquitos, where population rarely moves more than 1 km away from their homes [10] , however, some individuals recorded movements to very distant locations through the relatively short follow-up period. these movements were present in all three study groups and exceeded a 100-km radius from the center of the study, covering the neighboring states of morelos, state of mexico, puebla, mexico city, tlaxcala and hidalgo. all of these states are located in the mexican central plateau, which is also the best connected region in the country and therefore it is not difficult to reach those destinations by commute travel [20] . surprisingly, spatial mobility in humans was not geographically symmetrical in our study, since no movements were recorded to state of guerrero, which is a coastal, highly endemic area for dengue and also a popular destination for leisure activities. as for the reason why the mobility of the individuals is biased towards central plains in mexico and practically absent towards the southern regions, it was a surprising finding also for us, but we think that it has to do with two factors: first the economic activities in axochiapan are mainly related to agriculture, trade and services which are strongly influenced by the needs of mexico city and its metropolitan area, comprised also by the states of morelos, puebla, méxico and hidalgo. the main cities of these states were those that were visited by the cases and in a lesser extent by the controls. secondly, we did not perform any follow-up during summer and christmas holidays, which in mexico are specific periods for leisure. these activities are usually performed in places that might be different that those observed in our study, including the beaches in the southern coast. it is possible that had we performed our follow-up in vacation periods the observed mobility might have been different, and also leave us with a very interesting research question for the future. the large size of the area covered by these few individuals from a small locality (axochiapan has roughly 17,000 inhabitants) is of capital importance, given the fact that in mexico, and probably in many other places, epidemiological surveillance, prevention and control activities for df are mainly planned, supported and executed by local health authorities, who rely on the information generated by a number of systems, most of them automated [14] , but that are usually restricted to their local administrative limits, namely municipality, sanitary jurisdiction or state at best. thus when df outbreaks overcome those limits and a wider coordination is needed, it is probably that the outbreaks are already established and the window of time for effectively applying control measures has been lost. our data show that the cases group had the largest difference on the time spent in the home area with strong age dependence. older cases spent less time in their homes compared to younger cases. as far as the number of visits is concerned, subjects in the cases group, especially those aged over 25, performed many and more distant visits, than subjects in the intradomestic control group. this difference with the population control group was less marked. this scenario suggests that the population aged over 25 might play an important role in denv persistence and dispersion perhaps working as geographic spreaders. our group previously determined dengue incidence for this age group and proposed a dynamic model which seems to be corroborated by the results presented here [13] . infected individuals, both symptomatic but also asymptomatic [21] , may facilitate the infection of extradomestic mosquito populations in a local scale as models have suggested [8] , or at a regional scale introducing or exporting the virus. given the fact that we performed an uninterrupted 24-hour follow up, we were capable to register the time when individuals left their homes for whatever activity they performed. to our surprise, we recorded waypoints out of the participant's homes virtually at any hour of the day. although the majority of records show that people in axochiapan have a day-light pattern of activities, we recorded waypoints from cases, between 00:00 and 05:00 am, which were consistent with participants' declared jobs, which were related to nocturnal activities such as bakers and workers from the local stone processing plant. the dispersion patterns described above, both in space and time, might be of importance in the results obtained in the control of denv transmission in this and similar small localities. the usual schedule considered by local health authorities for applying preventive measures, which favors early hours for insecticide spraying and the visits by entomological control brigades, in a geographically focalized strategy might hinder the efficacy of the actions by the mere fact that people moves from their homes and remain away during the time these actions are normally applied. in our data, the hourly pattern for activity suggests that cases might leave earlier their homes during weekdays, and thus their homes might have a higher probability to remain closed by the time health authorities apply preventive or control measures. it is important to notice the high mean age of the cases in axochiapan, in both the participants in the study and those recorded historically in the state of morelos, in comparison to other endemic areas from mexico and the americas. this is however, consistent with a previous work in the area [13] , and is probably due to the fact that most (35 out of 42 members of the cases group) of the cases that we studied were asymptomatic, also, although not statistically significant, the mean age of the asymptomatic individuals was higher than that from symptomatic individuals (mean age asymptomatic: 31.54 vs mean age symptomatic: 17.43, t test p = 0.0535). thus, suggesting a possible stronger role of asymptomatic population with age above 25 as spreaders for denv transmission. previous studies have described that visiting other cases' households is a risk factor for denv dispersion [22] ; working sites have also been suggested as possible transmission sources outside of cases' households [23] . models have shown that sites outside homes can play a role in denv transmission and infection [7] , and a recently published work showed positive correlations in thailand between the aedes spp. house index and specific landscape features [24] . our findings are consistent with these data since cases coincided in houses different to their own in at least five different geographical locations. additionally we found cases that coincided in four potential working places, and a soccer field. as far as we know, this is the first time that leisure sites have been documented as possible areas for denv transmission. the lack of study of such sites has previously been pointed out as a weakness in the study of human mobility [25] . as for the common visit sites for controls, we identified 38 cells that were visited only by individuals from both control groups but not by cases. these cells included only one potential working site; whereas the cells commonly visited by cases included several likely workplaces. the identification and study of the extradomestic sites where people coincide is relevant: a recent simulation model has concluded that the selection of areas for df control out of the cases' homes is important not only in terms of the time the subjects spend in them, but also because of the local vector:host ratio, and the other habitual destinations of people that visit the same area [8] . it is possible that much of the movement in a given society is driven by specific population needs and the possibility to fulfill them within or outside from their own locality. axochiapan is a fairly small and well connected locality to other small cities, all of them endemic for df. it is not unrealistic to think that some of the population needs can be readily fulfilled within the same locality whereas other cannot, compelling the population to move away in a permanent or transitory fashion. if a specific population such as that with age older than 25 becomes infected and effectively play a role as spreaders, then perhaps small and peripheral localities to larger cities might have a key importance in sustaining denv transmission across large geographic areas. the assessment of such situations requires a critical review of existing data and the generation of specific studies that may help us to recast current models and more importantly, to completely understand urban denv transmission [26] . we have identified some weaknesses in our study. the most relevant is our small sample size which might have hindered our capability to identify clear patterns in the mobility from this mexican community. there is a possibility that any individual belonging to either control group might have get infected during the follow up; thus making her/him eligible to become a case, therefore disqualifying him/her for being a suitable control and consequently introducing an information bias; nonetheless, we believe that, although a possibility, this was negligible due to two reasons: first, the follow-up of 15 days was very short for this event to occur, and secondly because while recovering each gps, we asked all individuals whether they had experienced fever or any other symptom suggesting dengue infection during the follow-up. none of the participants reported any change in their health status. although we understand that a more robust argument to ensure the infected/uninfected status might be performing an elisa to each control after finishing their follow-up in order to be certain about their exposure, financial constrains made impossible this procedure. possible future improvements in our study are: increase the limited sample size, the inclusion of adequate representation for the population under the age of 12, which probably is a relevant group for transmission during the initial and focalized phases of a df outbreak. although no schools could not be identified in our study as a common site visited by cases, this observation needs to be taken cautiously since our study did not consider the follow-up of children usually studying elementary education. thus, we cannot rule out any role of these sites in dengue transmission at younger ages. additionally, extending the duration of the follow up might improve the chances of successful identification of patterns whose frequencies are longer than a week, such as wage collection, bill payments, and communitarian meetings, among others. this last topic is essential; nonetheless it is limited by technical issues that might be addressed as technology for massive and continuous long-lasting follow up becomes available. finally, the main reasons for which the participants move were not deeply explored in our study, thus we cannot be certain whether the recorded movements indeed depend on non-satisfied needs or on leisure activities. we can only assume that at least those movements performed during the mornings and afternoons between monday and friday correspond to real needs such as employment, education, and supply acquisition, and those performed during weekends are related to leisure. some causes for loss of gps information in field studies have been recently described [27] . although some of these causes might be present in our study, we believe they did not represent significant sources of bias or information loss, since we took some specific measures. for example, people were prevented of accidentally turning the gps off by strapping a tape in the controls. in order to diminish the probability that the participants could forget their gps units at home we performed a weekly phone call reminding them the importance of the usage attachment according the protocol during each individual follow-up. barriers to signal were not important in the studied area since it is located in a plateau with few elevations, and buildings taller than 3-stories are practically absent. finally, the gps equipment used in the study had battery autonomy of up to 32 straight hours and enough memory for recording up to five times the mean number of waypoints programmed to collect in each subject. based on our own data and that from recently published works we conclude that gpsbased technology is a solid tool for the study of detailed human mobility in denv transmission or other infectious diseases, which can and must be adopted in public health and epidemiology as a basic instrument. the important geographic dispersion in our results demonstrates the necessity for studying the potential role that human mobility has in denv transmission and outbreak duration and also a strong argument to study and clarify the role that asymptomatic cases might have in dengue virus dispersion. furthermore our data strongly suggest that the size of the areas considered for prevention and control of df outbreaks needs to be revised and that it is necessary to integrate this knowledge into the planning of preventive and control measures, which usually are prone to using basic shapes such as circles or squares as geographic references in order to define limits, ranges, trajectories and points of origin. it is clear that human populations move normally across geographical areas and not only during holidays or vacations. according to our data, the magnitude of these displacements is larger than that considered as an administrative responsibility for local health services providers. this is relevant for denv transmission if a large fraction of that mobile commuting population is also asymptomatic but viremic, facilitating with their movements the exposure 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methodology for data mining in large databases the use of a vest equipped with a global positioning system to assess water-contact patterns associated with schistosomiasis distribution and interspecies contact of feral swine and cattle on rangeland in south texas: implications for disease transmission health &demographic surveillance system profile: the kombewa health and demographic surveillance system (kombewa hdss) multiple outbreaks for the same pandemic: local transportation and social distancing explain the different "waves" of a-h1n1pdm cases observed in méxico during asymptomatic humans transmit dengue virus to mosquitoes house-to-house human movement drives denguevirus transmission epidemiology of dengue and dengue haemorrhagic fever in a cohort of adults living in bandung analyzing the spatio-temporal relationship between dengue vector larval density and land-use using factor analysis and spatial ring mapping population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and noncommuting dengue cases recasting the theory of mosquito-borne pathogen transmission dynamics and control strengths and weaknesses of global positioning system (gps) data-loggers and semi-structured interviews for capturing fine-scale human mobility: findings from iquitos authors wish to thank to servicios de salud de morelos for its support to this project. the authors have declare that no competing interests exist. key: cord-128436-xndrlnav authors: granozio, fabio miletto title: comparative analysis of the diffusion of covid-19 infection in different countries date: 2020-03-18 journal: nan doi: nan sha: doc_id: 128436 cord_uid: xndrlnav the sudden spread of covid-19 outside china has pushed on march 11 the world health organization to acknowledge the ongoing outbreak as a pandemic. it is crucial in this phase to understand what should countries which presently lag behind in the spread of the infection learn from countries where the infection spread earlier. the choice of this work is to prefer timeliness to comprehensiveness. by adopting a purely empirical approach, we will limit ourselves to identifying different phases in the plots of different countries, based on their different functional behaviour, and to make a comparative analysis. the comparative analysis of the registered cases curves highlights remarkable similarities, especially among western countries, together with some minor but crucial differences. we highlight how timeliness can largely reduce the size of the individual national outbreaks, ultimately limiting the final death toll. our data suggest that western governments have not unfortunately shown the capability to anticipate their decisions, based on the experience of countries hit earlier by the outbreak. italy is presently the hardest hit country. its death toll seems bound to rapidly overcome the chinese case. other western countries follow the same route. it is crucial in this phase to understand what countries presently lagging behind in the spread of the infection can learn from countries where the infection spread earlier. the first question we address is: which are the relevant numerical signatures to be monitored to check how effectively a country is acting, compared to other countries, in containing the infection? here we show that an answer to this question can be given not relying on specific epidemiological expertise, but based on a simple numerical analysis of public data available on the internet. it is widely acknowledged that comparison of curves from different countries is made difficult by the different ways the detection of the virus is addressed. in particular, the ratios of the tested population to the total population, among the countries addressed in this paper, range from about 5.000/million in the case of south korea, to 1.400/million for italy to less the 70/million in the case of usa 1 . therefore, not only the real number of infected people might largely exceed the registered cases, but such ratio (registered/total cases) might change country by country. this difference is partly mitigated by two observations: -in the specific spirit of this work, we will compare countries in the same stage of the epidemic. considering the delay of the outbreaks (korea is grossly 10 days ahead of italy and 20 days ahead of the states, as will be discussed later in detail) italy is grossly following the same testing curve as korea while the usa lag behind by less than one order of magnitude. -we should assume that in advanced countries most symptomatic patients are counted among the registered cases within a few days. these are exactly the cases we mostly want to focus on. the hidden background of asymptomatic patients, though playing a role in determining the disease spread, is a less relevant datum in foreseeing the final death count. at the end of this analysis, remarkable similarities are found, especially among western countries, together with some minor differences. the extent and relevance of the observed similarities for the case of western countries will justify, ex post, the adopted approach. epidemiological curves are typically believed to follow the stochastic logistic model. nevertheless, this assumption cannot take into account situations in which the infected population reacts by drastic changes of its collective behaviour, thus changing the virus reproductive number, in the course of the outbreak. a general model for covid-19 diffusion would require knowledge of all the specific virus containment measurements adopted in each single country, their dates, and their quantitative effect on the reproductive number. this is so far beyond our present understanding. the choice of this work is to prefer timeliness to comprehensiveness. by adopting a purely empirical approach, we will limit ourselves to identifying different phases in the plots of different countries, based on their different functional behaviour, and to make a comparative analysis. for more complex approaches, the human data analysis time could easily exceed the obsolescence time of the dataset, which is of the order of a couple of days. the case of countries beyond the initial phase of the outbreak the source of data for this work is the csse covid-19 dataset 2 . we analyse here the data of three of the countries that registered at the date of march 15 the highest cumulative number of registered cases, i.e. china, italy, and south korea. we neglect iran, also because of the lack of information about the number of tested people. the three countries are at different stages of the outbreak. china exceeded the number of 1000 registered cases in the hubei province on january 24 and presently reports few new cases per day. korea exceeded the number of 1000 registered cases on february 27 and presently reports few new cases per day. italy exceeded the number of 1000 registered cases only a few days later but, in spite of a recent slowdown, the end of the exponential phase, if confirmed, is happening in the present days. the chinese plot shown in fig. 1a stops on february 11. this is due to the change of criterium adopted in the counting of infected patients performed in china on february 12 3 . the reported data are fortunately widely sufficient for the purpose of this work. as for italy, last data are aligning to a linear curve, which might well be the inflection point, anticipating a smooth transition to a sublinear behaviour. the absolute number of the new daily cases in the country, about 3.500 on march 15, is still very high. by comparing the linear coefficient of the italian and korean blue curves, we observe in fact that the former exceeds the later by a factor 7. the comparison of the plots shows that, in spite of the extremely fast growth rate ( =2.4d, corresponding to a doubling time of one day) the rapid response of the korean society allowed to switch the growth to a slower rate before reaching 500 registered infected people. this rapidity is confirmed by the observation that at the time when only 21 infections were found, on february 18, the republic of korea had already tested over 8000 citizens 4 . italy had instead 322 registered cases by the time it reached the same number of tests on february 25 5 . the perduring fast growth rate in italy rises major concerns and suggests that the cumulative final number of cases might exceed the chinese case. comparison between western countries. the italian case seems to correspond to the worst-case scenario among the ones analysed above. it suggests that while korea implemented a faster reaction than china, profiting of the lesson learned by the experience of the neighbour country, italy apparently showed a longer response time than both asian countries, in terms either of diagnosis, or of governmental decision, or else of change of individual habits. it is of great importance to verify how well the italian lesson was learned by other western countries. the plots reported in fig. 2a this timescale seems to be characteristic of the covid-19 "free expansion" in all these countries or, to state it more prudently, of the rate at which they are detected. we remind in this context that, in absence of large-scale screening programs, most infected people are tested after showing symptoms, i.e. about 5 to 14 days after infection. therefore, the registered cases curves map the history of the past behaviours of the infected population. we observe that, still at the date of march 15, the spanish evolution is correctly described by the red exponential curve. the us curve shows a minor deviation, which might well indicate a switch to phase #2. france has switched to the second phase, with exponent  =4.5d. germany has also switched to the second phase, with exponent  =4.2d. the plot in fig. 3 gathers all the curves above in the same plot, comparing them with the italian curve. among the possible ways to plot the data together, the most significant one, by far, was found to apply a relative shift in time, in order to "synchronize" the different starting times of the outbreak. we remark that, by normalizing the number of infected people to the overall population, the us plot would have been shifted backwards by 5-6 days. when plotted with the appropriate relative time scale (it reference, de, fr -9d, es -10d, us -11d), the data show how early or late the different countries deviated from the red exponential "phase #1" curve with  ~2.0d, d ~2.0d. the violet curve fitting the italian "phase #2" is also shown. it can be qualitatively deduced that france, germany and probably the united states, on this particular conventional time scale, have switched to the violet curve grossly at the same time as italy did. spain is potentially running towards a worse scenario, although the last points hint to a possible alignment to the same violet curve, albeit a factor two above. the plot in fig. 4 graphically highlights the importance of the early reactions. the numerical history of the outbreak in korea is compared to the hypothetical outbreak evolution (dotted lines) in case a two-day delay in the transition from the red to the violet curve, rigidly reflected in a twoday shift in the transition to the blue curve, would have taken place. according to the estimation in fig. 4 , the transition to phase #3 would have taken place on march 3 with an infected population of about 14.500 people, about 3,5 times higher than the actual number of registered cases at the real transition, happened on march 1 st . the same scale factor would be applied today, within our hypotheses, to the actual infected population. we attempted an elementary, real-time analysis of the covid-19 diffusion data updated at march 16. timeliness was preferred to comprehensiveness. important information has been extracted by the data, but major caution is needed in deriving general and far-reaching conclusions. both the inhomogeneity in data acquisition rate in different countries and the huge background of undetected, presumably asymptomatic, infected patients, are two major sources of uncertainty. the criterion applied in the plot in fig. 3 is highly instructive but, to some extent, arbitrary. with all due prudence related to the uncertainties above, we believe the present analysis is an excellent and timely starting point for further studies on the delayed effects on the curves of the response adopted by different countries. such response includes both individual changes of habits (hands hygiene, social distancing by own choice) and restrictions imposed by the governments (closing of schools, constraints to the mobility of citizens). the korean example clearly shows that early diagnosis of the first infected patients and timeliness in the response can largely reduce the size of the outbreak, ultimately limiting the final death toll. our data suggest that western governments have not shown the capability to anticipate their decisions based on the experience of countries hit earlier by the outbreak. our hope is that this work can contribute to triggering early and appropriate responses to the covid-19 pandemic. how many tests for covid-19 are being performed around the world? our world in data coronavirus cases: statistics and charts -worldometer quanti test per il coronavirus abbiamo fatto key: cord-025337-lkv75bgf authors: vakkuri, ville; kemell, kai-kristian; jantunen, marianna; abrahamsson, pekka title: “this is just a prototype”: how ethics are ignored in software startup-like environments date: 2020-05-06 journal: agile processes in software engineering and extreme programming doi: 10.1007/978-3-030-49392-9_13 sha: doc_id: 25337 cord_uid: lkv75bgf artificial intelligence (ai) solutions are becoming increasingly common in software development endeavors, and consequently exert a growing societal influence as well. due to their unique nature, ai based systems influence a wide range of stakeholders with or without their consent, and thus the development of these systems necessitates a higher degree of ethical consideration than is currently carried out in most cases. various practical examples of ai failures have also highlighted this need. however, there is only limited research on methods and tools for implementing ai ethics in software development, and we currently have little knowledge of the state of practice. in this study, we explore the state of the art in startup-like environments where majority of the ai software today gets developed. based on a multiple case study, we discuss the current state of practice and highlight issues. the cases underline the complete ignorance of ethical consideration in ai endeavors. we also outline existing good practices that can already support the implementation of ai ethics, such as documentation and error handling. ai systems have become increasingly common in software engineering projects [1] . while much of the media attention is on flashier systems such as autonomous vehicles, less high-profile ai systems such as decision-making support systems have become increasingly widespread in various organizations. ai systems often operate under the surface in the form of e.g. recommendation algorithms, making the high-profile systems in the middle of the media hype only the tip of the iceberg. over the last two decades, progress on ai has been accelerating rapidly. ai systems are now widely used in various areas and for various purposes. examples include medical systems [2] , law enforcement [3] , and manufacturing industries and industry 4.0 [4] , among numerous others. as the field progresses, the already impressive potential of ai systems becomes even larger, including applications such as general ai systems, the likes of which are already being developed by the technology giants such as alphabet. it is exactly because of this impressive potential and impact of these systems, especially in the future, that their potential negative impacts should also discussed more. ai systems are ultimately still software. they are affected by largely the same requirements as any other software system. ai development projects are still for the most part conventional software engineering, with machine learning related tasks only comprising a small portion of these projects [5] . however, ai systems are unique in terms of their effects on various stakeholders to the point where they can even exert society-wide influence. moreover, these stakeholders often have little power in opting out of using these systems. e.g. it is difficult to avoid having a firm filter your job application using ai or trying to avoid being monitored using ai-based surveillance systems if such systems are in place in the area. various system failures have already highlighted some of the potential issues these systems can have in practice. past incidents that have received global media coverage, even smaller incidents can be costly for the affected organization(s). for example, the national finnish broadcasting company, yle 1 , utilized ai for moderation purposes in its services. having already changed its processes to suit the automation of the moderation, the organization ultimately ran into problems with the ai moderator system. though the software was working fine on the technical level, the socio-ethical issues forced the organization to revert back to human moderators. many of these issues are ultimately rooted in ethics. ai ethics has thus become a new non-functional requirement to address; an -ility among the likes of quality, maintainability, and scalability. existing methods have focused on tackling these functional and non-functional requirements. however, no such methods currently exist for ai ethics [6] , with the existing tools and methods largely being technical and limited to narrow contexts in ml as opposed to being project-level methods. in the absence of methods, how are ethics currently implemented? much of the current literature in the area has been theoretical, and our understanding of the state of practice in ai ethics is currently lacking. [6] ai ethics literature discusses various aspects of ai ethics that should be taken into account, but bridging the gap between research and practice in the area remains an on-going challenge [7, 8] . guidelines for implementing ai ethics exist, but their effect on the start of practice remains unknown. thus, to begin bridging this gap in the area, we conduct an empirical study to help us understand the current state of practice. we do so by means of a multiple case study of three projects focusing on healthcare systems. the goal of this study is two-fold: (1) to help us understand the current state of practice in ai ethics; and (2) to discover existing good practices that might help in implementing ai ethics. out of these two goals, the first is a theoretical contribution while the second one is a practical one. the specific research question of the paper is as follows: rq: how are ai ethics taken into consideration in software engineering projects when they are not formally considered? ethics in software development and interactive systems design in general has a history of over 30 years. for example, bynum [9] introduced the idea of adapting human values in design before the rise of human computer interaction and other human-centric paradigms. theoretically grounded approaches such as value sensitive design (vsd) and its variants have provided tools to design technology that takes into account human values in the design process [10, 11] . as more progress is made in the field of ai systems, old theoretical scenarios in ai ethics are slowly becoming reality. this calls for new methods to manage the ethical issues arising from these new systems [7, 12] . indeed, vallach and allen [12] argue that ai and ai-based systems produce new requirements to consider. specifically, they propose that designers implicitly embed values in the technologies they create [12] . ai and other complex systems force designers to consider what kind of values are embedded in the technologies and also how the practical implementation of these values could be carried out and how these systems could be governed [13] . yet, little is currently known about software development practices and methods in the context of ai ethics, as empirical studies in the area are scarce. our results from an existing study suggest that ai ethics are seldom formally implemented in se projects, [14] . similarly, there are currently no project-level methods that could aid in implementing ai ethics [6] . on the other hand, various tools that can support specific elements of ai ethics do exist, such as tools for managing machine learning [6] . however, they do not help developers implement ai ethics in general. in this light, it can be said that ai ethics has hardly been incorporated into mainstream se literature yet. the reason why ai ethics has received little attention in the prior engineering literature is three-fold: 1) prior research has been predominantly philosophical, 2) the field has not sensed the need to address ethical concerns and 3) thus it has not been part of the education system. though some practice-focused research does exist (e.g. [15] ), most of the research on ai ethics has been conceptual and theoretical in nature. these studies have e.g. focused on defining ai ethics in a practical manner through various constructs in the form of values. for the time being, this discussion on defining ai ethics has come to center around four values: transparency [16, 17] , accountability [8, 16] , responsibility [16] , and fairness (e.g. [18] ). not all four of these values are universally agreed to form the core of ai ethics, however, as we discuss in the following section while presenting our research framework. following various real-life incidents out on the field (e.g. amazon's biased recruitment ai 2 ), ai ethics has also begun to spawn public discussion. this has led to governments, standardization institutions, and practitioner organizations reacting by producing their own demands and guidelines for involving ethics into ai development, with many standards and regulations in the works. countries such as france [19] and germany [20] have emphasized the role of ethics in ai, and on an international level the eu began to draft its own ai ethics guidelines which were presented in april 2019 [21] . moreover, iso has founded its own ethical, trustworthy ai in iso/iec jtc 1/sc 42 artificial intelligence subcommittee [22] . finally, some larger practitioner organizations have also presented their own guidelines concerning ethics in ai (e.g. google [23] and microsoft [24] guidelines). thus far, these various attempts to bring this on-going academic discussion out on the field have been primarily made in the form of guidelines and principles. out of these guidelines, perhaps the most prominent ones up until now have been the ieee guidelines for ethically aligned design (ead), born from the ieee global initiative on ethics of autonomous and intelligent systems alongside its ieee p7000™ standards working groups, which were branded under the concept of ead [8] . existing literature has shown us that guidelines and principles in the field of ict ethics do not seem to be effective. mittelstadt [25] argue that ai developers lack the professional norms and methods to translate principles into practice in successful way. to this end, mcnamara et al. [26] also argue based on empirical data that the acm ethical guidelines 3 had ultimately had very little impact on developers, who had not changed their ways of working at all. in this light, this is likely to be the case with the aforementioned ai ethics guidelines as well, as mittelstadt suggest [25] . this notion is further supported by morley et al. [6] who argue that developers focused on practicality are unlikely to adopt them when the competitive advantage of ead is unclear. to assist in the data collection and analysis in this study, we devised a research framework based on prominent literature in the area. this research framework and the justifications behind it are further discussed in an existing paper [27] (fig. 1 ). as the basis of the framework, we utilized the art principles of dignum [16] , which consist of accountability, responsibility, and transparency. these have been central constructs in the area, having also been featured in the ead guidelines of ieee. transparency is required for accountability and responsibility (line 1.c), as we must understand why the system acts in a certain fashion, as well as who made what decisions during development in order to establish accountability [17] . whereas accountability can be considered to be externally motivated, closely related but separate construct responsibility is internally motivated. the concept of accountability holds a key role in aiming to prevent misuse of ai and in supporting wellbeing through ai [8] . accountability refers to determining who is accountable or liable for the decisions made by the ai. dignum [16] in their recent works defines accountability to be the explanation and justification of one's decisions and one's actions to the relevant stakeholders. in the context of this research framework, accountability is used not only in the context of systems, but also in a more general sense. we consider, e.g., how various accountability issues (legal, social) were considered during development. dignum [16] defines responsibility as a chain of responsibility that links the actions of the systems to all the decisions made by the stakeholders. we consider it to be the least accurately defined part of the art model, and thus have taken a more comprehensive approach to it in our research framework. according to the ead guidelines, responsibility can be considered to be an attitude or a moral obligation for acting responsibly [8] a simplified way of approaching responsibility would be for a developer to ask oneself e.g. "would i be fine with using my own system?". in addition to the art principles, we utilized the three ai ethics categories presented by dignum [28] to make these constructs more practical. dignum suggests that ai ethics can be divided into: • ethics by design (integration of ethical reasoning capabilities as a part of the behaviour of artificial autonomous system, e.g. ethical robots); • ethics in design (the regulatory and engineering methods supporting ethical implications of ai systems); and • ethics for design: (codes of conduct, standards, and certification processes that ensure the integrity of developers and users) [28] . in this paper, we focus on the ethically aligned development process, and therefore the last two categories were included into the research framework. finally, aspects of commitment were utilized in the framework to aid data analysis. specifically, we utilized the commitment net model of abrahamsson [29] to approach the implementation of ethics into practice and have an explaining theoretical framework to examine ethics role to developers. from this model, we focused on concerns and actions. concerns were analyzed to understand what ethical issues were of interest to the developers. actions were then studied to understand how these concerns were actually tackled, or whether they were tackled at all. in commitment net model, actions are connected to concerns because when actions are taken, they are always driven from concerns [29] . on the other hand, however, concerns can exist without any actions taken to address them. the dynamic between actions and concerns was considered a tangible way to approach the focus of this study: practices for implementing ai ethics. developers actions could be likened to practices that were taking during the development. on the other hand, analyzing the concerns that developers have opens a view to understanding e.g. whether the developers perhaps wanted to implement ethics but were unable to do so. this section is split into three subsections. first, we discuss the cases of the case study. in the second and third ones we discuss the data collection and analysis, respectively. we conducted a multiple case study featuring three case projects. in all of the case projects, ai systems were being developed for the healthcare sector. these cases are outlined in the table below (table 1) . we chose to utilize a qualitative case study approach due to the exploratory nature of the topic, as the research area is novel as far as empirical studies are concerned. healthcare cases were selected due to the assumption that ethical consideration would be more common in healthcare-related projects due to the nature of the area in closely dealing with human well-being (e.g. the tradition of bio and medical ethics). indeed, healthcare systems can, for example, influence the decisions made by doctors or their patients related to the health of the patients. moreover, due to the emphasis on taxfunded public healthcare in finland, where the cases were from, the area is particularly regulated. these regulations impose some ethical requirements on software systems as well, especially in relation to handling patient data, which is considered particularly sensitive data from a legal point of view. in the paper title, we characterize these case projects as being startup-like because the projects shared various characteristics typically associated with software startups. first, agile methods were commonly utilized in the projects. secondly, the projects were all characterized by notable time pressure. thirdly, the projects operated with scarce resources. fourthly, the cases were centered around the development of functional prototypes, which were intended to as proof-of-concept type artifacts. however, the prototypes were being developed with real customers and tested in practice. finally, the projects exhibited exploratory approaches that focused on experimentation. currently, much of the on-going ai development is happening in startups [1] , even if the multinational organizations receive much media coverage in relation to ai. in characterizing them as startup-like, we consider them to be representative of the current ai development projects. data from the cases were collected using semi-structured interviews [30] . this interview strategy enabled the interviews to be conducted in a way that allowed for flexibility from the interview questions, but without steering too far from the topic. the interview instrument used in the interviews can be found externally as a reference 4 . all interviews were conducted as f2f interviews and the audio was recorded for transcription. the analysis was conducted using the transcripts. the interviews were conducted in finnish. this was done so that the respondents would not give shorter responses due to being uncomfortable with communicating in english, especially while being on record. the respondents from the cases were either developers or managers. as we wanted to focus on development practices and project issues, we focused on the personnel directly involved with the practical development issues in the projects. the respondents are outlined in the table in the previous subsection. in terms of experience, respondents 4, 5, 7, and 8 were junior developers. respondents 3 and 6, on the other hand, were senior developers. respondent 1 was a junior data scientist. we analyzed the data in two phases. first, we utilized a grounded theory (heath [31] ) inspired approach to code the transcripts quote by quote for each interview. this process was carried out iteratively as the list of codes was updated during the process. this approach was chosen due to the lack of existing studies on the current state of practice in the area. in the second phase, we utilized the commitment net model of abrahamsson [29] to then further analyze and categorize the coded content. we utilized the model by focusing on the concerns and actions of the developers. the concerns and actions of each respondent were compared across cases in search of recurring concerns and actions between cases and respondents. by evaluating the relationships between the actions taken in development the development process and the concerns of the developers, we could better understand the motivation behind the actions. similarly, we could also see which concerns did not lead to any actions, pointing to a lack of commitment towards tackling those concerns. the data were then compared with the research framework again to evaluate how ai ethics were implemented in each project. actions were the emphasis here, as the focus of this study was on tangible implementation of ai ethics and how it was carried out in terms of tools, practices, or methods. however, we also highlighted interesting findings in relation to the mere concerns related to ai ethics. this section is split into four subsections. the first three feature the analysis split between the accountability, responsibility and transparency constructs. the final subsection summarizes the analysis. we highlight our findings as primary empirical conclusions (pecs). during the analysis, we use quotes from the interviews to elaborate on the topic at hand. however, it should be noted that the conclusions are not drawn merely based on these individual citations. the concerns of the developers related to responsibility were varied, but ultimately detached from practice as far as concerns related to ai ethics were considered. the concerns the developers discussed in relation to responsibility were simply very practical concerns related to internal project matters or delivering a high quality product: "responsibility on reporting and keeping the project on schedule" (r6) pec1. developers feel most responsibility towards tackling problems related to software development, such as finding bugs, meeting project goals. on the other hand, as the interviews progressed, the developers did also express some concerns towards various ethical issues. however, these concerns were detached from their current work. they did not affect the way they worked, and the developers felt that these types of concerns were not relevant during development. the presence of concerns in the absence of actions to address those concerns pointed towards a lack of commitment on this front. "it is just a prototype" (r8) "i do my best" (r5) "but this is a prototype, an experiment, just to show people that you can do this type of thing. this doesn't really have any responsibility issues in it." (r1) pec2. on a personal level, developers are concerned about the ethical aspects of product development. however, little is done to tackle these concerns. furthermore, it was evident that in none of the cases had the hypothetical effects of the system on the stakeholders been discussed. to give a practical example, a system potentially affecting memory illness diagnoses clearly has various effects on its potential users, especially when the test can be taken without supervision. yet, the developers of this particular tool also felt that their users would not be curious about the workings of the system. they considered it sufficient if the responsibility was outsourced to the user and it was underlined that the system does not make the diagnosis but simply advises doctors. the developers did not consider the potential harm of the system past the tangible, physical harm potential of the systems. for example, stress or other negative effects on users and other stakeholders were not considered. in all three cases, the respondents did not consider the system to have had any potential of causing physical harm, and thus did not consider the system to have any notable harm potential at all. "nobody wants to listen to ethics-related technical stuff. no five hour lectures about it. it's not relevant to the users" (r5) "i don't really understand what it [responsibility] has to do with product development. we developers are all responsible." (r7) "what could it affect… the distribution of funds in a region, or it could result in a school taking useless action… it does have its own risks, but no one is going to die because of it" (r1) pec3. responsibility of developers is unclear. case a highlighted the potential importance of mathematical expertise. the team had internal mathematical capabilities that allowed them to develop their own algorithms, as well as to better understand third party components, in order to have achieve a higher standard of transparency. they utilized algorithms they were familiar with and which they understood on an in-depth level. thus, the team considered themselves to be able to understand why the system made certain decisions in certain situations. this underlines the importance of mathematical skills in preventing the birth of black boxes in ai development. "in that sense it's not really a black box as we can understand what's going on in there just fine, and we can show the nodes and what affects them. it's a very transparent algorithm." (r3) the other two cases utilized existing ai solutions. they did not have an in-depth understanding of the technologies they were utilizing, which resulted in their systems being (partially) black boxes. they understood any components created by the team but did not have a full understanding of the third party components they had used as a base. this presents problems for feature traceability. even though transparency of algorithms and data was not present in two of the cases, the developers in case b nonetheless acknowledged its potential importance however, as it was not considered a formal requirement in the projects, the managers did not devote resources towards pursuing it. even in case a, transparency was not produced as a result of ethical goals but out of business reasons. "we have talked about the risks of decision-making support systems but it doesn't really affect what we do" (r5) pec5. developers recognize transparency as a goal, but it is not formally pursued. on the other hand, in relation to transparency of systems development, all three cases displayed transparency. by having formal decision-making strategies, they were able to keep track of higher-level decisions related to the system. through proper documentation, they were able to keep track of decisions made on the code level. version control also assisted in this regard, making it clear who made what changes and when in retrospect. there were thus various existing practices that produced transparency of systems development. two of the cases also acknowledged the effects of team size on transparency of systems development. they noted that, in addition to documentation practices, the small team size itself made it easy to keep track of the actions of individual developers even in an ad hoc manner. established se practices, such as code documentation and code review, support transparency of systems development. some aspects of accountability were clear points of focus in the projects, namely ones related to security in terms of general information security as well as data management. the respondents were aware of being in possession of personal data, given that they developed healthcare solutions, and were concerned with keeping it secure. they mentioned taking measures to keep the data secure from potentially malicious actors, and they were aware that they would have to take measures to act in accordance with laws and regulations in the area. however, in some cases they had not done so yet. "it's really important how you handle any kind of data, that you preserve it correctly, among researchers, and don't hand it out to any government actors. for example, many of the data packages have kind of interesting data and it can't get into the wrong hands. i personally can't see any way to harm anyone with the data we have though" (r2). "we haven't really paid much attention to the [data] safety aspects yet… it hasn't really been a main focus. there's probably a lot of things we have to take into account [eventually]" (r5). the ethical concerns they had in relation to accountability were in general largely related to existing areas of focus in software development. for example, error handling was one aspect of accountability the respondents were particularly concerned with. this was tied with their goal of making quality software, which they considered their responsibility as professionals. the respondents could, to this end, discuss what tangible practices they utilized to deal with error handling. pec7. developers feel accountable for error handling and have the means to deal with it. however, error handling was largely considered from the point of view of writing code and testing it in a laboratory setting. i.e. the system was considered error free if there were no red lines in the code in the ide during development. only case company b discussed measures they had taken to monitor errors in use. furthermore, potential misuse (e.g. a prankster drawing a horizontal white line on the pavement to intentionally confuse autonomous vehicles) and error scenarios during the operational life of the system had not been actively considered in any of the case projects. "the calculations are made in the algorithms, so it doesn't really make mistakes" (r2) pec8. product misuse and error scenarios are only considered during development. they are not considered in terms of the future operational life of the system out on the field. due to the nature of machine learning, ai systems learn as they are taught with new data or as they collect it themselves while operating out on the field. from this arises the potential issue of unexpected behavior as a result of machine learning. none of the respondents had made plans to tackle potential unexpected behavior during the operational life of their system, should such behavior arise. in only one of the projects was the possibility directly acknowledged: "we just put it up for end-users to test and note that this is still being developed" (r7). pec9. developers do not have plans to deal with unexpected behavior of the system resulting from e.g. machine learning or the future expansion of the use context of the system. past the art constructs, we highlight some commonalities between the cases on a more general level while summarizing our findings. in none of the cases were ethics implemented by following a formal method or tool, nor were ethical issues considered directly as ethical issues. rather, any ethical issues tackled in the projects were tackled for practical reasons (e.g. error free software is beneficial from the point of view of customer relations). nonetheless, some of the ethical issues such as error handling and transparency of systems development were tackled in a systematic manner through existing software engineering practices such as code documentation and version control. on the other hand, though ethics were not taken into consideration on a project level, the respondents still exhibited some concern towards the potential socio-ethical issues in the systems. when prompted, they were able to come up with various negative effects the systems could have on different stakeholders. they considered these to be potential real issues, but did not have a way to address these concerns in the absence of tools, practices, and methods for doing so. moreover, they seemed to realize these potential issues only after being directly asked about them in the interviews. this also points to a lack of tools to aid in ethical analyses. in this section, we have collected all the primary empirical conclusions (pec) outlined in preceding analysis section into table 2 . we relate each of these findings to existing literature and discuss their implications in this section. we classify each of these pecs based on their contribution into either novel findings, findings that (empirically) validated existing literature, or findings that contradict existing literature. many of our findings underline a gap between research and practice in the area. whereas research on ai ethics alongside various guidelines devised by researchers [8] and practitioners [23, 24] alike has discussed various ethical goals for ai systems, these goals have not been widely adopted out on the field. in this sense, we consider some of our findings (pecs 4, 5, 8, and 9) to contradict existing literature. for example, extant literature has highlighted the importance of transparency of algorithms and data [15] [16] [17] . without understanding how the system works, it is impossible to establish why it malfunctioned in a certain situation, which may e.g. be pivotal in understanding the causes of an accident that resulted in material damage [15] . our findings point towards transparency being largely ignored as a goal (pec5). existing system components are utilized as black boxes, and developers do not see this as a notable problem (pec4). we consider pec5 to contradict existing literature in that existing literature has, on multiple occasions, highlighted the importance of transparency in ai systems. yet, out on the field, this importance does not seem to be recognized to the point where it would result in changing development practices. the situation is similar for tackling potential misuse of the systems, error handling during system operations, and handling unexpected system behavior (pec8-9). these goals are included into the ieee ead guidelines [8] . however, none of the case companies took any measures to address these potential issues. on a further note of transparency, however, the lack of emphasis placed on it is also curious in relation to feature traceability in se. for decades, understanding the inner workings of the system was considered key in any se endeavor. yet, in the context of ai systems, the long-standing goal of feature traceability seems to be waning. our findings point towards this being at least partially a result of a lack of mathematical understanding, as the one case company that considered their system to be fully transparent also noted that they fully understood the mathematics behind the algorithms they utilized. in using existing components in their systems, developers may not always understand the algorithms in these components. indeed, in this vein, [32] noted that simply seeing the code is not enough if the algorithm is not understood, or the system is not understood as a whole. though we discovered various examples of ethics not being implemented, we also discovered that various existing and established se practices can be used to implement ai ethics. documentation, version control, and project management practices such as meeting transcripts produce transparency of systems development by tracking actions and decision-making (pec6). similarly, software quality practices help in error handling also in the context of ai ethics (pec7), although they do not specifically account for the errors autonomous systems may face while operating out on the field. while discussing responsibility with the respondents, we also discovered that most of their responsibility was related to producing quality software and meeting project requirements. this validates existing literature in the area of spi (e.g. unterkalmsteiner, [33] ). notably, we also discovered that the developers had ethical concerns towards their systems, which is a novel finding in this context (pec2). little is currently known about the state of practice out on the field, although a recent version of the ead guidelines speculated about a gap in the area, which our findings support in relation to most aspects of ai ethics. despite ai ethics largely not being implemented, our findings point towards it partially being a result of a lack of formal methods and tools to implement it. in our data, the reason given by multiple respondents for not actively considering ethical issues was that they were developing a prototype. however, prototypes do influence the final product or service developed based by them, as shown by existing studies [34] . ai ethical issues should be tackled during earlier stages of development as well, seeing as many of them are higher-level design decisions (such as how to carry out machine learning in the system [15] ), which can be difficult to undo later. following this study, as well as a past case study [14] , we suggest that future research seek to tackle the lack of methods and tooling in the area. though developers may be concerned about ethical issues, they lack the means to address these concerns. on the other hand, methods can also raise the awareness of developers in relation to ai ethics, creating concerns where there now are none. in creating these methods, we suggest exploring existing practices that can be used as is or tailored to implement ai ethics, as we have discussed here. given the amount of activity in ai ethics currently, with many governmental actors drafting their own ai ethics guidelines, likely followed by regulations, methods and tools will likely have practical demand in the future. thus, even if one barrier to implementing ai ethics is currently the fact that it is seldom considered a requirement on a project level, regulations and laws can force organizations to take ethics into account. this would inevitably result in a demand for methods in this area, as well as the birth of various in-house ones. finally, in terms of limitations, the most notable limitations of the study stem from the data and the research approach. the qualitative multiple case study approach always poses problems for the generalizability of the data. we acknowledge this as a limitation, although we also refer to eisenhardt [35] in arguing in favor of qualitative case studies, especially in the case of novel research areas. ai ethics, as far as empirical data goes, is a novel area of research. moreover, the multiple case study approach adds some further validity to the data, as we do not base our arguments on a single case. nonetheless, another limitation in the data is also that all the cases were based on finland. for example, the implementation of ai ethics can be more of a focus in us-based companies, as much of the current discussion on ai ethics also originates from the us. one other limitation in the data is that the interviews were conducted in finnish. the constructs such as transparency may not carry the same connotations in finnish as they do in english. this is especially the case with accountability and responsibility, which may not translate in a straightforward manner. however, during the interviews, we sought to clear any misunderstandings related to the constructs with the respondents. the research framework can also be argued to be a limitation. as ai ethics is a currently active field in terms of theoretical discussion, the constructs in the area are constantly evolving. the art principles and ead chosen as a basis of the framework were, at the time of writing, some of the most prominent works in the area. the framework ultimately presents but one way of perceiving ai ethics. this paper furthers our understanding of the current state of practice in the field of ai ethics. by means of a multiple case study, we studied the way ai ethics is currently implemented in practice, if it is implemented at all, when it is not formally or systematically implemented in software engineering projects. our findings can be summarized through the following two key takeaways: • even when ethics are not particularly considered, some currently commonly used software development practices, such as documentation, support ead. this is also the case with focusing on information security. • while the developers speculate potential socioethical impacts of the resulting system, they do not have means to address them. thus, from the point of view of software engineering methods and practices, this highlights a gap in the area. while some of the existing common practices support the implementation of some aspects of ai ethics, there are no methods or practices that help implement it on a project-level. further studies on the topic should seek to assist in the practical implementation of ai ethics. singular practices and especially project-level methods are needed to bridge the gap between research and practice in the area. this lack of higher-level methods was also highlighted in a review of tools and methods in the area [6] . open access this chapter is licensed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ai 50: america's most promising artificial intelligence companies artificial intelligence in medicine artificial intelligence for law enforcement: challenges and opportunities industrial artificial intelligence for industry 4.0-based manufacturing systems hidden technical debt in machine learning systems from what to how: an initial review of publicly available ai ethics tools, methods and research to translate principles in-to practices towards moral autonomous systems ethically aligned design: a vision for prioritizing human well-being with autonomous and intelligent systems, first edition flourishing ethics value-sensitive design value sensitive design: applications, adaptations, and critiques. in: van den hoven why machine ethics? incorporating ethics into artificial intelligence ethically aligned design of autonomous systems: industry viewpoint and an empirical study stop explaining black box machine learning models for high stakes decisions and use interpretable models instead the ethics of information transparency false positives, false negatives, and false analyses: a rejoinder to "machine bias: there's software used across the country to predict future criminals, and it's biased against blacks for a meaningful artificial intelligence: towards a french and european strategy german federal ministry of transport and digital infrastructure: automated and con-nected driving ethics guidelines for trustworthy ai iso/iec jtc 1/sc 42 artificial intelligence responsible bots: 10 guidelines for developers of conversational ai principles alone cannot guarantee ethical ai does acm's code of ethics change ethical decision making in software development? ai ethics in industry: a research framework ethics in artificial intelligence: introduction to the special issue commitment nets in software process improvement mastering the semi-structured interview and beyond: from research design to analysis and publication developing a grounded theory approach: a comparison of glaser and strauss seeing without knowing: limitations of the transparency ideal and its application to algorithmic accountability evaluation and measurement of software process improvementa systematic literature review minimum viable product or multiple facet product? the role of mvp in software startups building theories from case study research. acad key: cord-025886-259357pg authors: mehrotra, sanjay; rahimian, hamed; barah, masoud; luo, fengqiao; schantz, karolina title: a model of supply‐chain decisions for resource sharing with an application to ventilator allocation to combat covid‐19 date: 2020-05-02 journal: nan doi: 10.1002/nav.21905 sha: doc_id: 25886 cord_uid: 259357pg we present a stochastic optimization model for allocating and sharing a critical resource in the case of a pandemic. the demand for different entities peaks at different times, and an initial inventory for a central agency are to be allocated. the entities (states) may share the critical resource with a different state under a risk‐averse condition. the model is applied to study the allocation of ventilator inventory in the covid‐19 pandemic by fema to different u.s. states. findings suggest that if less than 60% of the ventilator inventory is available for non‐covid‐19 patients, fema's stockpile of 20 000 ventilators (as of march 23, 2020) would be nearly adequate to meet the projected needs in slightly above average demand scenarios. however, when more than 75% of the available ventilator inventory must be reserved for non‐covid‐19 patients, various degrees of shortfall are expected. in a severe case, where the demand is concentrated in the top‐most quartile of the forecast confidence interval and states are not willing to share their stockpile of ventilators, the total shortfall over the planning horizon (until may 31, 2020) is about 232 000 ventilator days, with a peak shortfall of 17 200 ventilators on april 19, 2020. results are also reported for a worst‐case where the demand is at the upper limit of the 95% confidence interval. an important finding of this study is that a central agency (fema) can act as a coordinator for sharing critical resources that are in short supply over time to add efficiency in the system. moreover, through properly managing risk‐aversion of different entities (states) additional efficiency can be gained. an additional implication is that ramping up production early in the planning cycle allows to reduce shortfall significantly. an optimal timing of this production ramp‐up consideration can be based on a cost‐benefit analysis. while approximately 80% of covid-19 cases are mild, the most severe cases of covid-19 can result in respiratory failure, with approximately 5% of patients requiring treatment in an intensive care unit (icu) with mechanical ventilation (wu & mcgoogan, 2020) . mechanical ventilation is used to save the lives of patients whose lungs are so damaged that they can no longer pump enough oxygen into the blood to sustain organ function. it provides more oxygen than can be delivered through a nasal cannula or face mask, allowing the patient's lungs time to recover and fight off the infection. physicians in italy have indicated that critical covid-19 patients often need to be intubated for a prolonged period of time (15-20 days) (rosenbaum, 2020) , further exacerbating ventilator scarcity. limiting the death toll within the united states depends on the ability to allocate sufficient numbers of ventilators to hard hit areas of the country before infections peak and ensuring that the inventory does not run out. harder hit states (such as new york, michigan, and louisiana) are desperately trying to acquire additional ventilators in anticipation of significant shortages in the near future. yet in the absence of a coordinated federal response, reports have emerged of states finding themselves forced to compete with each other in order to obtain ventilators from manufacturers (state health systems, 2020) . according to new york's governor cuomo, the state has ordered 17 000 ventilators at the cost of $25 000/ventilator, but is expected to receive only 2500 over the next 2 weeks (ny governor, 2020). as of march 31, 2020, according to the u.s. presidential news briefing, more than 8100 ventilators have been allocated by fema around the nation. of these, 400 ventilators have been allocated to michigan, 300 to new jersey, 150 to louisiana, 50 to connecticut, and 450 to illinois, in addition to the 4400 given to new york (march 31 white house briefing, 2020) . going forward, the federal response to the covid-19 pandemic will require centralized decision-making around how to equitably allocate, and reallocate, limited supplies of ventilators to states in need. projections from the institute for health metrics and evaluation at the university of washington, which assume that all states will institute strict social distancing practices and maintain them until after infections peak, show states will hit their peak demand at different time points throughout the months of april and may. many states are predicted to experience a significant gap in icu capacity, and similar, if not greater, gaps in ventilator capacity, with the time point at which needs will begin to exceed current capacity varying by state (ihme, 2020). in response to the above problem, this paper presents a model for allocation and possible reallocation of ventilators that are available in the national stockpile. importantly, computational results from the model also provide estimates of the shortfall of ventilators in each state under different future demand scenarios. this modeling framework can be used to develop master plans that will allocate part of the ventilator inventory here-and-now, while allocating and reallocating the available ventilators in the future. the modeling framework incorporates conditions under which part of the historically available ventilator inventory is used for non-covid-19 patients, who also present themselves for treatment along with covid-19 patients. thus, only a fraction of the historical ventilator inventory is available to treat covid-19 patients. the remaining demand needs are met by allocation and re-allocation of available ventilators from fema and availability of additional ventilators through planned production. fema is assumed as the central agency that coordinates state-to-state ventilator sharing. the availability of inventory from a state for re-allocation incorporates a certain risk-aversion parameter. we present results while performing a what-if analysis under realistically generated demand scenarios using available ventilator demand data and ventilator availability data for different u.s. states. an online planning tool is also developed and made available for use at https://covid-19.iems.northwestern.edu (covid-19 planning tool, 2020). this paper is organized as follows. a review of the related literature is provided in section 2. we present our resource allocation planning model, and its re-formulation in section 3. section 4 presents our computational results under different mechanical ventilator demand scenarios for the covid-19 pandemic in the united states. in section 5, we introduce our companion online covid-19 ventilator allocation and sharing planning tool. we end the paper with some discussion and concluding remarks. a medical resource allocation problem in a disaster is considered in xiang and zhang (2016) . victims' deteriorating health conditions are modeled as a markov chain, and the resources are allocated to optimize the total expected health recovery rate and reduce the total waiting time. certain illustrative examples in a queuing network setting are also given in xiang and zhang (2016) . the problem of scarce medical resource allocation after a natural disaster using a discrete event simulation approach is investigated in cao and huang (2012) . specifically, the authors in cao and huang (2012) investigate four resource-rationing principles: first come-first served, random, most serious first, and least serious first. it is found that without ethical constraints, the least serious first principle exhibits the highest efficiency. however, a random selection provides a relatively fairer allocation of services and a better trade-off with ethical considerations. resource allocation in an emergency department in a multiobjective and simulation-optimization framework is studied in feng, wu, and chen (2017) . simulation and queuing models for bed allocation are studied in vasilakis and ei-darzi (2001) and gorunescu, mcclean, and millard (2002) . the problem of determining the levels of contact tracing to control spread of infectious disease using a simulation approach to a social network model is considered in armbruster and brandeau (2007) . a linear programming model is used in investigating the allocation of hiv prevention funds across states (earnshaw, hicks, richter, & honeycut, 2007) . this paper suggests that in the optimal allocation, the funds are not distributed in an equitable manner. a linear programming model to derive an optimal allocation of healthcare resources in developing countries is studied in flessa (2000) . differential equation-based systems modeling approach is used in araz, galvani, and meyers (2012) to find a geographic and demographic dependent way of distributing pandemic influenza vaccines based on a case study of a/h1n1 pandemic. in a more recent covid-19-related study, kaplan (2020) proposes a probability model to estimate the effectiveness of quarantine and isolation on controlling the spread of covid-19. in the context of ventilator allocation, a conceptual framework for allocating ventilators in a public emergency is proposed in zaza et al. (2016) . the problem of estimating mechanical ventilator demand in the united states during an influenza pandemic was considered in meltzer, patel, ajao, nystrom, and koonin (2015) . in a high severity pandemic scenario, a need of 35 000-60 500 additional ventilators to avert 178 000-308 000 deaths was estimated. robust models for emergency staff deployment in the event of a flu pandemic were studied in bienstock and zenteno (2015) . specifically, the authors focused on managing critical staff levels during such an event, with the goal of minimizing the impact of the pandemic. effectiveness of the approach was demonstrated through experiments using realistic data. a method for optimizing stockpiles of mechanical ventilators, which are critical for treating hospitalized influenza patients in respiratory failure, is introduced in huang et al. (2017) . in a case-study, mild, moderate, and severe pandemic conditions are considered for the state of texas. optimal allocations prioritize local over central storage, even though the latter can be deployed adaptively, on the basis of real-time needs. similar to this paper, the model in huang et al. (2017) uses an expected shortfall of ventilators in the objective function, while also considering a second criteria of total cost of ventilator stockpiling. however, the model in huang et al. (2017) does not consider distribution of ventilators over time. in the case of covid-19, the ventilator demand is expected to peak at different times in different states, as the demand for each state has different trajectories. only forecasts are available on how the demand might evolve in the future. in this paper, we assume that the planning horizon is finite, and for simplicity we assume that reallocation decisions will be made at discrete times (days). under certain demand conditions, the ventilators may be in short supply to be able to meet the demand. our model is formulated as a stochastic program, and for the purpose of this paper, we reformulate and solve the developed model in its extensive form. we refer the reader to birge and louveaux (2011) and shapiro, dentcheva, and ruszczyński (2014) for a general description of this topic. in this section, we present a multiperiod planning model to allocate ventilators to different regions, based on their needs, for the treatment of critical patients. we assume that the demand for ventilators at each planning period is stochastic. we further assume that there is a central agency that coordinates the ventilator (re)location decisions. the ventilators' (re)location is executed at the beginning of each time period. once these decisions are made and executed, the states can use their inventory to treat patients. both the federal agency and the states have to decide whether to reserve their inventory in anticipation of future demand or share it with other entities. before presenting the formulation, we list the sets, parameters, and decision variables that are used in the model. - : states (regions), indexed by n ∈  ≔{1, … , | |}, - : planning periods, indexed by t ∈  ≔{1, … , | |}, -ω: ventilators' demand scenarios, indexed by ∈ ω ≔ {1, … , |ω|}, • deterministic parameters -y n : the initial inventory of ventilators in region n ∈  at time period t = 0, -i: the initial inventory of ventilators in the central agency at the beginning of time period t = 0, -q t : the number of ventilators produced during the time period t − 1 that become available at the beginning of time period t ∈  , for t ≥ 1, n : the percentage of the initial inventory of ventilators in region n ∈  that cannot be used to care for the patients at the critical level, n : the percentage of the initial inventory of ventilators in region n ∈  that the region is willing to share with other regions, among those that can be used to care for patients at the critical level, n : the risk-aversion of region n ∈  to send their idle ventilators to the central agency to be shared with other regions, • stochastic parameters d n,t : the number of patients in region n ∈  at the critical level that need a ventilator at the beginning of time period t ∈  under scenario ∈ ω, p : probability of scenario ∈ ω, • decision variables x n, t : the number of ventilators reallocated to region n ∈  by the central agency at the beginning of time period t ∈  , z n,t : the number of ventilators reallocated to the central agency by region n ∈  at the beginning of time period t ∈  under scenario ∈ ω, y n,t : the number of ventilators at region n ∈  that can be used to care for the patients at the critical level at the end of time period t ∈ {0} ∪  under scenario ∈ ω, s t : the number of ventilators at the central agency at the end of time period t ∈ {0} ∪  under scenario ∈ ω. for notational convenience, we also define the vector we might drop the superscript ∈ ω from this notation and use the same symbol with a tilde to denote that these parameters are stochastic. for example, we might used. similarly, we define the deciin this section, we assume that there is no lead time between sending a ventilator by an entity (a region or the central agency) and delivery by another entity. with this assumption, the planning model to minimize the expected shortage of ventilators in order to treat patients at the critical level is formulated as a two-stage stochastic program as follows: where we now explain the model in detail. in the first stage, the central agency makes the "here-and-now" decisions x before the stochastic parametersd are realized. as captured in (1a), the goal of the central agency is to minimize the expected total shortage of ventilators over all time periods t ∈  and all regions n ∈  . the objective also includes a cost, parameterized by of allocating a ventilator by the central agency to a state at a given time. this cost can be set to zero, or set to a small value. in our computations we set = 0.01. in the second stage, once the stochastic parametersd are realized, the "wait-and-see" decisions z n, t , y n, t , s t , n ∈  , and t ∈  , are made. these decisions are scenario-specific, and are indicated by the superscript ∈ ω, in the extensive formulation given in (3). constraints (2b) and (2c) ensure the conservation of ventilators for the regions and the central agency at each time period, respectively. constraint (2d) enforces that at each time period, a region is not sending out any ventilator to the central agency if its in-hand inventory is lower than its safety stock, where the safety stock is determined as ndn,t , for t ∈  and n ∈  . constraint (2e) ensures that at each time period, the total number of outgoing ventilators from the central agency to the regions cannot be larger than the available inventory, after incorporating the newly produced ventilators and the incoming ones from other regions. constraints (2f) and (2g) set the initial inventory at the regions and central agency, respectively. the remaining constraints ensure the nonnegativity of decision variables. note that the objective function (2a) and constraints (2d) are not linear. by introducing an additional variable, the term (d n,t − y n,t ) + in the objective function, for n ∈  , t ∈  , and ∈ ω, can be linearized as e n,t ≥d n,t − y n,t , e n,t ≥ 0. furthermore, for each region n ∈  and time period t ∈  , constraint (2d) can be linearized as where m is a big number. by incorporating the finiteness of the support ofd, a linearized reformulation of model (1) can be written as a mixed-binary linear program in the following extensive form: where d n,t denotes the number of patients at the critical level in regions n ∈  that need a ventilator at the beginning of time period t ∈  under scenario ∈ ω. note that all second-stage variables z n,t , y n,t , and s t , n ∈  , and t ∈  in model (3) have superscript to indicate their dependence to scenario ∈ ω. it is worth noting that (1) (and (3) as well) considers multiperiod decisions. in the model, the central agency will make decisions for the entire planning horizon using the information that is available at the beginning of planning. for our numerical experiments in section 4, we used a commercial mixed-integer programming solver to obtain the results. furthermore, we used i + n y n,0 + ∑ t ′ ≤t q t as a big-m for n ∈  and t ∈  . in this section, we assume that there is a lead time of l time periods between sending a ventilator by an entity (a region or the central agency) and delivery by another entity. with this assumption, (1) can be generalized as follows: where note that model (4) is obtained by revisiting constraints (2b), (2c), and (2e) to incorporate lead time in the planning. constraints (5b) and (5c) require the conservation of ventilators for the regions at each time period, where a ventilator sent by the federal agency to a region at time period t − l, t > l, will become available for the region at time period t. constraints (5d) and (5e) ensure the conservation of ventilators for the central agency, respectively, where a ventilator sent by a region to the federal agency at time period t − l, t > l, will become available for the central agency at time period t. constraints (5g) and (5h) enforce that the total number of outgoing ventilators from the central agency to the regions cannot be larger than the available inventory, after incorporating the newly produced ventilators and the incoming ones from other regions. similar to (2b) and (2c), constraint (2e) is also divided into sets for t ≤ l and t > l in (5g) and (5h). by incorporating the finiteness of the support ofd, a linearized reformulation of model (1) can be written as a mixed-binary linear program in the following extensive form: similar to (3), model (4) can be written as a mixed-binary linear program in the following extensive form: y n,t−1 + x n,t−l − z n,t = y n,t , ∀ ∈ ω, ∀n ∈  , z n,t ≤ y n,t − (1 − )y n,0 − n d n,t + m(1 − g n,t ), z n,t ≤ n,t , ∀ ∈ ω, ∀n ∈  , ∀t ∈  , e n,t ≥ d n,t − y n,t , ∀ ∈ ω, ∀n ∈  , ∀t ∈  , (6m) the ventilator allocation model (3), described in section 3, was implemented in python 3.7. all computations were performed using gurobi 9.0.1, on a linux ubuntu environment on two machines. in the first machine, we used 14 cores, with 3.4 ghz processor and 128 gb of ram, and set the time limit to 2 hours. in the second machine, we used 64 cores, with 2.2 ghz processor and 128 gb of ram, and set the time limit to 3 hours. since projected ventilator need is a key input for the model, it is important to use accurate estimates of the demand forecasts. the forecasts of ventilator needs generated by ihme (2020) were used in our computational study. these forecasts were first made available on march 26, 2020, and used the most recent epidemiological data and advanced modeling techniques. the available information closely tracks the real-time data (ihme covid-19 projections, 2020 we considered a 70-day planning period, starting from march 23, 2020 and ending on may 31, 2020. we generated the random demands in ways that correspond to projected future demands under different mitigation effects. more precisely, we considered six different cases to generate random samples for the number of ventilators needed to care for covid-19 patients. these cases are listed below: case i. average-i: each of the demand scenarios has equal probability and the distribution is uniform over the range of the ci provided in ihme (2020) we further discuss the demand generation procedure. a demand scenario contains the demand data for all days and states. in all cases i-vi, we assumed that the forecast ci provided in ihme (2020), for each day and for each state, represents the support of the demand distribution. cases i and ii are generated to develop average demand scenario representations that use the information provided in the ci given in ihme (2020) in two different ways. in case i, it is assumed that the mean is the median of the demand distribution (ie, the right-and left-tail of the demand distribution have 0.5 probability). we randomly generated a number to indicate which tail to sample from, where both tails have the same 0.5 probability of being chosen. once the tail is determined, we divided the tail into 50 equally distanced partitions, and chose a random partition to uniformly sample from. we repeated this process for all days and states. in order to capture the spatiotemporal correlation between demand realizations, we sampled from the same tail and partition for all days and states, although the range from which we sample depends on the ci. in this case, all scenarios are equally likely. in case ii, we randomly generated a number to indicate which tail to sample from, where the top 25% of the ci (ie, the right tail) has a 0.25 probability and the bottom 75% (ie, the left tail) has a 0.75 probability of being chosen. if the right tail is chosen, we set the weight of the scenario to 0.25, and we set it to 0.75 otherwise. the rest of the procedure is similar to case i. in order to determine the probability of scenarios, we normalized the weights. demand scenarios in cases iii-v are generated in the same fashion as in case ii, where the only difference is in the probability of which tail to choose from, which is determined by the sampling scheme described in the definition of the case. cases i and ii are intended to parameterize the model to capture the average case. these two cases were considered because our data analysis showed that the confidence intervals on the forecasts provided by ihme covid-19 projections (2020) were not symmetric. we attempted a log-transformation of the confidence intervals, but found that the log-transforms also provided asymmetric confidence intervals. hence, it was considered more appropriate to generate the demand scenarios using two different sampling schemes. for cases i-vi, we generated 24 scenarios, while in case vi, there is only one scenario which happens at the upper limit of ci. note that in each case, different quantities for the random demandd n,t , t ∈  , n ∈  , and ∈ ω, might be generated. an illustration of the trajectory of demand scenarios over time is given in figures 1-3 for the us and the states of new york and california. the y-axis in these figures represents the demand realization in each sampled scenario. another key input to the planning model is the initial ventilator inventory. as of march 23, 2020, before the rapid rise of covid-19 cases in the state of new york, fema had about 20 000 ventilators in reserve, that is, i = 20 000. we used this for our model which suggests ventilator allocation decisions from march 23, 2020. estimates for the initial inventory of ventilators at different states were obtained from mapping us health system (2020). these estimates are based on a hospital survey (rubinson et al., 2010; united states resource, 2020) . the estimates for new ventilator production were obtained based on information provided at the us presidential briefings on march 27, 2020 (coronavirus outbreak, 2020 . these estimates suggest that the normal yearly ventilator production capacity is about 30 000 ventilators/year. however, under the u.s. defense production act, with the participation of additional companies, production of approximately 10 000 ventilators/month could be possible (coronavirus outbreak, 2020) . using this information, for the baseline case we assumed that the current daily ventilator production rate is q t = 80 ventilators/day; and it will be increased to q t = 320 ventilators/day starting on april 15, 2020. we refer to this case, as "baseline production," and analyzed in section 4.5.1. we also analyze the case that the ramp-up in production happens on april 1, 2020 or april 7, 2020 in section 4.5.2. recall that in the model, parameter is used to indicate the fraction of ventilators used to care for non-covid-19 patients. additionally, a parameter is used in the model to estimate a state's willingness to share the fraction of their initial covid-19-use ventilators. similarly, the parameter is used to control the state's risk-aversion to sending their idle ventilators to fema for use in a different state. we suppose that for all states n, n ∈  , we have n = , n = , and n = . in order to systematically study the ventilator allocations and shortfalls, we used the following parameters: ∈ {50%, 60%, 75%}, ∈ {1.25, 1.5, 3}, and ∈ {0%, 10%, 25%}. in this section, we present and discuss the numerical results for the case that there is no lead-time, that is, l = 0 or there is a lead-time of 1 day. for most instances, we observed that even obtaining an integer feasible solution to (3) and (6) in the time limit was not possible. therefore, we replaced these models with their expected value problem, where the stochastic demand is substituted with the expected demand. then, we solved the resulting model. this heuristic yields an integer feasible solution to model (3) and (6) for all instances we tested in the time limit, and we report those results here. in section 4.5.1, we provide the results on ventilator shortage and inflow/outflow from/to fema for the case that there is no lead-time. we also analyze the effect of early ramp-up in production and lead-time on ventilators' shortage in sections 4.5.2 and 4.5.3, respectively. in this section, for each setting ( , , ), we solved the expected value problem of model (3) under cases i-vi. a summary of ventilators' shortage results is reported in tables 1-3. we briefly describe the columns in these tables. column "total" denotes the expected total shortage, and is calculated as where w n,t ≔ (d t,n − i t,n ) + , d t,n ≔ ∑ ∈ω p d n,t , i t,n ≔ y 0,n + fema t,n , and fema t,n ≔ min { ∑ t ′ ≤t x t,n , (d t,n − y 0,n ) + } . quantity "worst day" in column "worst day (t)" denotes the expected shortage in the worst day, and is calculated as where t denotes a day that the worst expected shortage happens, that is, t ∈ arg max t∈ ∑ n∈ w n,t . moreover, quantity "worst day-state" in column "worst day-state (t)" denotes the expected shortage in the worst day and state, and is calculated as where (t, n) ∈ arg max t∈ arg max n∈ w n,t . the results in tables 1-3 suggest that when up to 60% of a state's ventilator inventory is used for non-covid-19 patients, fema's current stockpile of 20 000 ventilators is nearly sufficient to meet the demand imposed by covid-19 patients in mild cases (ie, cases i-iii). the ventilator availability situation gets worse in the case where 75% (or greater %) of the available ventilators must be used for non-covid-19 patients and states' risk-aversion parameter to send the idle ventilators to fema to be used in a different state is 3. in this case, if states are willing to share up to 50% of their excess inventory with other states, then 12 700 number of ventilators will be required beyond fema's current stockpile to meet demand in cases i-iv. however, if no such sharing is considered, then the need for ventilators would increase to 14 200. this situation gets even worse for cases v and vi, where the inventory shortfall on the worst day (april 19, 2020) is between 17 200 and 30 600. this shortfall decreases moderately to 15 900-28 000 if states are willing to share part of their initial ventilator inventory. if parameter goes down to 1.25, the inventory shortfall on the worst day (april 19, 2020) is between 13 800 and 22 800. this shortfall decreases moderately to 12 800-21 300 if states are willing to share part of their initial ventilator inventory. we also analyzed the ventilators' reallocation to/from different states for the setting ( , , ) = (0.75, 3, 0), which is the most dramatic case we considered from the inventory and stockpile perspectives. we report a summary of results in table 4 under the two worst demand situations, cases v (severe) and vi (extreme). column "total inflow" in this table denotes the total incoming ventilators to a state n ∈  from fema, and is calculated as similarly, column "total outflow" denotes the expected total outgoing ventilators from a state n ∈  to fema in order to be shared with other states to be used to treat covid-19 patients, and is calculated as also, column "net flow" represents the difference between "total inflow" and "total outflow." the results in table 4 indicate that in cases vi (severe) and v (extreme), the state of new york requires between 11 100 and 17 500 additional ventilators for covid-19 patients during its peak demand. however, between 400 and 17 000 of these ventilators can be given to a different state after the peak demand in the state of new york has subsided. the insights about other states can also be obtained from this table. the effect of early ramp-up in production on ventilators' shortage in this section, we consider the cases that the ramp-up in production happens on april 1, 2020 or april 7, 2020, as opposed to the baseline production, where the ramp-up in production happens on april 15, 2020. a summary of ventilators' shortage is given in table 5 for the parameter setting ( , , ) = (0.75, 3.00, 0), under the two worst demand situations, cases v (severe) and vi (extreme). as it is evident from table 5 , early ramp-up in production could save up more than 80 000 and 100 000 lives in case v (severe) and case vi (extreme), respectively. in this section, we analyze ventilators' shortage for the case that there is a lead-time of 1 day. a summary of results under case vi is presented in table 6 . it can be seen from this table that, as expected, the inventory shortfall increases with an increase in the lead-time (approximately up to 500 on the worst day). a companion online planning tool is developed in order to view the outputs on the number of ventilators needed and the shortage that might happen under various conditions (covid-19 planning tool, 2020) . this website is available at https://covid-19.iems.northwestern.edu. the users can choose the demand scenario (cases i-vi) and choose different options for parameter , the fraction of ventilators used to care for non-covid patients, parameter , state's willingness to share the fraction of their initial covid-19-use ventilators, parameter , the state's risk-aversion to sending their idle ventilators to fema for use in a different state, and parameter l for lead-time. the results on the website are shown in interactive graphical and tabular formats. a snippet of this online planning tool is given in figure 4 . interested readers can refer to this online companion for more details and analysis beyond what is presented in this paper. the results on covid-19 planning tool (2020) will be updated as additional computations are conducted and new forecast confidence intervals become available. we have presented a model for procuring and sharing life-saving resources whose demand is stochastic. the demand arising from different entities (states) peaks at different times, and it is important to meet as much of this demand as possible to save lives. each participating state is risk averse to sharing their excess inventory at any given time, and this risk-aversion is captured by using a safety threshold parameter. specifically, the developed model is applicable to the current covid-19 pandemic, where many u.s. states are in dire need of mechanical ventilators to provide life-support to severely and critically ill patients. computations were performed using realistic ventilator need forecasts and availability under a wide combination of parameter settings. our findings suggest that the fraction of currently available ventilators that are to be used for non-covid-19 patients strongly impacts state and national ability to meet demand arising from covid-19 patients. when more than 40% of the existing inventory is available for covid-19 patients, the national stockpile is nearly sufficient to meet the demand in mild cases. however, if less than 25% of the existing inventory is available for covid-19 patients, the current national stockpile and the anticipated production may not be sufficient under extreme demand scenarios. as expected, the magnitude of this shortfall increases when one considers more and more extreme demand scenarios. overall, the model developed in this paper can be used as a planning tool/framework by state and federal agencies in acquiring and allocating ventilators to meet national demand. the results reported in this paper can also provide a guide to states in planning for their ventilator needs. we, however, emphasize that these results are based on certain modeling assumptions. this includes the process of demand forecast scenario generation, estimates of initial ventilator inventory, and future production quantities. each one of these, as well as other model parameters, can be changed in the model input to obtain more refined results. nevertheless, an important finding is that a state's willingness to share its idle inventory can help address overall shortfall. while this paper has focused on ventilator needs in the united states, such a model can also be adapted for use in international supply-chain coordination of equipment such as ventilators across countries. covid-19 is expected to have different peak dates and demand cycles in other countries, and one or two additional disease spread cycles are likely until an effective vaccine becomes available. in conclusion, we point out that the model developed in this paper has a one-time planning decision, that is, there are no "wait-and-see" decisions in the model over time. one can also formulate the ventilator allocation problem as a time-dynamic multistage stochastic program, where the decision maker can make recourse decisions as time evolves based on the information available so far on the stochastic demands and past decisions. we are currently working on such an extension. in addition to the model being a one-time planning decision model, the model and its output have some additional limitations. first, the model may have multiple optimal solutions. in a resource constrained environment, alternative solutions may allocate the same number of ventilators differently. the solutions reported in the tables are only one such solution. moreover, these solutions were obtained from solving the models approximately with a prespecified time limit. the solutions from the optimization model presented in this paper depend on the accuracy of ventilator need forecasts. these forecasts are being revised regularly, as additional data based on state-specific mitigation efforts is becoming available. second, the objective function in the model treats the shortfall in large and small states equally. state-specific consideration may allow further refinements to the model. specifically, instead of formulating the objective as an expected value minimization model, we can formulate the objective as a minmax objective of shortfall for each state-thus minimizing the maximum shortfall to any state. such a model is expected to yield a more equitable solution. third, the model in section 4.5.3 assumes a constant lead time. we can modify this model to allow for state-specific lead times. such a modification will allow one to systematically study the effect of state-specific lead time on the overall allocation efficiency. moreover, if shipment times are of concern and a secondary coordination to a stocking depot is required, the model can be adapted to allow for creation of transshipment depots (warehouses) that serve a cluster of states. in this case, the central agency will first ship the inventory to the warehouse, who will further distribute it to the states in need. in all of these aspects the modeling framework presented in this paper should be considered as a first step in the direction of developing planning models that allow for critical resource sharing over time. nevertheless, the overall conclusions based on the model remain valid: in a resource-constrained environment where the demand of different entities peaks at different time points, it is possible to achieve improved efficiency in resource utilization through supply-demand matching over time. risk aversion to sharing excess supply in anticipation of future demand reduces the efficiency resulting from such sharing. this work has been partially supported by the national science foundation through grant cmmi-1763035. the authors would like to thank ebru bish and nan liu for the constructive comments and suggestions. the authors thank ming hu for getting an expedited review of this paper, and making several suggestions that helped improve an earlier draft of this paper. specifically, the lead time model, and the sensitivity analysis for the production function was added in response to his suggestions. orcid sanjay mehrotra https://orcid.org/00000003-1106-1901 geographic prioritization of distributing pandemic influenza vaccines contact tracing to control infectious disease: when enough is enough. health care management science models for managing the impact of an epidemic introduction to stochastic programming principles of scarce medical resource allocation in natural disaster relief: a simulation approach coronavirus outbreak: trump 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stockpiled ventilators during large-scale public health emergencies a model of supply-chain decisions for resource sharing with an application to ventilator allocation to combat covid-19 key: cord-008686-9ybxuy00 authors: everett, tom; douglas, jenny; may, shoshanna; horne, simon; marquis, peter; cunningham, richard; tang, julian w title: poor transmission of seasonal cold viruses in a british antarctic survey base date: 2019-03-14 journal: j infect doi: 10.1016/j.jinf.2019.03.007 sha: doc_id: 8686 cord_uid: 9ybxuy00 nan recently, it is reported in journal of infection that h5n6 1 and h7n9 2 subtype avian influenza virus may have an increased pathogenicity to humans.the h1n1 subtype influenza virus has emerged in china. not only the h1n1 (95%) subtype but also several h3n2 (5%) subtype influenza viruses have been detected in samples. according to chinese national influenza data ( http: //ivdc.chinacdc.cn/ ), the h1n1 subtype influenza virus was prevalent from the end of 2018 to the beginning of 2019. the h1n1 and h3n2 subtypes of influenza virus are resistant to adamantanes (amantadine and rimantadine), and a small number of h1n1 strains have been found to be less sensitive to na inhibitors (nais; oseltamivir, zanamivir, and peramivir). in this study, we briefly evaluated the evolution patterns of the h1n1 influenza virus and the h3n2 influenza virus. we collected non-repeat 674 h1n1 and h3n2 subtype influenza virus sequences isolated in china over the course of nearly 5 years from the global initiative on sharing avian influenza data (gisaid) database ( www.gisaid.org ) and national center for biotechnology information (ncbi) ( www.ncbi.nlm.nih.gov/genomes/flu ). the haplotype network map shows that the 2014 h1n1 strain has a node in common with the 2016/17 h3n2 strain ( fig. 1 ) , and h1n1 and h3n2 often co-infect the same patients. when multiple strains of influenza infect the same host, they may undergo recombination and reassortment of the gene fragment, which greatly changes the pathogenicity and epidemiological characteristics of the virus. currently, the h1n1 subtype influenza virus is widespread in the population, and the number of children with neurological symptoms increased significantly this year. 3 the influenza virus has shown some variation, and whether this variation occurs along h1n1 and h3n2 lines remains to be seen. when multiple viruses co-infection occurs, it becomes possible for the viruses to undergo genetic communication, which may change the direction of viral evolution and so deserves our attention. we calculated the average gene evolution rate (nucleotide replacement rate) of the h1n1 influenza virus and the h3n2 influenza virus between different years from 2013 to 2019. it can be seen that the genetic evolution rate of the h1n1 influenza virus and the h3n2 influenza virus is 2.91e −5 -4.03e −4 and 2.72e −5 -1.05e −4 ( table 1 ) , respectively, in the same year and there are up and down fluctuations that may be related to the subtypes that were prevalent that year. additionally, we calculated the evolution rate of the h1n1 influenza virus from the end of 2018 to the beginning of 2019 (1.13e −3 ). a large increase in the rate of advancement indicates a rapid change in the virus in the short term, triggering changes in the replication, resistance, and transmission of the virus. the type a influenza virus emerges periodically every year,influenza undergoes continuous evolution, and different lineages have appeared. 4 at the same time, under the action of vaccines and drugs, the antigenicity and antigenic site of the virus are transformed and resistant. 5 however, medical science has also improved. it is necessary to determine the frequency of gene exchange between different subtypes and be alert to possible variations in gene communication between different subtypes. pu et al. 6 and shi et al. 7 reported that duck-derived virus h7nx and recombination of h7n9 can produce new h7n2 that can cause disease death in waterfowl. this recombination event may have occurred in 2013 or earlier, but the recombinant virus has a distinct evolutionary advantage given the use of a vaccine. exchange between h1n1 and h3n2 may allow them to give each other different viral characteristics, hence, ongoing surveillance of h1n1 and h3n2 subtypes of influenza is warranted. the authors declare not conflict of interest. recent studies in this journal revealed that some h7n9 viruses reassorted with duck aivs, and then attained the ability to efficiently infect ducks. 1,2 h7n9 aivs have been endemic in chicken since their emergence in china in february 2013. 3 after its emergence, h7n9 viruses have evolved substantially, and have frequently reassorted, acquiring internal genes from other chicken h9n2 viruses, increasing the genetic diversity of h7n9 viruses. 4 this raises the concern that whether h7n9 can attain internal genes from other aivs. thus, we collected all available h7n9 sequences to detect potential novel reassortments of the h7n9 aivs, and found evidences that three human-isolated h7n9 isolates attained internal genes from duck and human aivs. all available sequences of h7n9 aivs were downloaded from the ncbi ( https://www.ncbi.nlm.nih.gov ), gisaid ( https://www.gisaid.org ) and fludb ( https://www.fludb.org ) public databases. then, phylogenetic trees for ha, mp, np, ns, pa, pb1, pb2 and na genes were reconstructed, respectively, using raxml v.8.0.24 with gtrgamma model, and 10 0 0 bootstrap tests. phylogenetic analyses revealed that five genes (np, ns, pa, pb1, and pb2) of a/fujian/33845/2017(h7n9), mp gene of a/gd-66/2014/h7n9/2014-01-29, and two genes (pb1 and pb2) of a/zhejiang/9/2014(h7n9) did not clustered with chicken h7n9 aivs, respectively ( fig. 1 and supplementary fig. 1 ). further, blastn ( https://blast.ncbi.nlm.nih.gov/blast.cgi ) was used to search the homology sequences of these three abnormal strains ( 5 while domestic ducks act as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem. the 2013 h7n9 viruses cannot replicate efficiently in ducks in the first four waves. however, studies have indicates that the highly pathogenic h7n9 virus has extended its host range by acquiring genes from duck influenza viruses and has now adapted to ducks. 1, 2, 6 the reassortments between 2013 h7n9 and duck aivs would further raise the diversity and spread of the h7n9. 7 in addition to our finding of the reassortments between duck aivs and the human-isolated h7n9 viruses suggest that surveillance and control of duck aivs is critical for the control of h7n9 viruses, which was almost ignored previously. it's surprising that the pb1 and pb2 genes of a/zhejiang/9/2014 (h7n9) showed the most closed relationship with human h3n2 viruses (89% and 88% identity respectively, table 1 ). reassortments between human and avian aivs can make the reassortant viruses replicate efficiently in mammalian hosts. 8 it's very possible that the reassortment between h7n9 and human h3n2, may make the reassortant virus more adaptive to human, even attain the ability to efficiently transmit between humans, and thus raise great thread to the public health. historically, several pandemic human influenza viruses were derived from reassortant virus between avian and human influenza viruses. for example, the h1n1/pdm2009 virus, which was a swine reassortant virus that attained the pb1 from human h3n2, was rapidly transmitted between humans and then, globally circulates as a seasonal virus, posing a substantial risk to human. 9 h7n9 aivs have the genetic makeup associated with human infections, 10 in addition to our finding that the reassortant human-isolated h7n9 virus attained the pb1 and pb2 from human h3n2, possibility like the h1n1/pdm2009 virus, the reassortant virus would become more invasive to humans, and thus pose serious pandemic threat to humans. h7n9 is a novel reassortant aiv subtype, which has surpassed h5n1 in laboratory-confirmed human infections despite its limited dissemination outside of china. thus, whether this subtype could acquire the ability to efficiently human-to-human transmission, and become a new influenza pandemic raise great attention. although a h5/h7 vaccination in chicken has successfully decreased the prevalence of the h7n9 viruses in chicken, our findings that h7n9 viruses attained internal genes from human and duck aivs raise concerns about the potential ability of the viruses to increase their diversity and spread, and especially the possibility to develop better ability to infect human, and eventually attain efficient human-human infections. we note with interest these previous studies into household and hospital influenza outbreaks. 1,2 such community and hospitalbased respiratory virus transmission and outbreak investigations often suffer from the potential confounding arising from possible exposures to undiagnosed index cases outside of the outbreak cohort, leading to an overestimate of virus transmissibility, and potentially unnecessary costly and restrictive infection control interventions. to avoid such confounding, we performed a small pilot study in a closed population of adult research scientists ( n = 43 out of a possible 48). all participants signed informed consent forms, following ethical approval from plymouth university ethics committee. these scientists were confined to a british antarctic survey base for 1 month (march 2017), during which no personnel entered nor left the base. therefore any detectable human respiratory viruses could only have been brought into the base by personnel at the beginning of this 'closed period'. participants were given anonymous codes to maintain confidentiality. each agreed to give nasal swabs (collected in virus transport medium, virocult, medical wire and equipment ltd, corsham, wiltshire, england) upon entry (day 0, 14/3/17), then at days 4 (18/3/17), 10 (24/3/17) and 17 (31/3/17) post-entry. all viral swabs were stored at −80 °c until they could be shipped back to the uk and tested at the leicester royal infirmary. this was performed using a respiratory multiplex pcr assay (16-well, ausdiagnostics uk ltd., chesham, uk) that could detect any of: influenza a, b, respiratory syncytial virus (rsv), parainfluenza (piv) types 1-4, human metapneumo (hmpv)-, entero-/rhino-, corona-(229e, oc43, nl63, hku1) and adeno-viruses. no specific instructions about infection control were given to the participants. they were left to act as they would normally behave throughout the period of the study. any participants who developed any of 9, self-assessed, influenza-like symptoms (fever, cough, stuffy nose and/or sinuses, headache, sore throat, myalgia, fatigue, shortness of breath, nausea or vomiting) would complete a tick-box questionnaire (on a scale of 1-'very mild' to 5-'very severe') to describe the relative severity of their symptoms. this same questionnaire also requested the contact intensity (i.e. number, nature and frequency) of their daily contacts with other participants as a self-assessed, linear graded score (from 1-'sharing just one meal together' to 5-'spending the majority of the day and evening with the other person'), depending on the frequency of contact whilst working, eating meals and socialising together. the daily location of all personnel in any of the four station zones at 0830, 110 0, 140 0, 1630 and 20 0 0 h was also recorded routinely for safety and security, using a 'tagboard' system. out of the 43 participants who consented, 3 later declined to have any viral swabs taken, and of the resulting 160 (i.e. 40 × 4 swabbing time-points) possible swabs, 153 were successfully collected and stored for testing. testing the incubation period of human coronaviruses is around 2-5 days, 3 which can be used to link symptomatic cases together, epidemiologically, 4 with viral shedding being reported for up to 6 days post-symptom onset. 5 so the symptoms and positive nl63 and oc43 results for participants 21 and 74, respectively, could have been acquired from participants 40 and 47, who may have been the original index cases (sources) for these viruses. although no respiratory virus was detected in their samples, participants 40, 47 and 107 all reported similar symptoms to those of 21 and 74 during the study period, which were typical common cold symptoms. note that the participants' self-reported contact intensities were not entirely robust, e.g. both participants 40 and 47 list participant 21 as a contact, so could both have been index cases for him/her. however, 21 did not list either 40 or 47 as a contact (such contacts should be reciprocal). this may have just been a simple oversight, but it makes the contact link less reliable. similarly, participant 74 could have served as the index case for participant 107, but neither lists the other as a contact. regardless of the contact intensities reported in the questionnaires, there were no secondary cases of either nl63 or oc43 coronaviruses detected in any of the other study participants' weekly swabs. one possible explanation for this may have been an insufficient sensitivity of the assay to detect low levels of these respiratory viruses. the limit of detection (lod) for the ausdiagnostics assay varies significantly with each virus (as given in the kit insert, all in copies/ml): influenza a (1900-2375), b (525), rsv (50-2125), piv types 1-4 (50-250 0), hmpv (10 0-625), entero-/rhino-(75-1025), corona-(229e, oc43, nl63, hku1) (1350-4175) and adeno-(1075) viruses. however, in the acute infection stage respiratory viruses are generally present in relatively high copy numbers, with median values of mostly 4-8 log 10 (i.e. 10,0 0 0-10 0,0 0 0,0 0 0 copies/ml) for adeno-, corona-, hmpv, influenza, piv and rsv, as reported in one comprehensive paediatric study. 6 although children generally shed higher viral loads than adults, it is likely that the coronavirus loads in acutely infected adults would still be mostly detectable on this assay, which is approved (i.e. ce-marked) for routine diagnostic testing. yet, it is still possible for viruses that are infecting individuals at the lowest loads within these ranges, to fail to be detected by this assay. given the results that are currently available from this study, one of the key questions is: from where did the nl63 and oc43 coronaviruses arise? in addition, the lack of any secondary nl63 or oc43 coronaviruses cases (symptomatic or asymptomatic) arising from the known positive sources (participants 21 and 74), suggests that the transmissibility of these common cold viruses may be limited. this seems unexpected, given the potential stress on the body immune system whilst living and working in such an extreme environment. however, such relatively poor transmission of respiratory viruses has been previously described in antarctic base personnel for rhinovirus and adenovirus, 7, 8 for reasons that are still unclear. another potential confounding factor is the unknown status of the 5 individuals who were also present at the base (mostly base personnel) but who declined to participate in the study. it is possible that one or more of these non-participants could have been the original sources (i.e. index cases) of the nl63 and oc43 coronaviruses at the start of the study. whilst there are some limitations to this study, there are plans to repeat this on a larger scale, over a longer 'closed' period, with the use of real-time, point-of-care testing (poct) to detect such respiratory viruses. although previous respiratory virus outbreaks have been described in remote research bases, 7,8 these were unable to utilise the greater sensitivity and spectrum of respiratory viral targets provided by modern, molecular, diagnostic tools. 9, 10 thus, some positive cases in these earlier studies may have been missed, leading to an underestimate of the transmissibility of these respiratory viruses in these populations. respiratory infections in research personnel can impact significantly on their productivity, an important consideration when their time at such remote research bases is limited. this and future studies will enable medical teams to enhance the healthcare of research base personnel to optimise their precious research time spent there. none of the authors have any conflicts of interests to declare. we thank the following for their support of this study: uk clinical virology network (cvn), for general funding support; medical wire & equipment ltd., for donating some of the sampling swabs; ausdiagnostics uk ltd., for donating the respiratory multiplex pcr tests. none of these companies were involved in the writing of this article. we read with interest, the article by poller et al. 1 , in this journal, entitled "a unified personal protective equipment….". we understand the importance of proper personal protective equipment(ppe) as an integral component of healthcare workers(hcw) protection in outbreak situations of infections with possible high consequence. but at times, such an outbreak occurs in an unsuspected region, when initial cases present in early course of illness before the development of ominous clinical features. medical staff, particularly in busy rural set ups of resource-poor developing countries may discover that they have been exposed to a high consequence infectious disease after the event of exposure, particularly if these centres are unaware, reluctant or unequipped regarding routine use of ppe. a similar situation occurred in a rural healthcare setting of kerala, india, during the may-2018 outbreak of nipah virus (niv), killing 21 out of 23 reported cases. 2 a 26-year-old male ( index case of the outbreak report 2 ) from kerala's perambra town died undiagnosed with fever, en-cephalitis and respiratory distress in government medical college kozhikode(gmck), after being transferred from taluk hospital, perambra(thp). another 47-year-old male patient ( case-10 ) 2 was admitted in thp for an acute febrile illness and recovered while the unsuspected index case was being treated there in the adjacent bed. two weeks later, case-10 presented to taluk hospital, balussery(thb) (the setting of our intervention), with complaints of fever, headache and vomiting of 4 days duration. he was treated there as inpatient for about 24 h after which he was referred to gmck, owing to clinical deterioration. the next day he developed altered sensorium, respiratory distress and expired. till then, there was no suspicion about the niv outbreak situation, as kerala is at least 2500 km away from the last known outbreak in the indian subcontinent in 2012 3, 4 . in the meanwhile, the brother, father and aunt of the index case , and a nurse, who cared for him at thp, developed similar clinical features of acute encephalitis with respiratory distress and got admitted. all of their samples, along with that of case-10 , were tested positive for niv from the reference laboratory. the state public health authorities swiftly declared the outbreak and ensured containment and protective measures. however, by this time, 17 out of 18 confirmed niv cases were already infected and fell ill, being epidemiologically related as contacts of the index case in family, during transit to healthcare facilities or in hospital 2 . here, we report our experience with eight hcw including two doctors (authors aps and mb of this correspondence) and six nurses working in thb, who had unsuspected, inadvertent, yet significant exposure to case-10 when he was admitted there, without any ppe. both the doctors had closely clinically examined case-10 and the six nursing-staff had repeated bare-hand, unmasked contacts with him ( table 1 ). all of them were extremely panicked once the outbreak notification was out and beseeched aps and mb for an immediate solution. aps and mb contacted the other authors for advice regarding any possible post-exposure prophylaxis(pep). considering the possibility of human-to-human (airborne / contact) transmission of niv, 3, 4 in-vitro 5 and in-vivo 6 effects of ribavirin on niv, evidence of safety and efficacy of short-course high-dose ribavirin pep(rpep) used for lassa fever 7 and unavailability and inexperience of any other alternatives (favipiravir or monoclonal antibody m102.4), were discussed by vkmn, sb, ms, nw and ab, and a consensus opinion of rpep as the only available and reasonably safe option was placed before aps and mb. the importance of psychological factors 8 in the hcws were also considered seriously. the suggested dose was 10 0 0 mg thrice daily for 14 days(cumulative 42,0 0 0 mg) in congruence to the lassa fever recommendation. 7 all the contacts started rpep within 72 h of exposure. the mean cumulative dose of rpep taken by the contacts was 28,750 mg(17,60 0-40,0 0 0 mg) and the mean duration was 12.5 days(11-14 days, table 1 ). their clinical and laboratory parameters were monitored for the next 6 months. mean age of the 8 hcws was 35.4 years(30-43 years). two were males and rest females; none were pregnant ( table 1 ) . most of them experienced minor side effects like fatigue, headache, nausea, dry mouth and palpitations. there was a mean drop of 2.82 g/dl of haemoglobin, predominantly between days 17 and 21 after starting rpep, which started rising in all within a week of stopping rpep. bilirubin levels rose by a mean of 1.65 (range: 0.0 -3.6) mg/dl in 7 of the 8 hcw ( fig. 1 ) . none of them ultimately contracted niv disease. interestingly, one 25-year-old male patient ( case -23 of the outbreak report 2 ), was admitted at an adjacent bed in thb with dysentery, while case-10 was admitted. he was present within a distance of 1 metre for more than 5 h. there was apparently no direct contact between them, except that same hcw served both of them sharing non-critical medical devices. after recovery, case -23 was discharged from thb, to return after a week to gmck with high grade fever, developing encephalopathy and respiratory distress, diagnosed to have niv infection and succumbed to it. in the current outbreak, 3 family members, one staff nurse, one trainee nurse, one radiology assistant who took care of the index case and 13 hospital contacts contracted the infection, proving human-to-human transmission. respiratory aerosols and fomites cause human-to-human spread. 3 the mortality rate, in all recent niv outbreaks in the indian subcontinent is 75-100% with the bangladeshi strain 3 (niv-b) and its close relative in the recent kerala outbreak, 2 has been consistently almost double compared to ebola. further that case -23 getting infected from case-10 in the same premises of thb, makes our case for rpep stronger. the 2 survivors out of 23 infected patients in this outbreak have also received ribavirin. 9 given the recent findings of widespread presence of niv among pteropus bats in india, 10 another outbreak might be just a matter of time. our field notes from emergency, voluntary, off-label rpep among hcw provides evidence, albeit low-quality, of its safety and probable efficacy, strongly suggesting a pre-planned trial for pep to be started immediately once such an explosive outbreak of niv is notified. none. we note the previous report describing a decreasing incidence of eosinophilia in returning travellers by barrett and colleagues. 1 in contrast, another type of hazard reported by returning travellers -monkey bites -appears to be increasing. this makes it necessary for our frontline medical staff to be aware of the potential risks from rabies and simian herpes b virus (shbv or cercopithecine herpesvirus 1 -cehv-1) associated with this type of exposure. 2 although infections are rare as a consequence of bites, 3,4 both viruses can result in very high (80-100%) mortality if the appropriate post-exposure prophylaxis is not initiated promptly. in view of these serious consequences, post-exposure protocols have been developed to reduce likelihood of infection. 2 , 5-7 while this is agreed for rabies, 5, 8 post-exposure prophylaxis is not uniformly recommended for shbv, as cases have only been reported with captive monkeys, 3,9 despite numerous monkey bite exposures in regions where animals are thought to be infected. assessing risks versus benefits obviously needs to be done judiciously in each case, and national public health specialists can be consulted to support decision making. 8, 9 the main risk is primarily from monkey bites from macaque monkeys (genus macaca ), which are now encountered relatively frequently in various tourist areas in southeast asia (e.g. philippines, indonesia, malaysia, cambodia, vietnam, thailand). after a monkey bite, the patient should perform immediate wound cleansing: irrigation with soap and water, or other skincleansing detergent, or sterile water alone, for at least 15 min. later, when the patient presents to the emergency department (ed), all medical staff need to be aware of both the rabies and shbv post-exposure protocols (peps) associated with such bites. whilst most ed teams will likely know of the rabies pep protocol, 5 fewer will be aware of the guidelines for shbv pep. 6 along with the wound cleansing and post-exposure rabies immunoglobulin (rig) and vaccination, any risk of shbv requires that high dose acyclovir (preferably valaciclovir 1 g tds po; or acyclovir 800 mg 5 times daily po, for adults) pep for at least 14 days should be considered. immediate pcr and later serological testing for signs of shbv infection are possible. however, recommendations for such testing are somewhat variable, with some advising testing in symptomatic cases only, whilst others will test all potentially exposed cases, regardless of symptoms. 6, 7 symptoms of possible shbv disease include vesicular lesions, pain and itching near the bite site, local lymphadenopathy, flulike illness (fever, headache, myalgia, fatigue), and any focal or progressive neurological symptoms, including dyspnoea. outcomes are generally fatal (80% mortality without any treatment), once there is central nervous system involvement. 2, 7 however, with antiviral prophylaxis and treatment, such fatal outcomes are rarer. bacterial infections (e.g. staphylococcus and streptococcus spp.) can also arise from the bite itself, especially in children, for which systemic antibiotics can be given, 10 and tetanus vaccination. to highlight this issue, we present three cases of returning travellers with monkey bites. case 1: a 24-year old male was admitted with headache, lethargy and myalgia following a trip to indonesia (monkey forest, ubud, bali), where he sustained a penetrating bite to his right shoulder from a macaque monkey. there were no immediate post-bite complications. however, 16 days later, he developed paresthesia and neuropathic pain in his right thigh. after seeing a local physician, oral aciclovir 800 mg 5 times daily for 14 days was prescribed, as prophylaxis for possible shbv. one day later he developed a vesicular rash on his right thigh, which subsequently resolved on the antiviral therapy. on return to the uk, given this history, he was admitted and started rabies post-exposure immunisation, without rabies immunoglobulin (rig). he was then extensively investigated for possible shbv infection. he had five days of intravenous aciclovir and a further nine days of oral valaciclovir, whilst awaiting investigation results. diagnostic testing performed at the department of viroscience, erasmus medical centre (m/c), rotterdam, the netherlands on cerebrospinal fluid (csf), blood, lesion swab and saliva by shbv pcr showed no evidence of infection at that time. at outpatient review, two months later, there had been no further history of any rash or neurological symptoms, though the patient did mention several recurring episodes of genital herpes, for which he was given a 10-day course of oral valaciclovir 500 mg bd po. by this time, the risk of latent shbv was considered negligible and he was discharged from clinic. case 2: a 28-year old male was seen in clinic who gave a history of receiving an unprovoked, penetrating bite on his right upper arm from a vervet monkey ( chlorocebus pygerythrus , previously classified as cercopithecus aethiops ), one week earlier whilst on holiday in barbados. after immediate wound care, he was seen in a local clinic and received tetanus vaccine and oral antibiotics. no aciclovir shbv pep was commenced at this time. the bite wounds healed without complication. after returning to the uk a week later, an outpatient review revealed that he was still asymptomatic for any clinical features of shbv, rabies or other travel-associated illnesses. however, as a precaution, valaciclovir 1 g tds po, for 21 days was prescribed as shbv prophylaxis, due to the possibility of an incubating shbv infection. 3 blood and saliva samples were sent to viroscience for pcr testing to check for any residual shbv, dengue, chikungunya or zika virus infections. all tests were negative. the patient continued to remain asymptomatic, so was eventually discharged from outpatient follow-up. case 3: a 31-year old female was seen in clinic upon return from southeast asia, with a history of receiving a penetrating bite to her right upper arm from a macaque monkey, whilst visiting monkey island, vietnam, 18 days previously. she received her first dose of rabies vaccination (without rig) at a local clinic within four hours of the bite. a week later, whilst still in vietnam, she received a second dose of rabies vaccine from a different clinic, which also started her on acyclovir post-exposure prophylaxis for shbv. the bite wounds healed without sequelae. at her 18-day clinic review once back in the uk, she was still asymptomatic. baseline saliva and blood samples were taken and stored but not tested for shbv. she continued both the shbv and rabies pep whilst continuing her travels a week later, and remained asymptomatic six weeks post-exposure. this small case series demonstrates a diversity of presentations and follow-up management for these patients, notably: case 1 likely presented with genital herpes; case 2 sustained a bite from a vervet, not a macque, monkey; case 3 did not have any shbv testing. to our knowledge, all of these cases remain well. as there are no consensus guidelines available for managing such monkey bites, we suggest a precautionary approach and that to be safe, assume that both rabies and shbv are potential risks, regardless of monkey species. therefore, in the event of a monkey bite, where, after discussion with the patient (based on their individual clinical assessment), a decision is made to give prophylaxis: (i) immediately cleanse the wound for 15 mins with clean water + / − soap or detergent. consider appropriate antibiotic therapy to prevent skin infection (e.g. co-amoxiclav or amoxicillin), and tetanus vaccination. (ii) seek competent clinical help and obtain the first dose (day 0) of rabies vaccine + / − rig, depending on the risk assessment (in the uk, public health england tel: 0208 327 6204, 9-5 pm mon-fri). 8 complete post-exposure rabies vaccination with further doses on days 3, 7 and 21, post-exposure. 5 (iii) start acyclovir (1 g valacyclovir tds po or 800 mg acyclovir 5 times daily po -depending on local availability, for adults. adjust the dose as appropriate for children) as soon as possible after the bite, for at least 14 days, as post-exposure prophylaxis against shbv. (iv) if symptoms compatible with shbv develop within the next 2-4 weeks (e.g. vesicular lesions, pain, itching around the bite, local lymphadenopathy, flu-like illness, focal or progressive neurological symptoms), continue the acyclovir and seek further expert advice. (v) baseline samples (serum, saliva, wound swabs) can be taken and stored for comparison. if acute illness develops, repeat samples (including cerebrospinal fluid -csf) should be taken for diagnostic testing (by pcr) to check for shbv dna, and serology for shbv antibodies -if such testing is available. recent article in this journal has reported dengue patients were associated with a higher risk of autoimmune diseases than nondengue patients 1 . dengue has been a serious public health prob-lem in the world, the number of dengue epidemics has been on the rise worldwide. before 1970, only nine countries had experienced severe dengue epidemics; however, currently more than 100 countries have been severely affected by dengue 2 . after the first dengue-fever epidemic in china, which occurred in may 1978 in foshan, guangdong province, there have been regional outbreaks of dengue every year and the number of cases has increased. guangdong province is the area most seriously affected by dengue in china. the number of cases in guangdong accounts for more than 90% of the total number of cases in china 3,4 . however, the epidemic characteristics of dengue fever in guangdong province have not been reported since 2010 5 . a dengue epidemic is closely related to various factors, such as environmental conditions, imported cases, and migration; thus, its epidemic characteristics and patterns change rapidly. especially after the outbreak of dengue in guangdong province in 2014, the epidemic patterns have changed greatly. therefore, exploring the changing patterns of dengue outbreaks and epidemics in guangdong is of great significance to the prevention and control of dengue. this study aimed to investigate the changing patterns of the epidemic characteristics of dengue in guangdong province and to propose prevention and control measures. we collected dengue cases from 2008 to 2017 from the web-based disease reporting information system of the chinese national center for disease control and prevention. a total of 52,792 cases were reported for this period. population data were obtained from the statistics bureau of guangdong province ( http://www.gdstats.gov.cn/ ). a chi-square test was used to determine spatial differences in cities. this study was approved by center for disease control and prevention of pla review board. in terms of temporal distribution ( fig. 1 ) , dengue cases showed an increasing trend from 2008 to 2014 in guangdong province, and a decreasing trend from 2014 to 2017 (93 cases in 2008, 45,170 cases in 2014, and 1662 cases in 2017). the dengue outbreak in 2014, with 45,170 cases and 6 deaths, was the largest dengue outbreak in china in the past 20 years. before the outbreak in 2014, an average of 617 people was infected with dengue annually. following the outbreak, an average of 1305 people were infected with dengue annually-nearly twice the number of cases per year compared to that before 2014. dengue fever had typical characteristics in population distribution. adults aged 20-65 years accounted for 76.76% of patients, and children aged 0-10 years only accounted for 4.16% of patients. in terms of distribution across occupations, housekeepers and the unemployed (23.1%), retired individuals (12.82%), those involved in commercial services (11.52%), industrial worker (9.56%), and students (7.49%) formed the majority of patients with dengue fever. these five groups accounted for 64.48% of the total number of cases. the number of male patients was comparable to that of the number of female patients (male: female = 1:1.01), and the mortality rate of dengue was low at only 0.11 ‰ . monitoring data of the past 10 years showed that although the number of cases increased greatly after the outbreak in 2014, the population distribution did not change significantly. the key population for the prevention and control of dengue fever were adults over the age of 20 years. although cases have been reported in various regions, there are significant differences ( p < 0.001) in the distribution of dengue among the different cities in the guangdong province ( fig. 2 ) . incident cases were mainly concentrated in guangzhou city and foshan city. guangzhou city had a total of 40,170 cases during 2008-2017, which accounted for 76.09% of cases among the different cities. foshan city had a total of 4660 cases, which accounted for 8.83% of cases. however, according to annual data trends, 2014 was the turning point in spatial distribution patterns for dengue fever. from 2008-2013, the incidence of dengue was concentrated in foshan city, guangzhou city, jiangmen city, and zhongshan city (93.14% of cases). however, by 2014, except for meizhou city, there were case reports from all 20 cities in the province. among them, there were 13 cities with more than 200 cases each. after 2014, dengue fever was prevalent throughout the province. even in zhanjiang city, zhaoqing city and jieyang city, which are distant from guangzhou city, and the number of cases in remote areas increased significantly. this paper is the first to analyze the characteristics of the change in dengue epidemics in guangdong province in the past 10 years. in general, the incidence of dengue fever has typical spatial differences. the cases were mainly concentrated in urban areas with large populations and developed economies, such as guangzhou city and foshan city. theinflux of many migrants, especially those from southeast asia, could have caused the higher incidence of dengue fever in these areas. studies have shown that dengue epidemics in china were initiated by imported cases and then became prevalent in the local areas 6 . however, attention should be given to the large number of dengue epidemics in remote areas, which appeared after 2014. a possible reason for this could be global climate warming, which has caused the natural environments in some remote areas to become suitable for mosquito breeding. moreover, rapid development in tourism and trade has also led to an increase in local and imported cases. in summary, after 2014, the epidemic characteristics of dengue fever in guangdong province have undergone major changes. the key areas for prevention and control are no longer confined to traditional epidemic areas, such as guangzhou city and foshan city. instead, dengue prevention and control should be conducted throughout the province. in addition, according to the national dengue surveillance in 2018, dengue fever has shown a trend of "moving up north" in the country 7 . therefore, the changing characteristics of this epidemic warrant high attention of relevant departments. the authors declare that they have no competing interests. this work was supported by national key r&d program of china ( 2016yfc120 070 0 ), beijing nova program ( z17110 0 0 01117102 ) and military medical innovation project ( 16cxz046 ) and pla youth training project for medical science ( 16qnp127 ). the funders had no role in the study design, 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and therapy for exposure to b virus (cercopithecine herpesvirus 1) monkey bites in travelers: should we think of herpes b virus rabies and immunoglobulins service follow-up of monkey bites. phe publications gateway number keep children away from macaque monkeys! increased risk of autoimmune diseases in dengue patients: a population-based cohort study developing a time series predictive model for dengue in zhongshan, china based on weather and guangzhou dengue surveillance data the changing epidemiology of dengue in china, 1990-kk2014: a descriptive analysis of 25 years of nationwide surveillance data spatial and temporal patterns of dengue in guangdong province of china clinical and epidemiological features of the 2014 large-scale dengue outbreak in guangzhou city center for disease control and prevention of chinese people's liberation army, 20 dongdajie street xinying du center for disease control and prevention of chinese people's liberation army, 20 dongdajie street beijing 10 0 071, china hongbin song * center for disease control and prevention of chinese people's liberation army the authors declare not conflict of interest. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.03.008 . we thank the following for their support of this study: uk clinical virology network (cvn), for general funding support; medical wire & equipment ltd., for donating some of the sampling swabs; ausdiagnostics uk ltd., for donating the respiratory multiplex pcr tests. none of the authors have any conflicts of interest to declare. key: cord-249166-0w0t631x authors: booss-bavnbek, bernhelm; krickeberg, klaus title: dynamics and control of covid-19: comments by two mathematicians date: 2020-08-17 journal: nan doi: nan sha: doc_id: 249166 cord_uid: 0w0t631x we are asking: why are the dynamics and control of covid-19 most interesting for mathematicians and why are mathematicians urgently needed for controlling the pandemic? first we present our comments in a bottom-up approach, i.e., following the events from their beginning as they evolved through time. they happened differently in different countries, and the main objective of the first part is to compare these evolutions in a few selected countries with each other. the second part of the article is not"country-oriented"but"problem-oriented". from a given problem we go top-down to its solutions and their applications in concrete situations. we have organized this part by the mathematical methods that play a role in their solution. we give an overview of the main branches of mathematics that play a role and sketch the most frequent applications, emphasising mathematical pattern analysis in laboratory work and statistical-mathematical models in judging the quality of tests; demographic methods in the collection of data; different ways to model the evolution of the pandemic mathematically; and clinical epidemiology in attempts to develop a vaccine. since the covid-19 pandemic is not over it may appear to be premature to draw some conclusions. however, it may be, as well, just in time to recapitulate some lessons we as mathematicians should have learned and are urged to apply now. thus we are asking: why are the dynamics and control of covid-19 most interesting for mathematicians and why are mathematicians urgently needed for controlling the pandemic? we shall first present our comments in a bottom-up approach, i.e., following the events from their beginning as they evolved through time. they happened differently in different countries, and the main objective of this first part is to compare these evolutions in a few selected countries with each other. still, there are some general features, which we present separately as we are used to do in mathematics. they include the history of certain epidemics which have influenced the reactions of people in many countries, and some basic mathematical tools. in addition there is a common factor, which one of the present authors (kk) has defined on the 12th march 2020 in an e-mail to a german health office: "the extension and evolution of covid-19 in various countries and regions reflects the state of their health systems. this was for instance already very obvious in the case of ebola." it is in fact the public health component of the health system that plays a crucial role. the second part of the article is not "country-oriented" but "problem-oriented". from a given problem we go "top-down" to its solutions and their applications in concrete situations. we have organized this part by the mathematical methods that play a role in their solution. here is an example where specially much mathematics is needed: to develop a vaccine and the strategy for applying it without loosing sight of basic ethical principles. in the following the gentle reader may consult when necessary the book [kpp] for the basic concepts of epidemiology. demography as a mathematical subject area was already developed centuries ago well beyond its elementary beginnings. for a long time it remained the only mathematical tool in the study of the evolution of infectious diseases. here is a famous early example. in china, india and europe one tried to confer immunity against smallpox by infecting individuals slightly so they would contract a mild form of the disease and be immune afterwards. some of them died by this procedure but in 1766 the swiss mathematician daniel bernoulli showed by a demographical approach that the procedure would increase life expectancy if applied to everybody [di1] . nowadays evaluating the costeffectiveness of a public health measure is being done widely; it is based on methods of mathematical economy. the 19 th century saw the discovery of microorganisms as pathogens of many diseases and their study by mainly microbiological methods. the mathematical tools for following up an epidemic remained essentially demographical well into the 20 th century. a few physicians suggested that every epidemic ends because there are finally not enough people left to be infected, which is a naïve predecessor to the mathematical-epidemiologic concept of herd immunity (see sect. 8). nevertheless even the abundant literature on the influenza pandemic of 1918-19, wrongly called spanish flu, discusses only two possible ways for its ending: better clinical treatment and mutations of the pathogen. seen from a virological viewpoint the spanish flu was an extreme form of the so-called seasonal influenza. the virus which causes them can be one of a large variety, its genus being denoted by a, b, c or d, where some of them include several species. a is the most serious one; is has subtypes a(hxny), x = 1,...,18 and y = 1,...,11, where x and y represent proteins on the surface of the virus. the strategy for controlling the "normal" seasonal influenza epidemic is widely known even among laymen: identify the strain of the virus in autumn, develop a vaccine as fast as possible, and vaccinate people thought to be at risk. nevertheless the number of infections and of deaths by a seasonal influenza can be as high as those by some of the pandemics to be described now. the spanish flu was due to a(h1n1). pictures from that time show people wearing masks that resembled those used now. in the years 1957-58 another "digression" from seasonal influenza occurred, called the asian flu and caused by a(h2n2). it started in china and then became a pandemic, passing from neighbouring states through the uk and the usa. estimations of the number of cases vary around 500 millions and of the number of deaths around 3 millions. its beginnings looked much like those of the spanish flu but towards the end a vaccine became available, a predecessor to the ones being used now routinely against the seasonal flu. the hong kong influenza of 1968-69, generated by the virus a(h3n2), had similar characteristics and will not be described further. parallel to the entering the scene of these and other epidemics, and partly motivated by them, basically new mathematical tools of public health emerged in the first part of the 20 th century, preceded by a few studies in the late 19 th . they were twofold. the first tool was called a "statistical-mathematical model". its aim is the study of the influence of factors, also called determinants, on the health of people. such factors may for instance be a lack of hygiene or a polluted environment. a factor can also be a preventive or curative treatment by an immunization or a drug, respectively; in that case the main objective of a study is to estimate the efficacy of the treatment. sampling plans are statistical-mathematical models of a different but related kind. they form the basis of sample surveys, which are being done in profusion about covid-19, too, and not always very illuminating. the second tool is called "mathematical modelling of the evolution of an epidemic", or briefly "mathematical modelling". there are two kinds of it. first, one may aim at the epidemic curve, which is the cumulated number of cases up to a moment t as a function of covid-19 booß-bavnbek &krickeberg, 17 august 2020 5 t . in that case mathematical modelling serves to estimate or predict this curve under various assumptions on the infectivity of infected subjects. early predecessors are presented in [fin] , see figure 1 ; the question whether the infectivity remains constant or decreases played already a role. refined versions are still being used, in particular for covid-19 (sect. 7). early numerical simulation of an epidemic curve by j. brownlee, 1907, discussed in [fin] second, one may build so-called compartmental models (sect. 8). the first one, for measles, was published in 1889 by p.d. en'ko; see [di2] . around the year 1900 compartmental models for malaria appeared. then in the 1920s new models for the evolution of measles in closed populations were defined and intensively studied. they became very influential because they displayed already many basic features that reappeared later in mathematical models of epidemics in other and more complex settings. such tools found many applications. dealing with large epidemics mathematically was no longer a matter of demography alone, although that continued to be the main tool for estimating number of cases and deaths. statistical-mathematical models were employed to estimate the efficacy of antiviral drugs, for instance against hiv-infections, and the efficacy of various immunizations including those against forms of influenza. mathematical modelling of epidemics was used in planning strategies to eradicate smallpox, poliomyelitis, measles and perhaps others. the first articles on modelling influenza epidemics appeared in the scientific literature. planning a vaccination strategy involves both statisticalmathematical and mathematical models [hal] . these roads to progress may have produced a general feeling of success in dealing with epidemics. then in the period from 2002 to 2019 a few events occurred that evoked memories of previous pandemics and undermined such believes. in finally another zoonotic influenza appeared, popularly called bird flu and in scientific language highly pathogenic avian influenza (hpai). the main pathogen was an a(h5n1) influenza virus. it had been known long ago but reached a peak in the years 2013-2017. whether there existed an airborne transmission from poultry to humans was a hotly debated question with obvious economic consequences. the bird flu spread widely over the whole world but the number of known human cases remained small, just over 70. in addition to various forms of influenza and the epidemics generated by the corona virus sars-cov-1, sars-cov-2 or mers-cov, other epidemics occurred. it is instructive to compare them with those just mentioned, applying in addition mathematical yardsticks. we shall restrict ourselves to ebola epidemics. their most widespread outbreak was the western african ebola virus epidemic from 2013 to 2016, which caused 28,646 cases and 11,323 deaths. there is a fundamental difference in the evolution of a case of influenza or sars-cov-1 or sars-cov-2 on the one hand and of an ebola case on the other, which leads to a basic difference in their mathematical modelling (sects. 4 and 8). a carrier of an influenza or corona virus can transmit it to other persons well before the first symptoms appear, that is well before the end of the incubation period. a subject infected by ebola will become infective only around the end of the incubation period. he (if it is a man) could then be immediately isolated together with his latest contacts in order to avoid further extension of the infection, provided that there is a health service nearby to do it. therefore ebola did not spread to countries that have a sufficiently dense primary health care network but it caused much suffering in countries that do not have it. the strategy of who to control the epidemic was wrong. it insisted on drugs and the search for a vaccine (which became available only in december 2019) but neglected primary health care. for the present purpose it would even have been most useful to rapidly train village health workers and "barefoot doctors" as it had been done decades ago. only very few countries profited from the experiences of these premonitory 18 years to prepare much in advance for a possible, and probable, new outbreak of an epidemic. some others took appropriate measures only at the first signs of covid-19, and many started planning when the epidemic had almost reached its zenith. we shall sketch some examples. for simplicity we shall always describe the result of the strategy of a country by indicating its cumulated numbers of confirmed cases and deaths around the 1st june 2020. regarding the reliability of these data see sects. 5 and 6. we begin with those that had planned early. , the year after the sars-epidemic outbreak, the government established the national health command center (nhcc), which was to prepare the country for a possible new epidemic. from 2017 on it was headed by the popular minister of health, chen shih-chung, who had studied dentistry at the taipei medical college. the vice-president of taiwan from 2016 to 2020, chen chien-jen, had been minister of health from 2003 to 2005 after having studied human genetics, public health, and epidemiology at the national taiwan university and the johns hopkins university in the usa, followed by research. thus decisions about the control of covid-19 were taken by politicians competent in matters of health including public health. taiwan counts 23 million inhabitants and many of them travelled from and to china. from the 31st december 2019 on when who was notified of the epidemic in wuhan all incoming flights from there were checked, followed by controls of passengers arriving from anywhere. an "action table" was produced in the period 20th january to 24th february 2020, which listed 124 measures to be taken. the public obtained daily revised clear information by all existing means. "contact tracing", which means repeated followup of symptomatic persons, of confirmed cases and of all of their contacts, was rapidly established on the basis of the electronic health insurance card that everybody has. the virological pcr-tests used (sect. 4) were already available and quarantines well organised. in late january rules about the wearing of masks were edited; a sufficient supply existed already. as a result 442 confirmed cases had been found and 7 deaths recorded up to the 1st june. the vietnamese strategy resembles the taiwanese one in almost all aspects, with the exception of contact tracing. a steering committee to deal with new epidemics existed within the ministry of health. it put into effect its plan right after the 23rd january when the first infected persons arrived at vietnamese airports, among them a vietnamese returning from the uk. all schools were closed on the 25th january, and since the 1st february everybody entering vietnam must spend two weeks in quarantine. other measures were imposed or relieved in accordance with the evolution of the epidemic, for instance a limited confinement or the wearing of masks. the ministry of health issued regular precise and clear information for the entire population by all available means including smartphones. in addition there is a personalized information system by so-called "survival guides" given to everybody. every survival guide defines three categories of persons: f0: a confirmed case; f1: suspected to be infected or having had contact with an infected person; f2: having had contact with a person in f1. each person is expected to find the category to which it belongs. the survival guide then provides printed information about what she or he must do as a function of her or his category, for example to submit to a test. only pcrtests are being used. in contrast to taiwan contact tracing does not use electronic tools. it is being done by the population itself, aided by the survival guides, together with a large number of well-trained members of the health services, for example university lecturers. at the end of 2019 vietnam had 98,257,747 inhabitants. on the 1st of june there had been 328 confirmed cases and 0 deaths. these data are based on a strong demographic section of the "general statistical office" and on several health information systems [kkr] and can hardly be contested. the preceding sketch of control measures in taiwan and vietnam has shown us the three main components of their epidemiologic side: contact tracing; lock-down, that is physical, or social, distancing in the wide sense including quarantine and border controls; wearing of masks. we may call this the "surveillancecontainment strategy". in addition there is the medical-clinical side, from primary health care such as general practitioners up to large hospitals. its state is crucial to the number of deaths caused by the virus sars-cov-2. in contrast to taiwan and vietnam it seems that all other countries of the world were unprepared at the end of december 2019. a few of them took fairly systematic and strict measures that covered the entire population as soon as the first cases had declared themselves. for a quick overview see figure 2 . this was for example true for china at the end of january 2020, for slovakia and greece on the 27th and 28th february, for austria on the 10th march and for denmark on the 12th march. an alternative danish strategy, based on rigorous contact tracing and quarantine, but not implemented until now was argued for in [sia] . regarding the results the turbulent evolution in china is well known. in denmark, with a population of 5.806 million, about 12,000 cases had been confirmed and 593 deaths recorded, and the corresponding figures for austria are 8.86 million people, 16,979 cases and 672 deaths. the confrontation of slovakia, a country of around 5.5 million inhabitants, with greece, which counts 10.72 million people, is particularly striking because it makes visible the role of their physicians and hospitals. in slovakia there were 1,528 confirmed cases and 28 deaths. the corresponding data for greece are 3,058 and 183. the relatively much higher number of fatalities in greece, in spite of equally early reaction and almost the same number of cases per number of inhabitants, is no doubt due to the catastrophic state of its medical-clinical system caused mainly by the debt crisis from 2010 on. next we pass to a group of countries that reacted late and not systematically, applying the various measures in a haphazard way and only to part of the population. here are some of them with their numbers of inhabitants in million, cumulated numbers of confirmed cases and numbers of fatalities: the relatively low number of deaths in germany reflects mainly a sufficient medical-clinical system that could readily adapt itself to the epidemic. the opposite was true in france. there, about 100,000 hospital beds had been eliminated in the period between 1993 and 2018. an arbitrary strict "confinement" not determined by epidemiologic reasoning was imposed on the 17th march. finally there are countries that decided to do nothing, at least for a long while. their motivation, or pretext, was above all a belief in herd immunity (sects. this overview of strategies confirms that, as said in the introduction, the results depend indeed heavily on the state of public health. note that nowadays in every language of the world the concept "public health" is designated by a literal translation or a slight modification of this expression. for instance in danish it is "folkesundhed", that is, "health of the people". in this second part we shall sketch the scientific and in particular mathematical principles involved in the study of successive stages of the pandemics. in short: sect. 4: discovery of the new virus, basic properties, testing for its presence in a person. 5 and 6: data on the evolution of covid-19 in a population. 7: attempts at analysing mathematically and predicting such an evolution by representing it by an epidemic curve. 8: the analogous for a representation by a compartmental model. 9: trying to stop the epidemic by a vaccine. 10: what to learn and what to do? after the often-depicted outbreak in late december 2019 of cases of pneumonia of unknown aetiology around wuhan, in the course of january 2020 chinese scientists identified a new virus as the pathogen. they followed the usual procedures, i.e., they determined the load of 26 common respiratory pathogens in the patients. they found none of them in abundance. they suspected sars-cov but could not find it either. then they investigated all kinds of viral load that had a slight similarity (coincidence in a number of genomes) with sars-cov and detected a novel virus which displayed abundant virions in respiratory specimens from patients. electron microscopy and mathematical pattern analysis [mum, pev] showed that it belongs to the same species as sars-cov-1 and mers-cov (sect. 2); hence the name sars-cov-2. starting with this work in china a large number of publications about the peculiar properties of the pathogen and the ways it is acting have appeared. on the virological side its genetic sequence was determined. the new virus is believed to have zoonotic origins but human to human infection was rapidly established. the combination of sars and influenza features, that is intensive respiratory inhibition of patients and rapid transmission, make covid-19, the disease caused by sars-cov-2, particularly dangerous. for further work see [and] . in the clinical context, several periods in the evolution of a case were determined (see their definition in [kpp, sect. 5 .2]): the median incubation period is 5.2 days; the mean latency period is 4.6 days, i.e., in general the infectious period starts indeed before the prodromal phase. we have discussed the implications in sect. 2 by comparison with ebola. the mean length of the infectious period is 6 days for mild and asymptomatic cases; for severe and critical cases this period lasts on the average 22 days and ends only by recovery or death. the manifold applications to the control of the pandemic of both their virological and their clinical characteristics will appear in sects. 7, 8, 9 and 10. their study is still active and may even reverse former results; this happened for example recently about so-called cross-immunities. however, in this article we shall only treat applications to the basic element of well-designed control strategies, namely testing for infections. the first step of a test programme is to define the target population. who will be tested? subjects who had a contact with infected people? or those who complain about symptoms? or everybody coming from a region where cases exist? see the example of vietnam in sect. 3. next, what will be the objective? to discover the presence of the virus or that of some kind of antibodies? depending on the objective there exist virological and serological tests. the usual virological test is called the pcr-(polymerase chain reaction) test. dozens of serologic tests of varying quality have been and still are developed and even offered in some countries to the general public. recall that the characterization of a test with a given target population and a given objective is a classical subject of clinical epidemiology [kpp, sect. 19.2] . coming back to the fundamental role of testing in control strategies we only remark that in poor countries or in rich countries with inattentive public health officials, the target population was often determined by the shortage of test kits and by the influence of institutions that required them for themselves. this is classical medical statistics, which gives for a specific disease the number of cases and deaths together with the when and where and a few additional data such as sex, age and sometimes profession of the subjects. in principle the methods for finding the number of confirmed cases and of fatalities by covid-19 are the same as for any other disease. they fluctuate widely between countries. both the diagnosis of a case of a disease and the description of the cause of a death may be relatively correct or most unreliable. in particular finding a correct diagnosis for somebody who complains about acute health problems depends very much on the local contact tracing methods and on the state of the clinical-medical system. an additional difficulty arises form the existence of asymptomatic forms of the disease, that is, subjects infected by sars-cov-2 who display no symptoms. in sect. 3 we have mentioned vietnam, which uses its normal demographic and health information systems [kkr] . it includes in its statistics asymptomatic cases found by contact tracing. other countries obtain their morbidity and mortality data from a "health reporting system". such a system is partly based on sampling methods from various sources, for example hospitals and local health offices. in germany the robert koch-institute, a central institute mainly devoted to infectious diseases, reports on the results for covid-19. in the usa the johns hopkins university plays a similar role. still other countries use data from health insurance offices. however, many countries have neither a health information system nor a health reporting system, or they do not use it for covid-19. a host of alternative methods is being employed. for example france counts only hospitalized confirmed cases and only deaths which happen in a hospital or in a retirement home that is connected with a medical structure. summing up we may say that morbidity data and to a lesser degree mortality data for covid-19 that one finds in various periodic publications are fairly unreliable, with very few exceptions. the sources are not always clearly indicated. an important alternative idea is to compare the present situation with that in years past. speaking again naïvely we assume that the present higher case frequencies and death tolls, and only these, are the result of covid-19. given the diagnostic difficulties mentioned above this idea is mainly applied to fatalities and hardly to nonlethal cases. thus in the method of "excess mortality" we only measure how many more deaths by any cause happened this year than in the corresponding period in the past. for the uk we have for instance quoted in sect. 3 the figure of 41,128 deaths up to the 1st june as supplied by the national health service. by contrast the national statistical office advanced about 62,000 deaths as excess mortality! fig. 2 estimated number of infections on lock-down day and excess mortality for 16 selected countries. reproduced from [fit] , permission granted by finally, here is an interesting idea based on the most classical form of a statistical-mathematical model. a graphic in the paper [fit] (see figure 2 ) shows for every one of 16 selected countries the point in the plane whose coordinates are, respectively, the estimated number of infections per million inhabitants on lockdown day and the excess mortality. a short glance convinces us that they are positively correlated. a simple regression analysis based on this graphic would allow us to estimate one of these values by the other one for any other country, too. it goes in several directions beyond classical health statistics, all of them relevant to covid-19, too. firstly, sample surveys are conducted instead of using the data from the entire "target population". they have for example been used to study the influence of social factors on the evolution of various aspects of the disease. in particular the factor "to be an immigrant or to descend from them" was thoroughly investigated in some countries. secondly, more types of data about cases and deaths are collected, for example about morbidity and mortality by age groups. thirdly, data sets are not only being registered and perhaps published but also transformed and interpreted in various ways. here, standardization is the best-known procedure. a fictitious example would be the number of fatalities by covid-19 in denmark if denmark had the same age structure as vietnam and in each age group it had the same covid-19 mortality as in the same age group in vietnam. in sect. 9 we shall meet statistical-mathematical models as a basic mathematical tool in developing a preventive treatment of covid-19. with their help one studies in a clinical trial the influence of various factors on some outcome variable e of interest. here the idea of "controlling" for the influence of another factor, which might be a "confounder" in the study of the action of e , plays a role. it looks as if most demographers on the one hand, and most clinical epidemiologists on the other, ignore that the mathematical procedure of standardizing is the same as that of controlling for a confounder [kpp, lesson 21] . a mathematician will not be astonished, though! we have mentioned this classical concept in sect. 1; see [kpp, sect. 4.6 ]. let c be an epidemic, v a geographical region, t0 a moment of time which may be that of the first case of c in v , and f(t) for t ≥ t0 the number of observed and reported cases of c that had declared themselves in v before or at the instant t . then f is called the epidemic curve of c in v . in particular it needs to be said whether unconfirmed cases are included or not. measuring f(t) as the time t goes along is the task of the relevant demographic services (sects. 5 and 6). this process is therefore subject to all the deficiencies listed there. to get some knowledge about f for various regions v is of course one of the main concerns of the population of a country invaded by c . such knowledge is equally vital for health authorities who attempt to control c . however, much more knowledge is desirable. what can we learn about the mechanism of c by observing f(t) ? this was already the subject of the papers described in [fin] ; see sect. 1. in particular, is there a way to predict aspects of the future evolution of f , having observed the values f(t) for a while? answers to these questions are generally given by modelling f , that is by making certain assumptions about its shape and by estimating certain parameters in it. a very large number of papers was published about this issue. some of them use extrapolation methods known from mathematical economy. a recent survey on basic ideas and techniques can be found in [krm] where a model is described in terms of an integro-differential equation. we shall restrict ourselves to a discussion of an application, namely the so-called basic reproduction number r0 . it appears constantly in popular publications. to define it let us look at a subject s that is infected at a time t* ≥ t0 . let µ(s, t*) be the number of all subjects infected by s after t* in the form of secondary, tertiary etc. infections. then r0 is the average of µ(s, t*) over all s . thus it depends on t*. it is precisely this dependence in which people are interested: a value less than 1 is looked upon as predictor of the extinction of c after t*. in the case c = covid-19, values as high as 5.7 had been estimated in the beginning, that is, for t* close to the time of the first outbreak of c. the article [sia] presents an interesting factorisation of r0 in order to compare different approaches to control the size of it. we have sketched their historical origin in sect. 1. we distinguished between two ways of mathematically modelling the evolution of an epidemic. models of the first kind (sect. 7) represent the temporal evolution of the number of subjects in a certain state, for instance the state "to be infected". by contrast, compartmental models also represent changes of this state at some moments in the form of transitions of a subject from one compartment to another one. the sir-model, which we designated in sect. 1 as "intensively studied in the 1920s", is particularly simple and has served as a paragon for many others, in particular for those applied to covid-19. it involves three compartments: s are the susceptible, not yet infected subjects, i the infected ones, and r consists of subjects removed by recovery with immunity or death. the transitions between compartments are described by differential equations for the numbers s(t), i(t) and r(t) of subjects in the compartments as a function of time t . they involve certain parameters such as transition probabilities from one compartment to another one. under various assumptions the resulting system of differential equations for s , i and r can be solved explicitly or numerically. a first important application is to estimate the basic reproduction number r0 defined in sect. 7. it can be expressed by the basic parameters. secondly, it turns out that the limit s∞ of s(t) for t →∞ is strictly positive, which means that a certain part of the population will never be infected. this led to the concept of herd immunity, which, however, gave rise to much confusion among people who thought they had something to say about the matter. after the outbreak of covid-19 many more involved compartmental models were defined and analysed. their parameters represented among other features the underlying control strategy to be used. there was for instance the "do nothing" strategy and also the "mitigation" strategy, which consisted of the less stringent components of the "surveillancecontainment strategy" defined in sect. 3. in the much discussed paper [fer] neil ferguson and collaborators described the shape of the function i, that is the number of infected subjects, for the "do nothing" strategy. from the value 0 on it increases, reaches a maximum, decreases and finally reaches 0 at a certain moment thappy . this had apparently motivated the countries uk, usa, sweden and brazil to adopt this strategy for too long, ignoring that ferguson predicted (see figure 3 ) about 500,000 deaths caused by the epidemic in the uk and 2.2 million in the usa before extinction at the moment thappy. expected deaths caused by the epidemic for the do-nothing strategy, reproduced from [fer] with permission of school of public health, imperial college london at present compartmental models play hardly any practical role, mainly because they contain too many unknown parameters. some parameters such as infectivity are estimated with the help of a model of the epidemic curve, which seems to be a not very successful detour. it will hardly surprise that several pharmacological companies have started a run for developing curative and preventive treatments of various ailments, which sars-cov-2 may inflict on a person. up to now no curative treatment was found. there are only the wellknown methods to be used in the treatment of non-specific aspects of a case such as reducing pain, facilitate breathing or shorten the time to recovery by a antiviral drug. we shall therefore restrict ourselves to preventive treatments, that is, to immunizations. the objective of an immunization by a vaccine against a covid-19 connected health deficiency needs to be defined in the same way as for any other infectious disease. first the target population needs to be determined: whom do we intend to protect? next, what are the health deficiencies we want to prevent? for how long is the preventive effect to last? this is a particularly important aspect of the vaccine but is usually suppressed when a new one is announced. for instance the measles vaccination remains lifelong active in most subjects. for covid-19 the company which tries to develop the vaccine may be satisfied with a few months, hoping that sars-cov-2 will have disappeared after that. finally the efficacy needs to be found, which represents the part of the target population actually protected. it may also be defined in epidemiologic terms by regarding as "exposed" all subjects that had not obtained the treatment. then the efficacy is the "aetiological fraction among the exposed subjects". nowadays there is general agreement that the process of developing a vaccine against an infectious disease needs to run along a well-defined common line [kpp, lesson 18, and hal] . this ought to hold for covid-19, too, and we shall therefore recall it here. first, one or several substances are selected which, for some reasons whatsoever, usually virological ones, look like possible candidates for a vaccine. each of them needs to be submitted to a "clinical trial" in order to explore its most important properties. such a clinical trial consists of three "phases" i, ii and iii. phase i deals with various mainly pharmacologic aspects such as side effects for various possible dosages. statistical-mathematical models are the essential tools of the phases ii and iii. phase ii aims at providing a first idea of the efficacy of the selected vaccine. thus a relatively small target population is built artificially. here two basic problems arise. the first is the definition of the outcome variable of interest. often only the "immunogenicity" is being studied, which means the formation of antibodies, but not protection against the disease. it is a particularly complex and manifold problem in the case of covid-19. secondly, the target population needs to include among the vaccinated subjects a sufficient number of people who would attract the disease when not vaccinated. since covid-19 morbidity in the entire population of a country is small, such a group must be constructed by "challenge", that is, by infecting its members artificially. they are usually volunteers and their risk of dying is small except in the age groups where the lethality by the disease is high, that is, in the case of covid-19, for old people. faced with this ethical problem the usa used, for various previous infectious diseases, prison inmates whose terms were shortened as a reward. there was a time when vietnam, while developing a certain vaccine, sent its samples for the phase ii trial to the usa to be tested in this way because vietnamese ethical standards forbade all kinds of challenge. there are usually several phase ii trials in order to select the potential vaccine to be finally studied in a phase iii trial. this is a field trial in the sense that a sample of subjects is drawn from the entire population of interest, for instance from among all inhabitants of a country within a certain age group. the outcome variable is not immunogenicity but protection against the disease in the sense of the desired efficacy. the size of the sample is determined beforehand by the precision of the intended estimate of the efficacy. as noted above the decision about the duration of the trial is a crucial element. if high efficacy during the first two weeks after vaccination is considered sufficient, the trial may be stopped after two weeks; this philosophy underlies the vaccinations against the seasonal influenza. if we are interested in its efficacy during the first ten years after vaccination, it must last ten years. this has, in addition to other problems, caused the long delay in developing an ebola vaccine (end of sect. 2). we hope that it will not be glossed over by those who are trying to sell a covid-19 vaccine very soon. the pandemic has functioned like a magnifying glass. in some places, it showed a basically well-functioning society. in other places it revealed scandals and intolerable social inequalities. in particular it reflected the state of a country's public health system. the present article aimed at describing the role of mathematics in the pandemic. as said above there are two parts to this "outlook". let us take up the first one, namely: what can be learnt from the epidemic? in sect. 1 we gave an overview of the main branches of mathematics that play a role. then the sects. 4 -9 sketched the most frequent applications; their titles and their order correspond vaguely to the branches of mathematics concerned. thus there were mathematical pattern analysis in laboratory work and statistical-mathematical models in judging the quality of tests; demographic methods in the collection of data; different ways to model the evolution of the pandemic mathematically; and clinical epidemiology in attempts to develop a vaccine. in this way the article aimed at clarifying the potential role of mathematics in making decisions. on the one hand it turned out that in practise the role of epidemic curve or compartmental models is much more restricted than advertised in many publications. decisions based on them may even have disastrous consequences, for instance those based on the mathematical concept of herd immunity. thus blind trust in mathematical arguments is unjustified. on the other hand denying the existence of a valid mathematicalscientific foundation for a control strategy is just as detrimental. it was done in denmark with the "tracing and lock-down" strategy by a report of an "expert group" of health academics and officials, which reflected the interests of medical, industrial and governmental circles. this comment leads us to the second part of our "outlook", namely: what to do in the future? the authors of the present article started it in early may by "since the covid-19 pandemic is not over ...". while we are finally finishing our work in the middle of july, it is still not over! it is even very active but has taken a largely different form. hence it seems natural to analyse its present characteristics in the light of the facts we have described in the sections 4 -9 above and to ask ourselves: which lessons can we draw regarding the control strategies to be applied now? covid-19 does no longer surge from a single source. it reappears in small or large regions of many parts of the world, which may be of various forms and extensions: a single home for the elderly in france, two districts in germany, a large city like beijing, an entire province in spain, or a whole country like new zealand. we shall call them "nests" to distinguish them from "clusters", which denote certain discrete sets of people. a precise follow-up of the evolution of cases in these nests meets with the manifold difficulties explained in sects. 5 and 6 and will not be repeated here. a first natural question to ask is, then: why do "active" nests persist and reappear? sect. 3 presented three components of successful control strategies: contact tracing; lock-down; masks. while contact tracing continues reluctantly, lock-down and wearing masks were widely abandoned, often as a result of governmental policies seeking popularity. next, what should be done? in the sections 7, 8 and 9 we have explained, using in particular mathematical arguments, in how far the strategies of control treated there suffer from serious drawbacks. this leaves us with the combination of two measures: inside a nest a rigorous lock-down such as social distancing and preventing larger assemblies of people; at its borders: closing them or only allowing passage when combined with quarantine. for example new zealand regarded as a single nest has taken such rigorous measures. as a result there are now no new cases, except two cases around the 14th july in "managed isolation facilities". other nests will act similarly, we hope. the proximal origin of sars-cov-2 the first epidemic model: a historical note on p.d. en'ko daniel bernoulli's epidemiological model revisited impact of nonpharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand. mrc centre for global infectious disease analysis john brownlee and the measurement of infectiousness: an historical study in epidemic theory the risks of lifting lockdowns prematurely are very large design and analysis of vaccine studies principles of health information systems in developing countries key to public health a novel deterministic forecast model for covid-19 epidemic based on a single ordinary integro-differential equation pattern theory. the stochastic analysis of real-world signals an introduction to bioinformatics algorithms alternative corona strategy: we can beat the infection down for the count with quarantine and tracing of infectors the authors: bernhelm booß-bavnbek born in 1941, studied mathematics from 1960 to 1965 at bonn university. research, teaching and practical work first in econometrics and operations research and then in geometric analysis and membrane processes of cell physiology professor at several universities in europe and outside; research, teaching and practical work first in mathematics and then in epidemiology and public health. much of this was done in developing countries key: cord-018917-7px75s3c authors: hopkins, richard s.; magnuson, j. a. title: informatics in disease prevention and epidemiology date: 2013-07-29 journal: public health informatics and information systems doi: 10.1007/978-1-4471-4237-9_14 sha: doc_id: 18917 cord_uid: 7px75s3c this chapter provides a description of the components of disease prevention and control programs, and then focuses on information systems designed to support public health surveillance, epidemiologic investigation of cases and outbreaks, and case management. for each such system, we describe sources used to acquire necessary data for use by public health agencies, and the technology used to clean, manage, organize, and display the information. we discuss challenges and successes in sharing information among these various systems, and opportunities presented by emerging technologies. systems to support public health surveillance may support traditional passive case-reporting, as enhanced by electronic laboratory reporting and (emerging) direct reporting from electronic health records, and also a wide variety of different surveillance systems. we address syndromic surveillance and other novel approaches including registries for reporting and follow-up of cases of cancer, birth defects, lead poisoning, hepatitis b, etc., and population-based surveys (such as brfss or prams). systems to support epidemiologic investigation of outbreaks and clusters include generic tools such as excel, sas, spss, and r, and specialized tool-kits for epidemiologic analysis such as epi-info. in addition to supporting outbreak investigation, agencies also need systems to collect and manage summary information about outbreaks, investigations, and responses. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. public health programs to prevent disease typically have been designed and implemented one disease at a time. each disease has its own patterns of distribution in populations, risk factors, and optimal and practical intervention strategies that are effective in controlling, preventing, or even eliminating cases of the disease. for example, an important strategy to prevent measles is vaccination, the main strategy to prevent gonorrhea is antibiotic treatment of case contacts before they become ill themselves, an important strategy to prevent cervical cancer is screening with pap smears and treatment of preclinical disease, and the main strategy for prevention of neural tube defects is folic acid supplementation of selected foods. still, each disease prevention program's components are drawn from a relatively short list: • planning and evaluation • public health surveillance • outbreak or cluster recognition and response • policy and guidance development • clinical services -screening -immunization -prophylaxis -treatment • laboratory services • case-contact identifi cation and interventions • education and training for clinicians • public education • regulation (for example, of food services, drinking water, child-care centers, hospitals, etc.) • administration and fi nancial management ideally, program managers choose the most effective combination of these program components to prevent or control the disease or diseases they are charged with addressing. however, as this must be done within the constraints imposed by the available funds, cost-effectiveness is the usual criterion for choosing the preferred combination of program components. public health agencies typically are organized both by disease and by function. for example, each disease-specifi c program usually does not have its own laboratory, and a single public health clinical facility and its staff may provide varied services such as immunizations for well children, treatment of people with tuberculosis (tb) and their contacts, and pap smear services. to variable degrees, they may even combine activities in a single patient encounter, for example, testing women for gonorrhea and chlamydia trachomatis infections at the same visit where they get a pap smear, or offering hepatitis b vaccination during a visit for sexually transmitted diseases (std) treatment. as information technology has become more widely used in public health and replaced paper-based systems, it has typically been implemented program area by program area, as resources became available. this has led to the creation of information 'silos.' for example, laboratory information systems usually have developed in isolation from those to support clinical care or public health surveillance. information systems to support clinical operations of public health departments (for example, clinical services for stds, childhood immunizations, hiv/aids, tb, or family planning services) have characteristics similar to those of other electronic health record systems in ambulatory care. however, in some health departments, clinical information systems have been separated by disease or clinic. if one were to design information systems from scratch for a set of disease prevention programs, there would be potential savings and effi ciencies from identifying the ways that one program component depends on information from another, or can serve multiple programs, and then designing the system to provide that information seamlessly. one can identify potential effi ciencies from two perspectives: in reality, it is rare to have an opportunity to design such extensive information systems as a single project. one is dealing with numerous legacy systems that were designed to support program-specifi c workfl ows. so a key challenge for the public health informaticist is to help their agency make decisions about where information system 'integration' will yield substantial benefi ts and where it will not. for example, if it is desired to know (one time) how many people in the jurisdiction have been reported during a particular time interval with both syphilis and hepatitis b, one could do an ad hoc match of information in two independent surveillance information systems. this task might take an analyst a few days or weeks to accomplish -which is almost certainly inexpensive compared to the cost of building a new information system that could do this task almost immediately. for many purposes, it may be useful and suffi cient to be able to display multiple streams of surveillance or programmatic data in the same environment, on the same screen or even in the same chart. in florida, de-identifi ed reportable disease case information and death certifi cate information are imported into the essence analytic environment that was originally designed for syndromic surveillance [ 1 ] , so that trends for similar conditions by age, sex, and geographic area in the two data streams can be easily compared. on the other hand, if it is desired to have real-time information available to the std clinic staff about past diagnoses of hepatitis b, or about past receipt of hepatitis b vaccine, then information systems need to be designed to support this kind of look-up; the usual solution is a shared person index between the two systems. alternatively, a common data repository can be designed in which all information about each person is permanently linked. as mentioned earlier, there are a number of components common to disease control and prevention programs. in this chapter, we will address information systems designed to support the following: • public health surveillance • outbreak or cluster recognition and response • acquisition of laboratory information • case-contact identifi cation and intervention cdc defi nes public health surveillance as "the ongoing, systematic collection, analysis, and interpretation of health data, essential to the planning, implementation, and evaluation of public health practice, closely integrated with the dissemination of these data to those who need to know and linked to prevention and control" [ 2 ] . each word of this defi nition is carefully chosen, and has implications for the design of surveillance information systems. a one-time data collection activity is not surveillance. data collection for research purposes is not surveillance. surveillance data are collected to support public health action, and analyses and recommendations based on these data must be shared with those who provided the data and with others who need to know. objectives of surveillance systems differ at the local, state, and federal levels [ 3 ] . at the local level, immediate response to individual cases is relatively more important, while at the federal level the analysis of larger-scale patterns is the most important function of surveillance. for state health departments, both uses of surveillance data may be important, depending on the disease and the size of the state. public health surveillance systems may be based on data capture from a variety of sources, including case reports, population-based surveys, sentinel providers, electronic health records (including laboratory information management systems for elr and emergency department records for syndromic surveillance), or administrative data (like hospital or physician claims for reimbursement). for some noninfectious diseases, surveillance is carried out through registries (see below). information systems to support reportable disease surveillance contain records representing case reports that currently are, for the most part, entered manually into an application by public health staff, based on information received from doctors, infection control practitioners, hospitals, and laboratories. increasingly, the laboratory information in these records comes from electronic records transmitted by the public health laboratory, hospital laboratories, and commercial laboratories, when there is a positive result meeting certain reporting criteria (like a positive igm antibody test for hepatitis a). these records typically contain a combination of clinical, laboratory, and epidemiologic information about each case. in future, increasing proportions of these case reports will be entered directly into a website by the practitioner creating the case report, or be transmitted electronically from the practitioner's electronic health record (ehr) system. currently almost half the states in the us use the cdc-provided nedss base system (nbs) as their platform for managing case reports. the remainder use either a system developed in-house or one of several commercially-available solutions [ 4 ] . in case-based surveillance practice, there is usually a relatively short list of required elements in the initial case report. for some diseases this is the only information received on all cases. for other diseases, usually of more importance and with lower case numbers, an additional data collection form is initiated by the receiving health department, which gathers information as appropriate from the ill person, the treating physician, and health records. the optimum amount of information to collect in the initial case report, as opposed to the disease-specifi c case report form, is a matter of judgment and may change as technology changes. in a largely manual system, health departments typically desire to minimize barriers to reporting of cases, so the incentive is to keep the initial case report form short. if much of the information desired for the disease-specifi c case report form can in fact be extracted from an electronic medical record with no additional effort by the person making an electronic case report, then the balance changes. careful decisions are needed: for which cases of which diseases are follow-up interviews necessary [ 5 ] ? until very recently, virtually all of the case-based surveillance information used at the federal level was collected initially at the local (or sometimes state) level, where it was used in the fi rst instance for local response. as the case report information passes from the local to the state to the federal level, it is subjected to validation and cleaning: cases not meeting the surveillance case defi nition have been removed from the data submitted to the federal level, missing data have been fi lled in to the extent possible, and cases have been classifi ed as to whether they are confi rmed, probable, or suspected using standard national surveillance case defi nitions (these case defi nitions are developed by the council of state and territorial epidemiologists in consultation with cdc) [ 6 ] . more recently, advances in technology have allowed case reports, and the information on which they are based, to move almost instantaneously from electronic health record systems, maintained by doctors, hospitals, and laboratories, to public health authorities. there are no technical barriers to these data being available at the federal level essentially as early as they are at the local and state levels. this ready availability of unfi ltered clinical information may allow more rapid awareness by public health offi cials at all levels of individual cases of high-priority diseases (like botulism or hemorrhagic fevers like ebola virus infection), and thus lead to more rapid detection and characterization of likely outbreaks. the simultaneous availability of raw data to multiple agencies at different levels of government also presents certain challenges. the user at the local level will have ready access to information from many sources about local conditions and events, and can use this information to interpret local observations. they will be in a position to understand when an apparent anomaly in their surveillance data is due to an artifact or to local conditions that are not a cause for alarm. they will also know whether a problem is already under investigation. a user at a state or federal level will be able to see patterns over a larger area, and thus may be able to identify multijurisdictional outbreaks, patterns, or trends that are not evident at a local level. the fact that several users may be examining the same raw data at the same time requires that these multiple users be in frequent communication about what they are seeing in their data and which apparent anomalies are already explained or need further investigation. there is a danger that users at a higher level may prematurely disseminate or act on information that, while based on facts, is incomplete or misleading. similarly, users at a local level may not realize that what they are seeing is part of a larger phenomenon. in the syndromic surveillance domain, the biosense 2.0 governance group [ 7 ] has adopted a set of etiquette principles which participating jurisdictions will be required to agree to, that spell out the mutual obligations of analysts at each level of the system (scott gordon , association of state and territorial health offi cials, 2013, personal communication). from an information management perspective, an important question is where to put human review of case reports in this information fl ow. for example, it is becoming technically possible for likely cases of reportable diseases to be recognized automatically in health care electronic record systems. some of these could be passed on to public health authorities without human review, in the same way that reportable laboratory results are already passed on in electronic laboratory reporting (elr). for which constellations of fi ndings in the electronic health record would this be appropriate? should some electronic case reports generated by electronic health record systems be passed to state or even federal public health offi cials before they are reviewed and validated at the local or state levels? if so, which ones? as always, there is a tension between the speed of information fl ow and its quality and completeness. there is a need for research to determine which constellations of fi ndings in electronic health records have adequate specifi city and sensitivity to warrant automated identifi cation of a person as being likely to have a case of a reportable disease. the acceptable sensitivity and specifi city will vary by disease. in 2001, cdc published the updated guidelines for evaluating public health surveillance systems [ 8 ] . this document identifi es a set of key attributes of surveillance systems to be assessed during a surveillance system evaluation, including simplicity, fl exibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness, and stability. these are also useful attributes to consider when designing a surveillance information system [ 9 ] . the relative importance of these attributes will vary depending on the condition under surveillance and the main purposes for surveillance. for example, a surveillance system to detect cases of botulism for immediate public health response puts a high premium on timeliness, and its operators are likely to be willing to accept a modest number of false-positive reports (a lower positive predictive value ) in order to assure that reports are received very quickly. on the other hand, surveillance to support planning of cancer prevention programs and treatment services is less time-sensitive, given the quite long incubation periods for most cancers, and therefore more concerned with diagnostic accuracy of every case report than with speed of reporting. timeliness, positive predictive value, and sensitivity of a public health surveillance system are always in tension with each other; increasing two of these always compromises the third. in systems based on case-reporting from doctors, hospitals, and laboratories, and receipt of electronic health records from these same organizations, records for an individual can in principle be linked with records for that same individual in numerous public health information systems, including those supporting clinical service, immunization registries, case investigation, partner or contact identifi cation, partner or contact notifi cation, and provision of interventions to partners or contacts. sometimes this will be done best by automated messaging of structured data from one system to another, sometimes by supporting real-time look-up capabilities, and sometimes by development of a master person index to underlie some or all of these applications. one key decision is which application to consider as the hub for this information sharing, for example, the surveillance application itself or a clinical application. surveillance systems that are based on sample surveys (such as the behavioral risk factor surveillance system, brfss [ 10 ] ), on sentinel practices (such as ili-net for surveillance of infl uenza-like illness [ 11 ] ) or on syndromic surveillance do not have individual patient identifi ers, and so intrinsically cannot be linked at the individual level to information systems supporting other disease control program components. their data are typically managed in systems built on standard statistical software packages, or other independent systems. syndromic surveillance systems are based on rapid acquisition of unfi ltered, real-time, electronic records without individual identifi ers from hospital emergency rooms [ 12 ] and urgent care centers, and also, increasingly, from outpatient physicians' offi ces and from hospital admissions [ 13 ] . the primary purpose of these systems is to support detection and characterization of community disease outbreaks, as they are refl ected in care received at emergency departments, physicians' offi ces, or hospitals. each visit to an emergency department is assigned to a category or syndrome , based on words and strings contained in the patient's chief complaint and/or the triage nurse's notes. as the records received by the health department do not have individual identifi ers, they cannot be linked to records in other information systems. however, records received by the syndromic surveillance system should contain unique identifi ers that could allow the epidemiologist analyzing the data to work back through the sending facility to an identifi ed clinical record. this traceback might become necessary if the person appeared to have a case of a reportable disease or to be part of a signifi cant outbreak. adding outpatient visits and hospital admissions to the scope of syndromic surveillance is opening up additional uses for this technology, especially in the areas of real-time non-infectious disease surveillance. surveillance for cancers [ 14 ] , stroke [ 15 ] , birth defects [ 16 ] , and some other chronic diseases like amyotrophic lateral sclerosis (als) is carried out through registries. registries are usually established by specifi c legislation, and typically relate to a single topic -for example a registry of records for a disease, or of immunization records. registries may be restricted to a geographic region. a distinctive feature of registries is that individual case reports are kept open for long periods of time, up to several or many years, allowing additional information about treatment, hospitalization, and death or other outcomes to be added. registries thus serve as systems to monitor type, duration, and outcome of treatment for these diseases, in addition to the occurrence of new cases of disease (disease incidence ). they may also support outreach efforts to patients or their families, as a way to document that appropriate steps have been taken to link patients to needed types and sources of care. most cases recorded in state-level cancer registries are acquired from hospitallevel registries, using an electronic case report in a standardized format [ 17 ] . some case abstracts are obtained directly by registry personnel or contractors, when hospitals do not have suitable registries of their own. case reports require extensive review and abstraction of medical records by trained workers. birth defect registries may also be built by active search for cases in hospital and other medical records, and abstraction of those records to make case reports. they also may be built by electronically linking records from vital statistics (birth and death records), centralized hospital discharge record systems, and clinical service providers for children with birth defects (such as state programs for children with special medical needs) [ 18 ] . the latter are much less expensive to develop but cannot be assumed to have captured all cases of the disease under surveillance, or captured them correctly [ 19 ] . a disease outbreak is defi ned as a number of cases greater than the number expected during a particular time interval in a geographic area or population. this term usually is used for events due to infectious diseases, and sometimes for those of toxic origin. a similar increase above expected numbers for a non-infectious disease, such as birth defects or cancer, is usually called a cluster . outbreaks and clusters may be due to diseases for which individual cases are reportable (like shigellosis or breast cancer), or diseases for which they are not (like food poisoning due to staphylococcal or clostridium perfringens toxins in most states, sars when it was new, or multiple sclerosis). surveillance systems are designed to facilitate recognition of outbreaks or clusters by frequent examination of the most current information available. the design of the user interface is particularly important. the interface should allow users to: fl exibly display line lists, bar charts by date of event (epidemic curves), and maps of location of cases; fl exibly select subsets of cases for display; apply appropriate statistical tests to detect improbable increases in case counts; and display multiple streams of data on the same chart. for example, users may want to display the epidemic curve of an infl uenza outbreak for several different regions of a state or for several different age groups, or to display counts of positive infl uenza tests and emergency department visits for infl uenza-like illness on the same graph with different scales for each. syndromic surveillance systems have been leaders in developing and evaluating statistical algorithms for automated detection of anomalies which may, on investigation, turn out to be outbreaks. such algorithms have less frequently been applied for automated detection of possible outbreaks or clusters in reportable disease data streams. most outbreaks and clusters are in fact not recognized by examination of regularly-collected surveillance system data. instead, they are recognized by private citizens (such as the organizer of a social event, a teacher or school nurse, the manager of a child care center, the manager of a food service facility, an employer, or the ill people themselves) or by practicing doctors, and brought to public health attention via a phone call or e-mail or entry on a web site established for the purpose [ 20 ] . public health workers assess the information and make the decision whether or not to do a formal investigation of the outbreak. one part of such an assessment is to look at available streams of surveillance data and determine whether there is information supporting the occurrence of an outbreak. for example, a report of a possible infl uenza outbreak in a high school might prompt closer examination of syndromic surveillance data from nearby hospital emergency departments to determine whether there is a more general increase in visits for infl uenza-like illness. a report of a neighborhood cluster of brain cancers would prompt closer examination of available cancer registry information, which might or might not support an interim conclusion that such a cluster is real and statistically signifi cant. in order to be accountable for the effectiveness of their work, local and state health departments need to track the occurrence of outbreaks and the public health response to those outbreaks. since outbreaks can be due to reportable or nonreportable diseases, this cannot be done only by actions such as identifying some cases in the reportable disease data system as being part of an outbreak. systems to track the occurrence of outbreaks need to document the following: • time and date the fi rst and last cases occurred • total (estimated or counted) number of cases • population group most affected (by age, sex, location) • setting of the outbreak (school, workplace, restaurant, wedding, etc.) • suspected or confi rmed agent • most common clinical presentation • suspected or confi rmed source and mode of spread • methods used to investigate agent, source and mode of spread • control measures recommended • control measures implemented • lessons learned for prevention of future outbreaks and improved investigation and response in future events this information about outbreaks should be stored for ready retrieval, and to serve as a basis for quality improvement efforts. for quality improvement purposes, it is also helpful to document the content of the summary report written about each outbreak. when the outbreak is due to a reportable disease, individual cases in the reportable disease surveillance information system can be linked to the outbreak, for example by having an outbreak identifi er attached to their records. if preliminary information about outbreaks in a jurisdiction is entered into the outbreak information system in real time, as the investigation is proceeding, and if the outbreak database is readily searchable by all communicable disease investigators in the jurisdiction, then local investigators can use the outbreak database to help them with investigations of new illness or outbreak complaints [ 21 ] . for example, if they receive a complaint that illness has occurred in people who consumed a particular food product, they can look in the database and determine whether other recent or current complaints or outbreaks mention the same food product. if they receive a report about a gastroenteritis outbreak in a childcare center, they can determine what agents have been found to be responsible for recent or current similar outbreaks in nearby communities; this can help focus their laboratory testing and initial control strategies. some us states have had long-standing systems to document all outbreaks investigated by local or state personnel, but others have not. a major variable in the design of such systems is the state-local division of responsibilities in each state, including the degree of state oversight of 'routine' local outbreak investigations. the actual investigation of an outbreak or cluster may involve enhanced "active" case-fi nding, use of case-report forms, group surveys, and formal epidemiologic studies. active case-fi nding involves regular solicitation of case reports from doctors, hospitals, and laboratories. managing the reports of possible, probable, and confi rmed cases that are part of the outbreak is an important task. for a reportable disease, the jurisdiction's reportable disease surveillance system may be adequate to manage reported cases. it may be necessary, however, to create a continuouslyupdated line list of possible cases and their current status, which is outside the scope of the standard reportable disease application. outbreak investigation surveys will typically involve interviewing everyone with a possible exposure (like all attendees of a wedding reception), whether they were ill or not. formal studies may involve interviewing selected non-ill people, for example, as part of a case-control study. the investigation may also involve obtaining and sending to a laboratory a large number of specimens from ill persons, and sometimes from exposed non-ill persons and from environmental sources (food, water, air, soil, etc.). managing these disparate types of information is a challenge, especially in a large outbreak or one involving multiple jurisdictions. there is currently no one widely-accepted and satisfactory way to manage data in such settings. each investigation team typically uses the tools it is most familiar with, including some combination of data management tools like ms excel, ms access, or epiinfo [ 22 ] , and standard statistical packages. many health departments maintain libraries of standard questionnaires with associated empty data bases, for use during outbreak investigations. when cdc is involved in a multistate outbreak, the investigation team at the local or state level needs to be able to produce and transmit timely case report and other information in the format desired by cdc. the services of an experienced public health informaticist can be extremely helpful to the investigation team when outbreaks are large and multifocal. an ongoing challenge for cdc and the states is how to make the transition from specialized case reporting during an outbreak of a new disease, such as west nile virus encephalitis or sars, to routine case-based surveillance. if this transition is not well-managed, it is likely to result in the creation of a permanent stand-alone surveillance information system (or silo) for that disease. if the new disease is of national importance, cases should be made nationally notifi able and its surveillance should be incorporated into existing systems. laboratory information is a critical component of disease surveillance and prevention. laboratory data form the foundation of many surveillance systems. there are different types of laboratories involved in the public health data stream. laboratories providing data to public health fall into the general categories of commercial or private industry, hospital or clinical, and public health laboratories. public health laboratory information systems (lis) contain information about test results on specimens submitted for primary diagnosis, for confi rmation of a commercial or hospital laboratory's results, for identifi cation of unusual organisms, or for further characterization of organisms into subgroupings (like serotypes) that are of epidemiologic importance. in some states, all clinical laboratories must submit all isolates of certain organisms to the public health laboratory. many of the results obtained in a public health laboratory turn out to be for diseases that are not reportable and not targets of specifi c prevention programs. some of those results may, however, be for cases of non-reportable diseases that are historically rare in the jurisdiction but of great public health importance, or are new or newly-recognized. the main business of clinical laboratories (located both inside and outside hospitals) is to test specimens for pathogens or groups of pathogens specifi ed by the ordering physician, and return the results to the person who ordered the test. public health agencies have, since the early 1990s, asked or required such laboratories to also identify results meeting certain criteria (indicating the presence of a case of a reportable disease) and send a copy of the results to the public health agency for public health surveillance. initially, case reporting by laboratories was accomplished on paper forms, which were mailed or faxed to public health departments. some laboratories very soon moved to mailing printouts of relevant laboratory results, then to sending diskettes, then to transferring computerized fi les containing laboratory results by direct modem-to-modem transfer, and eventually to transferring such fi les via the internet using standard formats and vocabularies. in some states, public clinics (for example, std clinics) have used contract laboratories for their testing needs. in this situation, the outside laboratory supplies both positive and negative results to the public health agency, increasingly by transfer of electronic results in standard formats. laboratories provide data on reportable conditions to their local or state public health authority. reportable diseases are determined by each state; clinicians, hospitals, and/or laboratories must report to public health when these conditions are identifi ed. some reportable conditions are also nationally notifi able. deidentifi ed cases of these are voluntarily notifi ed by states and territories to cdc, which, in collaboration with the council of state and territorial epidemiologists, maintains a listing of nationally notifi able conditions that includes both infectious (e.g., rabies, tb) and non-infectious (e.g., blood lead, cancer) conditions [ 23 ] . the public health partnership with laboratories has led to the very successful and still increasing implementation of electronic laboratory reporting (elr) in the us. elr refers to the secure, electronic, standards-based reporting of laboratory data to public health. elr implementation has been steadily escalating since its inception around the year 2000, replacing previous reporting systems that relied on slower, more labor-intensive paper reporting. the elr national working group conducted annual surveys from 2004 to 2011 [ 24 ] which gathered data from all 50 states as well as from several territories and large metropolitan areas. these data were supplemented with data for years 2000-2004, retroactively gathered in the 2010 survey. the tracked growth of elr (fig. 14.1 ) illustrates its rapid rise in the us, from the start of early stage planning to fully operational elr [ 25 ] . the expected benefi ts of elr include more rapid reporting of reportable cases to public health departments, allowing faster recognition of priority cases and outbreaks for investigation and response, and thus more effective prevention and control [ 26 ] . elr also is expected to reduce the number of missed cases, as automated systems do not require laboratory staff to actively remember to make case reports, and to improve the item-level completeness and quality of case reports. although experience shows that the expected improvements in timeliness, sensitivity, completeness, and accuracy are generally being realized [ 27 ] , timeliness may not be improved substantially for those diseases where clinicians routinely report based on clinical suspicion without waiting for laboratory confi rmation (for example, meningococcal disease) [ 28 ] . in addition, laboratories (especially referral laboratories) often do not have access in their own information systems to home addresses for people whose specimens they are testing, and have struggled with providing complete demographic information to public health agencies. implementation of an operational elr system is not a trivial undertaking. laboratories must confi gure data into an acceptable message format, most commonly health level seven (hl7 ® ) [ 29 ] . laboratory tests and results should be reported with correlated vocabulary or content codes. two of the most common code systems used for laboratory tests and their associated results are logical observations identifi ers names and codes (loinc ® ) [ 30 ] and systematized nomenclature of medicine (snomed ct ® ) [ 31 ] . neither of these systems is suffi cient by itself to encode all the information needed for public health surveillance. public health jurisdictions have introduced elr to their partner laboratories using one or more of the following approaches: • the "charm" approach -relies on establishing goodwill and collaboration with laboratory partners. while this collegial approach is very appealing, it may be unable to overcome signifi cant barriers such as lack of laboratory funding or resources, and some facilities will supply data only in methods specifi cally required by law. • the incentive approach -involves offering either fi nancial or technical assistance to laboratory partners, assisting them in the startup process of elr. while this approach may be preferred by many laboratories, relatively few jurisdictions have the discretionary funds (or are able to receive federal assistance funds) to implement the approach. • the enforcement or legislative approach -requires reporting rules or legislation that requires laboratories to participate in elr. the most successful enforcement approach will include low-cost options for smaller laboratories, such as web data entry, so that they may benefi t from an elr -"lite" implementation [ 32 ] . the mainstreaming of elr systems in the us has pioneered a clear path forward for public health to begin maximizing its presence in the domain of electronic data interchange. at a local level, case reports for communicable diseases prompt action. although the specifi c action varies by disease, the general approach is the same. it starts with an interview of the ill person (or that person's parents or other surrogates) to determine who or what the person was in contact with in ways that facilitate transmission, both to determine a likely source of infection and to identify other people who may be at risk from exposure to this person. information systems to support contact tracing, partner notifi cation, and postexposure prophylaxis (for stds or tb, for example) contain records about all elicited contacts (exposed persons) for each reported case of the disease in question. these records contain information about each contact, such as whether they were located, whether they received post-exposure prophylaxis, and the results of any additional partner-elicitation interviews or clinical testing that were completed. information systems to support surveillance for other reportable diseases also increasingly contain information about what disease-appropriate action was taken in response to each case; such actions may include identifi cation of contacts, education of household members, vaccination or antibiotic prophylaxis of contacts, isolation of the case (including staying home from work or school), or quarantine of exposed people. std and tb information systems typically capture full locating information for contacts, and can be used both to support fi eld work and to generate statistics on effectiveness of partner notifi cation activities worker by worker and in the aggregate. systems for other reportable diseases may capture only the fact that various interventions were done, and the date that these were initiated. information about the timeliness of initiation of recommended control measures is now required as a performance measure for selected diseases by cdc's public health emergency preparedness cooperative agreement [ 33 ] . in the investigation of a case of meningococcal disease, contacts are people who had very close contact with the original person, for example a household member, boyfriend, or regular playmate. health department staff determines who the close contacts are. each will then be offered specifi c antibiotic treatment to prevent illness. for syphilis, contacts are people who have had sex with the original case. contacts will be examined by a clinician and assessed serologically to see if they are already infected, and offered appropriate prophylactic or curative antibiotic treatment. for measles, contacts may include anyone who spent even a few minutes in the same room as a case. contacts whose exposure was recent enough, and who are not fully immunized already, will receive a dose of measles-containing vaccine, and all contacts will be asked to self-isolate immediately if they develop symptoms of measles. in investigating a common-source outbreak of legionellosis, histoplasmosis, or anthrax, the local health department may want to locate everyone who had a specifi ed exposure to the apparent source of the infection. these exposed people may need antibiotic prophylaxis or may be advised to seek medical care promptly if they become ill. information systems to support this type of work typically have three purposes: 1. serve as a place for workers to record and look up information about people who are or may be contacts, and to track which contacts have and have not yet received needed interventions. 2. serve as a source of information for calculating indices of program or worker timeliness and performance, such as the average number of sexual contacts elicited per syphilis patient interviewed, or the percentage of measles contacts who were identifi ed in a timely way and who received post-exposure measles vaccine prophylaxis. 3. document the workload and effort put in by epidemiology and disease control fi eld staff it seems logical that the surveillance information system should serve as the basis for a system to support fi eld investigation, and this is often the case. the fact that the recommended interventions vary by disease makes designing a single system more complex. existing systems that track fi eld worker activities in detail are much more common for std and tb programs than for others. for general communicable disease fi eldwork, it is currently more common that the system simply documents which interventions were done and when, rather than use the application to track specifi c named contacts or exposed people. the public health informatics institute has published a detailed analysis [ 34 ] of the typical workfl ow involved in surveillance, investigation, and intervention for reportable diseases, and the corresponding information system requirements. the work group that phii convened had representatives of nine different state and local health departments, who were able to identify a large number of processes that were common to all nine jurisdictions, such as case-fi nding, case investigation, data analysis and visualization, monitoring and reporting, case/contact specifi c intervention, and others. these common processes can then serve as a basis for designing information systems to support case-reporting, surveillance, and case-based intervention work that are useable in multiple jurisdictions. consider existing or planned surveillance systems for multiple diseases and conditions. broadly, there are three functions in each of these systems -acquiring the raw data, cleaning and managing the data, and making the data available to users. each of these functions potentially can be integrated, to varying degrees. for example, multiple surveillance systems may benefi t from receiving electronic laboratory reports with a result indicating the presence of a case of a reportable disease. laboratories appreciate having a single set of instructions and a single destination for all their required reports, as this simplifi es their work. the laboratories then benefi t from the ability of the recipient health department to route the reports internally to the right surveillance information system. at the other end of the data pathway, users appreciate having a single interface with which to examine data about multiple conditions or diseases, using the same commands and defi nitions. the users do not have to understand how different surveillance information systems may internally code the same concept in different ways. they also appreciate being able to directly compare information that originally was submitted for the use of different program areas -for example, hepatitis b and gonorrhea in the same chart or table. in the short to medium term, it is not necessary to build a single integrated data repository or a master person index to achieve these goals, even if that is what one would have designed if one were starting from the beginning. however, if one wants to be able to see information about the same person that originates and is stored in multiple systems -for example, so that tb clinicians can see hiv data on their patients and vice versa -then an integrated data repository, or a master person index, or a query system that is extremely accurate in fi nding data on the right person, is needed. modifying existing systems to be able to carry out these functions is time consuming and expensive, so the business case and requirements need to be especially clear. florida's essence system: from syndromic surveillance to routine epidemiologic analysis across syndromic and nonsyndromic data sources (abstract) history of public health surveillance blueprint for a national public health surveillance system for the 21st century status of state electronic disease surveillance systems -united states prioritizing investigations of reported cases of selected enteric infections. paper presented at council of state and territorial epidemiologists nationally notifi able disease surveillance system case defi nitions association of state and territorial health offi cers. biosense 2.0 governance updated guidelines for evaluating public health surveillance systems, recommendations from the guidelines working group design and operation of local and state infectious disease surveillance systems oxford handbook of public health practice behavioral risk factor surveillance system overview of infl uenza surveillance in the united states international society for disease surveillance meaningful use workgroup. final recommendation: core processes and ehr requirements for public health syndromic surveillance electronic syndromic surveillance using hospital in patient and ambulatory clinical care electronic health record data national program of cancer registries coverdell national acute stroke registry surveillance -four states atlanta congenital defects program (macdp) north american association of central cancer registries, inc. (naaccr). implementation guidelines and recommendations report on birth defects in florida a comparison of two surveillance strategies for selected birth defects in florida online food and waterborne illness complaint form biosurveillance plan for human health, version 2.0. atlanta national notifi able diseases surveillance system (nndss), cdc. accessed at http:// wwwn.cdc.gov/nndss/ on available from www.coast2coastin-formatics.com national electronic laboratory reporting (elr) snapshot survey. available from www.coast2coastinformatics.com . cited statewide system of electronic notifi able disease reporting from clinical laboratories: comparing automated reporting with conventional methods a comparison of the completeness and timeliness of automated electronic laboratory reporting and spontaneous reporting of notifi able conditions potential effects of electronic laboratory reporting on improving timeliness of infectious disease notifi cation -florida health level seven (hl7 ® ) homepage. available at logical observation identifi ers names and codes (loinc ® ) systematized nomenclature of medicine-clinical terms (snomed ct ® ) see for example section 64d-3.031(5) of the florida administrative code: notifi cation by laboratories public health emergency preparedness cooperative agreement, budget period 1, performance measures specifi cation and implementation guidance, at-a-glance summary redesigning public health surveillance in an ehealth world on 31 1. what are some of the methods for surveillance besides case-reporting? 2. how are registries different from other surveillance information systems? 3. what are the advantages and disadvantages of building a master person index across surveillance information systems for multiple diseases? 4. what are the expected benefi ts of electronic laboratory reporting as a method to enhance surveillance? 5. what are the advantages and disadvantages of building a system to manage information about case contacts as part of the surveillance information system? 6. who determines for which diseases cases are nationally notifi able? key: cord-151030-5x3ztp1n authors: piasecki, tomasz; mucha, piotr b.; rosi'nska, magdalena title: a new seir type model including quarantine effects and its application to analysis of covid-19 pandemia in poland in march-april 2020 date: 2020-05-29 journal: nan doi: nan sha: doc_id: 151030 cord_uid: 5x3ztp1n contact tracing and quarantine are well established non-pharmaceutical epidemic control tools. the paper aims to clarify the impact of these measures in covid-19 epidemic. a new deterministic model is introduced (seirq: susceptible, exposed, infectious, removed, quarantined) with q compartment capturing individuals and releasing them with delay. we obtain a simple rule defining the reproduction number $mathcal{r}$ in terms of quarantine parameters, ratio of diagnosed cases and transmission parameters. the model is applied to the epidemic in poland in march april 2020, when social distancing measures were in place. we investigate 3 scenarios corresponding to different ratios of diagnosed cases. our results show that depending on the scenario contact tracing could have prevented from 50% to over 90% of cases. the effects of quarantine are limited by fraction of undiagnosed cases. taking into account the transmission intensity in poland prior to introduction of social restrictions it is unlikely that the control of the epidemic could be achieved without any social distancing measures. the epidemic of sars-cov-2 infection triggered an unprecedented public health response. given the lack of effective vaccine and treatment in 2020, this response included a variety of travel restrictions and social distancing measures [2] . while these measures help to slow down the epidemic they come at significant economical and societal cost [1] . as an alternative an approach focusing on rapid diagnosis is increasingly recommended [21] and prior to lifting social distancing measures largescale community testing should be in place [36] . testing efforts are complemented by identifying and quarantining contacts of the diagnosed cases. of note, by isolating the asymptomatic contacts from their social networks, this strategy takes into account the pre-symptomatic and asymptomatic spread of the infection [9, 10] , believed to be one of the key drivers of fast spread of covid-19. as an example, wide spread testing in general population followed by isolation of the infected helped to reduce covid-19 incidence by 90% in an italian village of vo'euganeo [14] . a modelling study in france offers similar conclusions arguing that relaxing social lock-down will be only feasible in case of extensive testing [22] . while there is already a number of studies estimating the effects of general social distancing measures [25, 2, 22, 26] , less is known about the impact of quarantine. hellewell et al. [27] investigated the potential of rapid isolation of cases and contact tracing to control the epidemic, finding that prohibitively high levels of timely contact tracing are necessary to achieve control. however, new technologies may offer sufficiently fast alternative to traditional contact tracing, in which case the epidemic could be still controlled by contact tracing [28] . our aim is to develop a seir-type model which incorporates the effects of quarantine and validate it in a setting in which measures to reduce contacts are in place. we apply it to investigate the role of quarantine in poland. the first case of covid-19 in poland was diagnosed on march 4th. social distancing measures were rapidly introduced during the week of 9 -13th march including closure of schools and universities, cancellation of mass events and closure of recreation facilities such as bars, restaurants, gyms etc. as well as shopping malls. religious gatherings were limited. finally, borders were closed for non-citizens [24] . these measures were fully in place on march 16th. further, beginning at march 25th restrictions on movement and travel were introduced (lockdown). wearing face covers became obligatory on april 14th. the restrictions were gradually lifted beginning at april 20th. we focus on modelling the time period when the social distancing measures were in place and then consider different scenarios of relaxation of the restrictions with possible improvement of testing and contact tracing. we note that the procedures for quarantine were in place even before the social distancing measures. they initially focused on individuals arriving from covid-19 affected areas in china. when the epidemic started spreading in european countries people who came back to poland from these countries were advised to immediately seek medical attention if they experienced any symptoms consistent with covid-19. however, adherence to these recommendations was not evaluated. as soon as the first case was diagnosed in poland quarantine for close contacts was also implemented. this paper aims to define a deterministic population model describing the epidemic in classical terms of susceptible, exposed, infectious, removed. in our model the quarantine becomes a separate state that removes individuals from susceptible and exposed states. we show that the reproductive number in our model is given by a simple formula referring to the parameters of transmission and transition, but also to parameters describing the quarantine. we demonstrate that in a real life scenario (case study of poland) the quarantine effectively reduces the growth of infectious compartment. increasing the efficiency of contact tracing and testing may may to some extent compensate lifting up the social distancing restrictions. we introduce a modification of the classical seir model including effects of quarantine. to underline importance of that extension we call it seirq. formally the model is described by a system of ordinary differential equations with delay dedicated to the quarantine. the following states are included in the model: s(t) -susceptible e(t) -exposed (infected, not infectious) i d (t) -infectious who will be diagnosed i u (t) -infectious who will not be diagnosed r d (t) -diagnosed and isolated r u (t) -spontaneously recovered without being diagnosed q(t) -quarantined the figure 2.1 presents the schematic representation of the model. a susceptible individual (state s), when becoming infected first moves to the state e, to model the initial period, when the infected individual is not yet infectious. next the cases progress to one of the infectious states i d , i u at the rates κσ and (1 − κ)σ, respectively. in general, moving through the i d pathway concerns these individuals who (independently of quarantine) would meet the testing criteria, as relevant to the local testing policy, e.g. testing of people with noticeable symptoms. we shall emphasize that from the point of view of analysis of spread of infection, the quantity i u shall be regarded rather as not recognized infections, not necessarily asymptomatic or mild. with this interpretation the value of κ can be influenced by intensity of testing. the creation of state e is via i d and i u with transmission rates β d and β u , respectively, normalized to the total population size n = s + e + i d + i u + r d + r u + q, which is assumed to be constant in time, births and deaths are neglected. the transition parameter σ is assumed identical for both groups, relating to the time between infection and becoming infectious. the infectious individuals then move to the state r d , which is the state of being diagnosed and isolated (and later recovered or deceased), with the rate γ d corresponding to the observed time between onset and diagnosis. on the other hand r u contains people who spontaneously recovered with rate γ u . our model includes an additional state of being quarantined (q). to mimic the situation of contact tracing, individuals can be put in quarantine from the state s (uninfected contacts) or the state e (infected contacts). these individuals stay in the quarantine for a predefined time period t . we assume that the number of people who will be quarantined depend on the number of individuals who are diagnosed. an average number of individuals quarantined per each diagnosed person is denoted as α. however, as the epidemic progresses some of the contacts could be identified among people who were already infected, but were not previously diagnosed, i.e. the state r u . we note that moving individuals between the states q and r u has no effect on the epidemic dynamics, therefore we assume that only individuals from s and e are quarantined and we reduce the average number of people put on quarantine by the factor s(t) s(t)+ru(t) . further, to acknowledge the capacity limits of the public health system to perform the contact tracing, we introduce a quantity k max , describing the maximum number of people who can be put in quarantine during one time step. we also assume that among the quarantined a proportion θ is infected. after the quarantine, the infected part θk(t − t ) goes to r d and the rest (1 − θ)k(t − t ) returns to s. taking all of the above into account, the model is described with the following seirq system: where k(t) = min{ s(t) s(t)+ru(t) αγ d i d (t), k max }, and α, β d , β u , γ d , γ u , θ, t ≥ 0. (2.1) we assume that the parameters α, β d , β u , θ, γ u and γ d depend on the country and time-specific public health interventions and may therefore change in time periods. due to proper interpretation of the equation on e we require that β d ≥ θαγ d to ensure positiveness of e. 2.2 basic reproductive number, critical transmission parameter β * . based on the general theory of seir type models [23] , we introduce the reproductive number it determines the stability of the system as r < 1 and instability for r > 1 (the growth/decrease of pandemia). this quantity not only explains the importance is testing (in terms of κ) and quarantine (in terms of α), but also gives an indication on levels of optimal testing and contact tracing. we underline that this formula works for the case when the capacity of the contact tracing has not been exceeded (k(t) < k max ). the details of derivation of (2.2) are provided in the appendix, section a.4. we shall emphasize the formal mathematical derivation holds for the case when i and e are small comparing to s. therefore the complete dynamics of the nonlinear system (2.1) is not fully determined by (2.2) . however in the regime of epidemic suppression, which is the case of covid-19 epidemic in poland, i and e are small compared to s and so the formula (2.2) reasonably prescribes spreading of infection in the population. the critical value r = 1 defines the level of transmission which is admissible, taking into account the existing quarantine policy, in order to control epidemic. as the level of transmission depends on the level of contacts, this provides information on the necessary level of social distancing measures. the formula (2.2) indicates that improving the contact tracing may compensate relaxation of contact restrictions. the key quantity is θα. indeed the system with the quarantine has the same stability properties as one without k, but with the new transmission rate β new d = β d − θαγ d . in order to guarantee the positiveness of e, β new d must be nonnegative. it generates the constraint the above condition also implies the theoretical maximal admissible level of quarantine. we define it by improving the targeting of the quarantine, i.e. by the highest possible level of θ: as long as the k max threshold is not exceeded the effect of the increase in θ or in α play the same role at the level of linearization (small i, e). however, in general it is not the case and for the purpose of our analysis we fix α. for our analysis we assume β d = β u = β. the reason is that, both i d and i u contain a mixture of asymptomatic and symptomatic cases and although there might be a difference we lack information to quantify this difference. then using formula (2.2) we compute critical values β * (κ, θ, α) defined as it shows the upper bound on transmission rate β which still guarantees the suppression of pandemic. we shall omit the dependence on γ d , γ u as these are fixed in our case, and denote briefly β * (κ, θ, α). in the case of maximal admissible quarantine (2.4) we obtain which can be regarded as theoretical upper bound for β if we assume "optimal admissible" quarantine for fixed κ, for which the epidemic could be still controlled. it must be kept in mind though that the condition (2.3) means that we are able to efficiently isolate all persons infected by every diagnosed, therefore is unrealistic. the resulting β * (θ max , κ) should be therefore considered as a theoretical limit for transmission rate. all simulations are performed using gnu octave (https://www.gnu.org/software/octave/). the underlying tool for all computations is a direct finite difference solver with a 1 day time step. basic assumptions for data fitting. we estimate the transmission rates β by fitting the model predictions to the data on the cumulative number of confirmed cases. since people with confirmed diagnosis are efficiently isolated, they are immediately included into r d . therefore, the quantity fitted to the data is r d (t). the crucial assumption behind our approach is that the parameter β changes twice during the period of analysis. the reason is that we can distinguish two important time points in the development of epidemic in poland. the first is initial restrictions including school closure effective march 12, which was accompanied with restrictions on other social activities. as we do not take migration into account in our model, we assume that the effect of border closing is reflected in β. the second turning point was a lockdown announced on march 25. for simplicity we comprise the effect of above measures in two jump changes in β in t ∈ {t 1 , t 2 } and choose t 1 = 14, t 2 = 28. with t = 1 corresponding to march 3 it means small delay with respect to the above dates which can be justified by the fact that new cases are reported with a delay of approximately 2 days. choice of fixed parameters (tab. 2.1). we assume that the parameters σ, γ u represent the natural course of infection and their values could be based on the existing literature. the parameter σ describes the rate of transition from non-infectious incubation state e into the infectious states i d or i u . the value of σ takes into account the incubation period and presymptomatic infectivity period. γ u relates to the period of infectivity, which we select based on the research regarding milder cases, assuming that serious cases are likely diagnosed. further, κ is a parameter related both to the proportion of asymptomatic infection and the local testing strategies. since the literature findings provide different possible figures, for κ we examine three different scenarios. parameters γ d , θ and α are fixed in our model for the purpose of data fitting, but informed by available data. one of the scenarios of future dynamics of the epidemic (section 3.3) considers possible increase of θ. parameter γ d was estimated basing on time from onset to diagnosis for diagnosed cases, and θ as rate of diagnosed among quarantined. furthermore we fix the parameter α by comparing the number of quarantined people obtained in simulations with actual data. the capacity level of public health services is set in terms of possible number of quarantined per day k max , as double the level observed so far. detailed justification of the values of fixed parameters collected in the following optimization algorithm. in order to fit the values β 1 , β 2 , β 3 we use a standard gradient descent algorithm. the error function is defined as mean square difference between the cumulative number of diagnoses and the r d (t) predicted from the model. for the initial values the error function is optimized only for a limited number of possible conditions, as these mostly impact β 1 , which is less relevant for future predictions. to estimate confidence intervals we use a method of parametric bootstrap. the optimisation procedures are described in the appendix, section a.1, where we also show precise errors of data fitting. dataset. the data series contains cumulative number of confirmed cases of covid-19 in poland from march 3 (first confirmed case in poland) till april 26, which amounts to 54 observations. the data are taken from official communications of the ministry of health. as explained in table 2.1 and appendix (section a.2 additional data sources were used for choosing θ, α and γ d . in table 3 .1 we show estimated values of β i , where i = 1, 2, 3 correspond to the time intervals when different measures were in place, and the r for the third time interval. given the social distancing measures in place early april 2020, as well as the quarantine levels, the reproductive number was below 1, independently of the value of κ, which relates to testing effectiveness. the figure 3.1 shows the fit of the models assuming different levels of κ. good fit is found for all three models although predictions start to differ in the middle-term prognosis. we proceed with predictions assuming that the restrictions are continued, i.e.keeping β = β 3 (note that the estimated β 3 is different for each κ). we calculate the epidemic duration (t max ), the peak number of infected (i max d , i max u ) and the final size of the epidemic (r d (t max ), r u (t max )). in order to show the influence of quarantine we compare the situation with quarantine, keeping the same θ, α, to the situation without quarantine, setting αθ = 0. the results of the development of the epidemic during the first 120 days are shown on for κ = 0.2 the difference between the scenarios with and without quarantine is visible but not striking. however for κ = 0.5 and κ = 0.8 a bifurcation in the number of new cases occurs around t = 40 leading to huge difference in the total time of epidemic and total number of cases. these values are summarized in the table 3.2. we note that given the epidemic state in the first half of april 2020 for all values of κ the model predicts epidemic extinction both with quarantine and without quarantine. however, since the epidemic is very near to the endemic state, the predicted duration is very long, especially if no quarantine is applied. using the formula (2.5) we can compute critical values β * . in table 3 .3 we show the values of β * (κ, 0.006, 75) and for convenience recall also estimated values of β 3 and r, listed already in table 3 .1. moreover we compute β * (κ, 0, 0), i.e. without quarantine and show values of r for our estimated values of β 3 and the same γ d , γ u but without quarantine. comparing the estimated values of β 3 (table 3. 3) for all cases of κ are only slightly below β * . eliminating the quarantine, for the estimated values of β 3 , we have different situations depending on the actual value of κ. in case κ = 0.2, so assuming that currently only 20% of infections are diagnosed, the low values of r are due to low β 3 rather than the effect of quarantine (controlling epidemic by social contact restrictions). in effect even if we remove the quarantine we have still r < 1, but very close to 1. on the other hand if κ = 0.5 or κ = 0.8 we estimate higher β 3 , which corresponds to the situation of controlling the epidemic by extensive testing and quarantine. for these cases, if we remove the quarantine, we end up with r > 1. the quantity β 3 − θαγ d represents effective transmission rate due to diagnosed cases. in particular it shows by how much the transmission could be reduced by improved contact tracing (θα) and faster diagnosis (γ d ). these results confirm that the higher is the ratio of undiagnosed infections, the weaker is influence of quarantine. in the next section we verify these results numerically. our second goal is to simulate loosening of restrictions. in particular we want to verify numerically the critical thresholds β * listed in table 3.3. for this purpose we assume that at t = 60 we change β. for each value of κ we consider 3 scenarios: (a) current level quarantine: i.e. quarantine parameters θ = 0.006, α = 75 are maintained; (b) no quarantine is applied starting from t = 60; (c) the maximal admissible quarantine is applied, meaning that θ max = β αγ d (see (2.2) ). in this case α = 75. as long as the limit k max is not reached there is no difference whether we increase α or θ, the decisive parameter is αθ. increasing α would lead to reaching k = k max earlier and hence worse outcomes. the results confirm that around β * a rapid increase in the total number of infected occurs, coinciding with the peak total epidemic duration. thus the numerical computations confirm that the critical β * calculated for the linear approximation in the section 2.2 are adequate, with a small bias towards lower values. the case κ = 0.2 shows that the influence of quarantine is not high, even for the maximal admissible case, when we are able to efficiently isolate all persons infected by every diagnosed. a striking feature in the behaviour of total number of infected are jumps for certain critical value of β observed for κ = 0.5 and κ = 0.8, both in case θ = 0.006 and θ = θ max . the values of r d and r u before and after these qualitative changes are summarized in table 3.4. a closer investigation for these values of β shows that in all 4 cases the jump occurs for the first value of β for which the limit number of quarantined, k max = 50000, is achieved. notice that immediately after passing the threshold the values become very close to those without quarantine. we propose a simple seir-type model (seirq), which includes the effects of testing and contact tracing. the model formulation allows to calculate an interpretable formula for the reproductive number r (2.2). as typical for this class of models, r depends on transmission parameters β. increasing β corresponding in e.g. to higher frequency of social contacts increases r. decreasing β, for example in consequence of widespread use of face masks, has the opposite effect. on the other hand γ d reflects the time to diagnosis and the formula indicates that more rapid diagnosis is associated with lower r. in addition, our model offers a clear interpretation of the quarantine effect. the transmission rate due to diagnosed cases, β d , is decreased by the factor θαγ d indicating that both the number of quarantined per diagnosed individual (α) and proper targeting of the quarantine (the infection rate among the quarantined θ) equally contribute to this factor. also the parameter related to testing: the delay in diagnosis, γ −1 d , plays similar role. this quantifies the potential of a wide range of interventions to improve testing and contact tracing, as outlined in e.g. in ecdc recommendations [31] . in particular, as the number of people put in quarantine per each case and the infection rate among the quarantined impact r in similar fashion, our results support the recommendations to focus on the high risk contacts when the resources do not allow to follow all contacts. our model takes into consideration only the effective contact tracing, i.e. the situation when the infected contacts are identified and put in quarantine before they become infectious. people who are identified later would be modelled as passing through one of the i states to the r states. this means that the number of quarantined in our model can be also increased by faster contact tracing. the timely identification of contacts may be a significant challenge in the quarantine approach given that the incubation time can be as short as 2 days in 25% of cases [3] . as mentioned by other authors [28] , the delays in manual contact tracing are usually at least 3 days and under such circumstances the contact tracing and quarantine alone may be insufficient to control the epidemic. this could be improved with digital contact tracing. notably, mixed contact tracing strategies implemented in south korea indeed helped to control the epidemic at the early stages [32] . the use of "smart contact tracing" with mobile phone location data and administrative databases were also key to rapid identification and self-quarantine of contacts in taiwan [33] and implementation of such strategy helped singapore to control the epidemic without major disruptions of social activities [34] . we note that the quarantine effect relates only to transmission due to diagnosed cases. as expected, in order to control the epidemic the transmission due to undiagnosed cases has to be negligible. this can be controlled by general measures such as lockdown, which universally decrease the frequency of social contacts and are therefore likely to reduce β u . in our model the part of r representing transmission due to undiagnosed cases is scaled by (1 − κ) , the parameter relating to the efficiency of the testing system. again, the examples of singapore as well as the italian village of vo'euganeo show that the widespread testing complementing the efficient contact tracing was essential to suppress epidemic. testing unrelated to epidemiological links decreases (1 − κ) factor, thus making the factors impacting transmission due to diagnosed cases, such as quarantine, more powerful to decrease r. further, our model allows to study the effect of the situation, in which the contact tracing capacities are exceeded. in this situation the epidemic is likely to quickly develop to the levels observed without quarantine. it is therefore quite crucial to implement the aggressive contact tracing system, when the epidemic is still at low levels and it is possible to bring the epidemic to suppression phase. we demonstrate the high impact of contact tracing and quarantine on the observed numbers of cases in poland. this effect was coupled with substantial reduction in the transmission parameter β resulting from social contact restrictions. depending on the scenario, β decreased by 76% to 84%, bringing r below 1. the estimated effect of the quarantine in poland would depend on which of the considered scenarios regarding testing efficiency was the most relevant to our situation. in our model the quarantine is estimated to be the most effective for the scenario in which most of the cases are diagnosed (κ = 0.8). testing strategies that comprise testing of all individuals with symptoms of respiratory illness could theoretically identify up to 82% of infected, assuming they would all present to medical care. this could be coupled with random screening of high risk individuals, in e.g. health care workers, or -in case of high incidence -even random screening of entire community to achieve the κ of the order of 0.8. the polish clinical recommendations specifically mention only testing all individuals with severe infections [35] . in addition testing is provided to health care workers. the severe course corresponds to approximately 18% of all infections [3] . therefore, the κ = 0.8 scenario is unlikely to be realistic in poland. we believe that the plausible current κ in our country lies between 0.2 and 0.5. for these scenarios the model shows that the control of the epidemic is largely achieved through suppression of β. in case of relaxation of social contact restrictions, the efforts should be focused on increasing the level of testing in order to decrease the proportion of undiagnosed cases as well as maintaining or increasing the effectiveness of quarantine. for smaller κ, even substantially increasing the effectiveness of quarantine does not allow to go back to the level of social contacts from before the epidemic (β 1 ). finally, the contact tracing effort was manageable in poland due to relatively small number of cases. should the case load increase substantially longer delays in contact tracing would occur, which can substantially decrease the effects of quarantine [27, 28] . limitations and future directions of research. we do not consider the likely reduced transmission from undiagnosed cases who are more likely to be asymptomatic or paucisymtopmatic cases (β u < β d ). the reduction factor for infectiousness of asymptomatic is still under investigation. one study found a 60-fold lower viral loads in asymptomatic cases [29] , but another estimated the transmissibility reduction by 50% [6] . moreover, we did not have sufficient data to include this additional parameter. we calibrated our model only to diagnosed cases, which were officially available. calibration to mortality data is another approach successfully implemented in e.g. [25] that potentially removes bias due to different testing policies. as there were relatively fewer fatalities in poland and little data on clinical progression we decided on simplified model without explicit modelling of the outcomes. furthermore, we did not consider the sub-optimal adherence to quarantine. it is likely that some individuals would not fully comply to strict quarantine rules. however, only anecdotal evidence for such phenomenon is available at this time. in our model it would decrease the effective αθ, which was chosen to fit to observed number of people put in quarantine. finally, the analysis of r is suitable for small size of epidemic, when s ≈ n . for other cases the results are still useful, but the approximation may be biased, as we have shown for β * . due to little available data and policy changes we did not have sufficient data to determine which κ scenario is the most appropriate. in conclusion we present a simple model, which allows to understand the effects of testing, contact tracing and quarantining of the contacts. we apply the model to the data in poland and we show that despite a substantial impact of contact tracing and quarantine, it is unlikely that the control of the epidemic could be achieved without any reduction of social contacts. [ a.1 optimization algorithm. in order to fit the values β 1 , β 2 , β 3 we use a standard gradient descent algorithm. namely, we define error function as where {r d (κ, t)} 54 t=1 is the vector of computed values of r d and {data(t)} 54 t=1 the vector of data (cumulative number of confirmed cases). at each step we approximate the gradient of the error function with respect to β 1 , β 2 , β 3 by differential quotients and move in the direction opposite to the gradient. the algorithm reveals a good performance provided we start sufficiently close to the minimum, which is not difficult to ensure in our case. it remains to choose the initial data. a closer look on results of simulations shows that the choice of initial data mostly influence the fitting in the beginning of period under consideration and hence the value of β 1 , while for analysis of future scenarios β 3 is the most important. taking all this into account we do not struggle for sharp optimization of data fitting with respect to initial data and restrict to the following heuristic choice. it is natural to assume i u (0) = 1−κ κ i d (0). concerning the choice of e(0) we assume it in a form e(0) = m(i d (0) + i u (0)). we set initial values i d (0) ∈ {10, 20, 30} and for each value we set i u (0) according to the above formula and three values of e(0) corresponding to m ∈ {2, 3, 4}. for each of these 9 combinations we run the optimization algorithm looking for the best fit of β i , i = 1 . . . 3. we have repeated this approach for κ ∈ {0.2, 0.5, 0.8}. it turns in that for all values of κ the best fit was obtained for i d (0) = 20 and m = 2. more careful analysis around i d (0) = 20 did not improve the quality of fitting, therefore: a.2 choice of fixed parameters 1. the parameter σ describes the rate of transition from non-infectious incubation state e into the infectious states i d or i u . the median incubation time from exposure till the onset of symptoms was estimated at 4 to 5 days [3, 4, 5] . however, there exists evidence that typically infectivity preceeds symptoms, by 1 to 3 days [7, 9, 10] . a modelling study identified the rate of transition between the non-infectious and infectious states at 1 3.69 [6] , which corresponds to an average time lag of 3.69 days untill the case becomes infectious. 2. the parameter γ u represents the period of infectivity during the natural course of disease. we discuss the period of infectivity, especially as applied to mild cases. the median duration of viral shedding was estimated among 113 chinese hospitalized patients. overall it was 17 days, but it was shorter among cases with milder clinical course [16] . a study among 23 patients in hong kong confirmed viral shedding longer than 20 days among a third of patients, although the peak level of shedding was noted during the first week of infection [17] . in the mission report from china who reports viral shedding in mild and moderate cases to last 7 -12 days from symptom onset. among younger and asymptomatic or mild cases the shedding may be shorter: in a study among 24 initially asymptomatic youngsters the median duration was 9.5 days [11] . 3. the value of κ generally depends on the testing policy. however, recommended testing policies often rely on the presence of respiratory symptoms. this is also the case in poland. it was observed that some infected people never develop symptoms, although the precised rate of such truly asymptomatic infections is still under investigation. some studies may be biased by a too short follow-up time. a small study among residents of a long-term care skilled nursing facility found that even though more than half of individuals with confirmed infection were asymptomatic at the time of test, majority of them subsequently developed symptoms. the proportion of people who remain asymptomatic may be higher among younger individuals [hu] . a study among japanese nationals repatriated from wuhan suggests the proportion of asymptomatic infections is about 30% [13] . an analysis among the passengers of diamond princess ship, where a covid-19 outbreak occurred, taking into account this delayed onset of symptoms estimated the proportion of asymptomatic infections to be about 18%, even though almost 50% were asymptomatic on initial test. in addition, large scale screening implemented in italian village vo'euganeo indicated that 50% to 75% of infected individuals did not report symptoms [14] . similarly, in population screening in iceland 50% were asymptomatic at the time of screening [15] . it may be stipulated that some of the people diagnosed through screening developed symptoms latter, consistently with the findings from the diamond princess study. on the other hand a sizable proportion of infected people, especially at younger ages, experience only mild symptoms, for which they may not seek medical attention. in the study of li [6] , the proportion of undocumented cases was estimated as 86%. 4. the parameter γ d was estimated basing on a sample of case-based data available in routine surveillance, by fitting gamma distribution to the time from onset to diagnosis, for cases who were not in quarantine before diagnosis. time from onset to diagnosis was estimated based on surveillance data available in the epidemiological reports registration system for covid-19, as of 28.04.2020.the system collects epidemiological data on cases diagnosed in poland and is operated by local public health departments. all cases eventually are entered into the database. however, substantial reporting delays are noted. there were altogether 4976 cases registered in the system, including 1995 (40.1%), who did not have symptoms at the time of diagnosis. plausible onset date and plausible diagnosis date were available for 2884 cases ( 96.7% of 2981 cases that were not asymptomatic) gamma distribution was fitted by maximum likelihood to cases who were not diagnosed in quarantine. the observed and fitted distributions are shown below (figure a.1). we next fitted gamma-regression model with week of diagnosis as an explanatory variable. we found no significant trend in time. we therefore adopted the average time from onset to diagnosis to be 4.6 days, and taking into account the probability of asymptomatic spread we assumed the parameter γ d to be 1/5.5. 5. next we base θ on available data. we calculate prevalence of infection among the quarantined individuals, according to data published by the chief sanitary inspectorate on the number of cases diagnosed among quarantined people and the total number of quarantined. we used a series of data 8.04 -20.04 to estimate a likely value of θ. we chose this time period due to data availability. data are shown on the figure below. during this time period there was an increasing trend in the proportion of diagnosed from 0.5% to 0.8% a.2. we presume that this parameter could change with changing procedures of contact tracing and testing. however, since no detailed data were available, for the modelling purposes we chose a simplifying assumption that θ is stable (i.e. we always take a similar group of contacts under quarantine) selecting an average value of 0.6%. this proportion could be also viewed as attack rate among the contacts of cases. the proportion in poland is in line with what was observed in korea, where an estimated attack rate was 0.55% [32] , although household attack rate was higher (> 19%) in other studies [20] . 6. furthermore we fix the parameter α. here we make another simplification assuming this parameter to be constant. the main difficulty is a lack of precise data concerning the number of newly quarantined people per day, distinguishing between reasons of quarantine (travel related or contact tracing relate). at the beginning of epidemic in poland the average amount of quarantined following one diagnosed case was definitely higher. moreover, people coming back from abroad were subject to obligatory quarantine starting from march 16 and constituted a considerable part of quarantined in the second half of march and beginning of april. in particular, around 54 000 polish citizens staying abroad came back within a special program of charter flights operated by polish airlines which ended on april 5. we can assume that after this date the ratio of people coming from abroad among all people subject to quarantine was negligible. as our model does not take migration into account, we have to take into account only quarantine from contact. for above reasons, for fitting α we restrict our analysis only to a period of 2 weeks of april. assuming already θ = 0.006 we then choose α minimizing the square error between the number of quarantined from the data and computed k(t). this way we obtain α = 75. taking these into consideration we set the values of parameters collected in table 2.1. bootstrap. to estimate confidence intervals we use a method of parametric bootstrap. we generate m = 200 sequences of perturbed data assuming that for each time t ∈ {1, 54} the increment of r (i.e. daily number of new diagnoses) is a random number from poisson distribution with mean value equal to increment of observed data. model parameters are also perturbed, see below. for each series of perturbed data we estimate the values of β i and take estimated confidence intervals as appropriate quantiles of obtained sets. in order to estimate confidence intervals for r d (t), shown on panel a of figure 3 .1, we proceed as follows. for each sequence of perturbed data we compute fitted r d (t). this way we obtain a set of curves {r distribution of parameters. following other authors [30] as well as experimental data, for the uncertainty analysis we used the following distributions of the parameters. 1. 1/γ d ∼ gamma(a 1 , b 1 ), where the shape parameter, a 1 = 1.05 and scale parameter b 1 = 5.23 2. 1/γ u ∼ gamma(a 2 , b 2 ), where the shape parameter, a 2 = 2 and scale parameter b 2 = 5 3. 1/σ ∼ gamma(a 3 , b 3 ), where the shape parameter, a 2 = 2 and scale parameter b 2 = 1.75 4. α ∼ p oisson(α 0 ), where we assume constant α 0 = 75; 5. θ -is not sampled for the uncertainty analysis. as the results depend on the quantity αθ, we rely on the distribution of α. we take n = 200 (approximate average of daily number of diagnosed cases from the data). we approximate the mean value of n samples from gamma distribution using central limit theorem. namely, we generate based on the classical approach to epidemiological models we address the basic question concerning the propagation of the disease. namely, how many persons are infected by one infectious individual, a quantity which is usually called reproductive number, r. in order to compute this quantity we use the approach from [23] . we look at the system assuming s ∼ n and e, i d , i u are close to zero, then we consider the following linearizatioṅ then one deduces (see [23] ) that if we define r = max{eigenvalues of t σ −1 } then the system is stable for r < 1 and it is unstable for r > 1. stability of system (a.5) means that the whole vector (e, i d , i u ) is going to zero, it follows that the main system (2.1) also tends to the zero solution for (e, i d , i u ). instability implies that for "almost all" small data, the vector (e, i d , i u ) is growing in time (exponentially fast), causing the nonlinear system also evolves far away from the trivial state, i.e. e, i d , i u rapidly grow. by (a.4) we have hence the stability of our system is determined by the following factor: to make a final comment, let us note that in case of spread of pandemia, as r d , r u grow, the above analysis become less reliable. recall that β is normalized by n , so as s/n is not close to one and the analysis of stability becomes more complex. this behavior is illustrated by figures 3.3, 3.4 and 3.5, where we observe deviation from predictions based on (2.2), covid-19) in the eu/eea and the uk -ninth update an investigation of transmission control measures 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determinants medrxiv 2020.04.11 the construction of next-generation matrices for compartmental epidemic models public health interventions to mitigate early spread of sars-cov-2 in poland estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries.imperial college london modelling the covid-19 epidemic and implementation of population-wide interventions in italy feasibility of controlling covid-19 outbreaks by isolation of cases and contacts quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing viral dynamics in mild and severe cases of covid-19 centre for mathematical modelling of infectious diseases covid-19 working group. early dynamics of transmission and control of covid-19: a mathematical modelling study contact tracing for covid-19: current evidence, options for scale-up and an assessment of resources needed epidemiology and case management team contact investigations of the first 30 cases in the republic of korea. osong public health res perspect containing covid-19 among 627,386 persons in contact with the diamond princess cruise ship passengers who disembarked in taiwan: big data analytics evaluation of the effectiveness of surveillance and containment measures for the first 100 patients with covid-19 in singapore acknowledgments. this work was partially supported by the polish national science centre's grant no2018/30/m/st1/00340 (harmonia). key: cord-221717-h1h2vd3r authors: scabini, leonardo f. s.; ribas, lucas c.; neiva, mariane b.; junior, altamir g. b.; farf'an, alex j. f.; bruno, odemir m. title: social interaction layers in complex networks for the dynamical epidemic modeling of covid-19 in brazil date: 2020-05-16 journal: nan doi: nan sha: doc_id: 221717 cord_uid: h1h2vd3r we are currently living in a state of uncertainty due to the pandemic caused by the sars-cov-2 virus. there are several factors involved in the epidemic spreading such as the individual characteristics of each city/country. the true shape of the epidemic dynamics is a large, complex system such as most of the social systems. in this context, complex networks are a great candidate to analyze these systems due to their ability to tackle structural and dynamical properties. therefore this study presents a new approach to model the covid-19 epidemic using a multi-layer complex network, where nodes represent people, edges are social contacts, and layers represent different social activities. the model improves the traditional sir and it is applied to study the brazilian epidemic by analyzing possible future actions and their consequences. the network is characterized using statistics of infection, death, and hospitalization time. to simulate isolation, social distancing, or precautionary measures we remove layers and/or reduce the intensity of social contacts. results show that even taking various optimistic assumptions, the current isolation levels in brazil still may lead to a critical scenario for the healthcare system and a considerable death toll (average of 149,000). if all activities return to normal, the epidemic growth may suffer a steep increase, and the demand for icu beds may surpass 3 times the country's capacity. this would surely lead to a catastrophic scenario, as our estimation reaches an average of 212,000 deaths even considering that all cases are effectively treated. the increase of isolation (up to a lockdown) shows to be the best option to keep the situation under the healthcare system capacity, aside from ensuring a faster decrease of new case occurrences (months of difference), and a significantly smaller death toll (average of 87,000). although we have experienced several pandemics throughout history, covid-19 is the first major pandemic in the modern era. the last critical global epidemic occurred in 1918 and became known as the spanish flu. but, in 1918, the reality was quite different. scientific and medical knowledge was much more limited, making it difficult to fight the disease. furthermore, the world was not globalized, the means of transport were not as agile as the current ones and the population was much smaller. the 21st century is marked by globalization and an intricate and intense social network, which connects in one way or another to everyone on the planet. the latter fact increases the danger that a local epidemic disease will rapidly evolve into a pandemic like what happened in wuhan, china, and now is all over the world. the form of propagation and contagion of the sars-cov-2 virus occurs by direct contact between individuals, through secretions, saliva, and especially by droplets expelled during breathing, speeching, coughing, or sneezing. the virus also spreads by indirect contact, when such secretions reach surfaces, food, and objects [41] . besides, infected people take a few days to manifest symptoms, which can be severe or as mild as a simple cold. there is even a large proportion of infected people who remain asymptomatic [37] . this makes it practically impossible to quickly identify the infected and apply effective measures to limit the spread of the disease. also, sars-cov-2 was discovered in december 2019, which makes it very recently in the face of the current epidemic. little is known about the covid-19 disease, which appears to be highly lethal, with no drugs to prevent or treat. the concern is greater since direct (individual -individual) and indirect (individual -objects -individual) social relations are the means of spreading the disease. thus, the social interaction structure is the key to create strategies and guide health organizations and governments to take appropriate actions to combat the disease. one of the main concerns is overloading the health system. the first case in brazil was confirmed on february 26, a 61-year-old man who traveled to the lombardy region in northern italy. now, in the middle of may, there are more than 200,000 cases and 14,000 deaths in all states of brazil [30] . the concern is even worse due to the country's social inequality, over 80% of the population relies solely on the public health system and this distribution is not uniform. according to [11] , there are only 9 hospital beds per 100,000 people in the north region while southeast accounts for 21 hospital beds. the treatment of severe cases requires the use of respirators/ventilation in intensive care units (icu), and if simultaneous infections occur there will be no beds to meet the demand and a possibly large number of victims. thus, it is urgent to develop models and analyses to try to predict the evolution of the virus. also, as noted in figure 1 , brazil is running towards being the next epicenter of the pandemic. it has already exceeded the number of cases in important countries such as germany, china, japan, italy, iran, south korea, and france (the rates consider the population size of each country and are on a logarithmic scale). [24] ). it is possible to notice that brazil is surpassing countries such as italy, south korea, japan, and china, and it is reaching the relative number of cases in the united kingdom and france. as of the date of this study, the united states is the epicenter of the pandemic. since covid-19 presents a unique and unprecedented situation, this work proposes a specific model for the current pandemic. based on the classic epidemic model sir, also extended to sid [35] , siasd [9] and siqr [14] , we propose a more realistic model to better represent the effects of the covid-19 disease by adding more infection states. the proposed approach also considers social structures and demographic data for complex network modeling. each individual is represented as a node and edges represent social interaction between them. the multi-layer structure is implemented by different edges representing specific social activities: home, work, transports, schools, religious activities, and random contacts. the probability of contagion is composed of a dynamic term, which depends on the circumstances of the social activity considered, and a global scaling factor β for controlling characteristics such as isolation, preventive measures, and social distancing. the proposed model can be used to analyze any society given sufficient demographic data, such as medium/big cities, countries, or regions. here we analyze in depth the brazilian data. the sir model is applied through the network using an agent model, and each iteration of the system is simulated using the 24-hour pattern, allowing us to understand the dynamics of the disease throughout the days. the results show the importance of social distancing recommendations to flatten the curve of infected people over time. this is currently maybe the only way to avoid a collapse of the health system in the country. the paper is divided as follows: section 2 presents important concepts about complex networks, the sir model and its applications. section 3 explains our proposed approach and sections 4 and 5 presents the results, discussion, and conclusions of the work. created from a mixture of graph theory, physics, and statistics, complex networks (cn) are capable to analyze not only the elements themselves but also their environment to find patterns and obtain information about the dynamics of a system. as most of the natural structures are composed of connected elements, graphs are suitable to analyze most of the real-world phenomena. over the past two decades researchers have been showing that many real networks do not present a random structure, and its emergent patterns can be used to understand and characterize a model [8, 39] . complex network analysis has then been applied to sociology, physics, nanotechnology, neuroscience, biology, among other areas [12, 13] . to start with a formal definition, a graph g is a set {v,e} where v is composed by n vertices (also known as nodes or elements) {v i , v n } and e is the set e(v i , v j ) of edges (or connections) among its elements. edges represents the relationships between two elements and its value can also represent the strength or weight of a connection if usually, applications with complex networks consist of two main steps: i) transform the real structure into a complex network, and ii) analyze the model and extract its features or understand its dynamics. one natural phenomenon that has a straight forward connection to a complex network in society. people are connected due to several aspects such as members of a family, religious groups, co-workers, members of the same school, or faculty, among other social relationships. therefore cns have been widely employed for social network analysis [38] . extended from social interactions, the epidemic spread has also been studied by researchers in the last decades. in this context, one of the best known and widely used epidemic models in infectious diseases is the susceptible-infectedrecovered (sir) model, which is composed of three categories of individuals [4, 7] • susceptible: the ones who are not infected but could change its status to a state to infected if in contact with a sick person combined with a probability β of contagion • infected: the ones that have the disease • recovered: usually after some time, a person recovers from the illness and it is not able to be infected again due to the immunity process (in this case, this is an assumption of the process). the recovery rate of infected people is aligned with a probability of γ also, the model can be described as where s, i and r represents the ratio of susceptible, infected and recovered people in the population, respectively. usually, the problem is solved with differential equations, however, agent-based techniques in networks can represent the nature of the spread of viral diseases in a more complex scenario. if a network is fully connected, meaning that e(v i , v j ) = {1, ∀ i,j 0 < i, j <= n }, equation 2 fits the structure perfectly. however, in the real world, not everyone is connected and people only contract the disease if in contact with an infected individual or object. this is why a complex network approximates the dynamics of real viruses and can help us to understand the disease behavior. there are various approaches to represent people and society as networks, named social network analysis. small world networks [32] can be used as a good approximation of the social connections. in 2000, moore [32] emphasized that the use of small-world networks, where the distance among two elements is usually small in comparison to the size of the population, showed a faster spread of the viral disease than classical diffusion methods. the approximation of real social phenomena was first explained by milgram [29] in [29] , the sociologist is the author of the well-known idea that there are up to six people separating any two individuals in the world, which reinforces the importance of analyzing the epidemic spread from a graph view. in [33] , the authors used small-world networks to simulate a sir model, however, they considered that every contact with an infected person resulted in contamination, which is not realistic. therefore, other researchers improved the model over the years, adding new constraints to approximate the simulation to real scenarios [15] . the sir model on networks works as follows: each node represents a person and, the elements are connected according to some criteria and the epidemic propagation happens through an agent-based approach. it starts from a random node, and for each time step nodes with the susceptible state can contract the disease from a linked infected node with a predefined probability. the same idea occurs with the recovered category. after a certain period, a node can recover or can be removed from the system (case of death) according to a certain probability. at the end of the evolution of a sir model applied to a network, the number of nodes in each sir category (susceptible, infected and recovered) can be calculated for each unit of time evaluated and then compare these data with real information, for example, the hospital capabilities of the health system. also, the probability of infection and recovery can be adjusted over time considering social distancing, hygiene, and health conditions. the proposed model extends the sir model to a more realistic scenario to achieve a better correlation to the covid-19 disease, since the model was created specifically for the disease, we named the model as complexvid-19. our strategy is based on a multi-layer network to represent the brazilian demography and its different characteristics of social relationships. each layer is composed of a set of groups representing how people interact in a given social context. in the network, a node represents a person and the edges are the social relationships between persons, and they are also the means through which the disease can be transmitted. the virus spreads from an infected node to neighboring nodes at each iteration step (1 step = 1 day), according to a given infection probability. first, we describe how the layers are built based on social data from brazil. to define the different social relations, the first information needed is the age distribution so that groups such as schools and work can be separated. we consider the brazilian age distribution in relation to the total population in 2019 [20] , details are given on table 1 . this distribution is used to define an age group for each node, which is then used to determine its social activities through the creation of edges on different layers. in this approach, each network-layer represents a kind of social relationship or activity that influences the transmission of the covid-19. in this way, it is possible to evaluate and understand what is the impact of each social activity in the epidemic propagation. basically, in this work, a network layer is represented by a set of edges connecting some nodes. the following social activities are considered, composing 6 different layers: • home: in this layer, all people that live in the same residence are connected. • work: connects people that work in the same environment/company. • transport: this layer represents people that eventually take the same vehicle at public transports. • school: represents the social contact of students that belong to the same school class. • religious activities: connects people of the same group of some religious activity. • random: this layer represents activities of smaller intensity, such as indirect contact (through objects/surfaces). the first layer represents home interactions and is composed of a set of groups with varying size which are fully connected internally. these groups have no external connections, i.e. the network starts with disconnected components representing each family. to create each group, we consider the brazilian family size distribution for 2010 [19] , the year with more detailed information on family sizes from 1 up to 14 members. we consider the probability of a family having sizes from 1 to 10, therefore the probability of a family having 10 persons is the sum of the higher sizes, the details of this distribution are given in table 1 . the first layer is then created following the family size distribution and ensuring that each family has at least 1 adult. figure 2 (a) shows the structure of such a layer built for a population of n = 100. a large fraction of the population in any country needs to work or practice some kind of economic activity, which also means interacting with other people. thus, work represents one of the most important factors of social relations, which is also very important in an epidemic scenario. to represent the work activity we propose a generic layer to connect people with ages from 18 to 59 years, i.e. 60% of the total population in the case of brazil. there is a wide variety of jobs and companies, therefore it is not trivial to create a connection rule that precisely reflects the real world. here, we consider an average scenario with random groups of sizes around [5, 30] , uniformly distributed, and internally connected (such as the "home" layer). an example of this layer is shown on figure 2 (b), using n = 100. although the nodes of a group are fully connected, the transmission of the virus depends directly on the edge weights, which we discuss in-depth on section 3.1.1. collective transports are essential in most cities, however, it is one of the most crowded environments and plays an important role in an epidemic scenario also due to the possibility of geographical spread, as vehicles are constantly moving around. the third layer we propose represents this kind of transports, such as public transports, and includes people that do not possess or use a personal vehicle. in brazil the number of people using public transport depends on the size of the city, with 64.98% in the capitals and 35.89% in other cities [23] , with an average use of around 1.2 hours a day 1 . here we consider the average of the population between the two cases (50%), randomly sampled, to participate in the "transports" layer. random groups are created with sizes between [10, 40] , uniformly sampled, and the nodes within each group are fully connected. this variation of sizes is considered to represent cases such as low and high commuting times, and also the differences between vehicle sizes. other factors such as agglomeration and contact intensity are discussed in section 3.1.1. this layer is illustrated on figure 2 (c). schools are another environment of great risk for epidemic propagation. the proposed layer considers the characteristics of schools from primary to high school and how children interact. we consider that all persons from 0 to 17 years (24% of the brazilian population) participate in this layer, and the size of the groups, which represents different school classes, varies uniformly between [16, 30] [21]. this layer is illustrated on figure 2 (d). brazil is a very religious country, in which by 2010 only around 16.2% of the population claimed not to belong to any religion [18] . 64.6% claimed to be catholic and 22.2% to be protestant, summing up to 86.8% of the total population. here we consider that nearly half of these people (40% of the total population) actively participate in religious activities (weekly). the distribution of religious temple sizes is defined as a pareto distribution in the interval [10, 100] . taking into account that wage distribution follows the pareto distribution approximately, we model real estate predominance according to their capacity. the assumption here is that building costs (for churches, offices, homes, etc.) have a linear relationship to their internal capacity, and thus any given capacity has a power-law relationship with the number of such buildings within a region. we consider a random layer to represent all kinds of contacts not related to the specific previous social layers. this includes small direct contacts (person-to-person) and indirect contacts (individual -objects -individual) that may happen throughout the week, such as random friend/neighbor meetings, shopping, and other activities that involve surface contacts. for that 5n new random edges are created, that can connect any node. on the one hand, this yields an average of 5 random connections to each node, which can randomly connect any other node. on the other hand, the impact of this layer on the epidemic is smaller than the others, as it represents rapid contacts in comparison to the other activities described, thus its infection probability is smaller. in the following section we discuss the details concerning this aspect, deriving from the edge weights of each layer. in figure 2 (f) an example of this layer is shown. the overall structure of social interactions in our model can be compared to the statistical analysis in [16] , however here we introduce a more detailed model of social contacts with specific layers and connection patterns to better fit the particularities of a given country or city. unlike the traditional sir model, which consists of a single β term to describe the probability of infection, here we propose a dynamic strategy to better represent the real world and the new covid-19 disease. the idea is to incorporate important characteristics in the context of epidemic propagation according to each layer. firstly, to a given layer a fixed probability term is calculated to represent its characteristic of social interaction. for this, we considered 3 local terms: the contact time per week, the average number of people close to each other (agglomeration level), and the total number of people involved in the respective activity. considering two nodes v x and v y , connected at group j of layer i, its edge weight is then defined by where t i represents the average weekly contact time on layer i, k i is the agglomeration level (average number of nearby people) and n ij represents the size of the group j in which the nodes participates on layer i. the first fraction represents the contact time normalized by the total time of the week (24 * 7 = 168), and the second fraction represents the proportion among the local people closest to the total number of people on that activity group. the first part of the infection probability equation is multiplied by a β term, which scales the original probability. the β term is then the only parameter to tune the infection rates for the entire network, and the other properties are specific for the studied society, based on its population characteristics and the nature of the activities (layers). table 2 shows these specific properties that we considered for the brazilian population, and how the infection probabilities are calculated for each layer. in the table, we have the following information: who or how many people are part of the activity represented by a layer (column "who", discussed in the previous section); contact time according to activity (column "time of contact"); the average number of people close to each other in each activity (column "nearest", represents the agglomeration level); the number of connections between people (column "group size"); the probability of infection (column "probability"). • susceptible: traditional case, it means that a person can be infected at any time. this is the initial state of every node. • infected -asymptomatic: people who do not show any symptoms (30% of the total cases of infection) and remain contagious for up to 18 days (they may recover after 8 days). this is the most dangerous case for the epidemic spreading because the person is not aware of its infection. • infected -mild: 55% of the cases, present mild and moderated symptoms with no need for hospitalization, remain contagious for up to 20 days, and may recover after 10 days of infection. • infected -severe: 10% of the cases, present strong symptoms, and need hospitalization, remain contagious for up to 25 days. has a death rate of 15% and may recover after 20 days. • infected -critical: present worst symptoms and remain contagious for up to 25 days, need icu and ventilation, have a death rate of 50% and may recover after 21 days. • recovered: people who went through one of the infection cases and overcame the disease, ceasing to contaminate and supposedly becoming immune. these nodes no longer interact with other nodes anymore and are therefore removed from the network. • dead: people who went through severe or critical cases and eventually died. these nodes are also removed from the network. estimates for the proportion of asymptomatic cases vary from 18% (95% confidence, [15.5, 20 .2%]) [31] to 34% (95% confidence, [8.3, 58 .3%]) [17] . considering the confidence intervals, here we roughly approximate it to an average of 30% of the total number of infected cases. however, it is very difficult to study asymptomatic cases due to several reasons, such as the lack of available tests and the difficulty in identifying potential cases, which would include every person who had contact with known symptomatic cases. some studies indicate that asymptomatic cases may remain contagious for up to 25 days, with an incubation period of 19 days [6] , but the viral load may be smaller at the end of the infection. here we take an optimistic approach considering that they may recover (become immune and cease to contaminate) uniformly after 8 days of infection, up to around 18 days. as for the recovered nodes, we are considering that people become immune or at least acquire a long-term resistance to the virus, up to a maximum of 300 days (limit of our simulations). however, this should be taken cautiously as these properties are not yet fully understood [26] . the infection grows through the contact (edges) between infected and susceptible nodes, and the probability of being infected is the edge weight. if infection occurs, then one of the 4 infection cases are chosen based on the probability described above (30%, 55%, 10% and 5%). this distribution plays an important role in the structure and dynamics of the network. the node structure of asymptomatic cases does not change during the simulation, except for the time it takes to cease contamination and recover. it means that as these persons are not aware of their contamination, they will remain acting normally on the network (according to the active layers and edge weights). their contagious time varies from 1 to 18 days after infection. concerning the other cases (mild, severe, and critical), we consider the incubation time of the virus, the recovery time, the contagion time, the death rates of each case, and the usual action taken by the infected person or health professionals at hospitals. various works [5, 27, 28] point out that the average incubation period of covid-19 is around 5 days, but some cases may take much less or more time. the official who report [40] states that the average incubation time is around 5 to 6 days, with cases up to 14 days. the results in [27] show that the average shape of the incubation time follows a log-normal distribution (weibull distribution) with an average of 6.4 days and a standard deviation of 2.3 days. in this context, we consider the day when an infected person begins to show symptoms by randomly sampling from this distribution (1000 repetitions), with cases varying from 2 to 14 days. for mild cases, the nodes are isolated at home, maintaining the connections of the first layer, and then only 20% of the cases are diagnosed. considering the ratio of diagnosed cases, patients who are asymptomatic or with mild symptoms of covid-19 may not seek health care, which leads to the underestimation of the burden of covid-19 [25] . moreover, our diagnosis rule is also based on the fact that ongoing tests in brazil are increasing more slowly than in most european countries and the usa (tests are being performed mostly on people that need hospitalization). if a given case is severe or critical, the patient goes to a hospital and is fully isolated, i.e. we remove all of its connections. this is a rather optimistic assumption, considering that these patients still may infect the hospital staff. concerning the time that patients usually stay at hospitalization/icu, the works [10, 44] points to an average of 14 days for all cases. for standard hospitalization, we considered a minimum of 6 days and a maximum of 16 days of stay, and for the icu/ventilation, a minimum of 7 and a maximum of 17 days of stay. the time of each case will depend on the day the symptoms start and the day of recovering/death. figure 3 illustrates all the infected states and mechanisms described here. this configuration results in an overall lethality of 4%. it is important to stress that here we consider a maximum of 25 days of infection time, which is the time frame based on most studies we have seen so far in the literature. we are still at the beginning of the pandemic and a better characterization of the long-term impact is very difficult. nonetheless, the available information allows to represent the most obvious features of the sars-cov-2 virus and to evaluate its main impacts on society. to simulate the reduction or increase of social distancing/quarantine, we remove/include some layers of the network, or change their edge weights. similarly to the approach on [16] to improve home contact when in quarantine, we increase the home layer edge weights by 20% for each removed layer. to balance that we considered a smaller number of hours of contact in the base calculation for the home layer (3 hours a day), also taking into consideration that this layer has full contact between people of the same family. when the home contacts are increased according to our approach of layer removal, the time/intensity of contacts may increase up to its double. for each experiment with the proposed model, we consider the average and standard deviation (error) of 100 random repetitions to extract statistics of infection, death, and hospitalization time. due to the random nature of these networks, it is possible that extreme cases occur within the repetitions, i.e. when the infection starts at a node that is not capable of further propagation, leading the epidemic to end at few iterations. considering the real data we know that this is not the case, at least not for brazil, therefore we manually remove these networks and they are not considered for the average/error calculations. it is important to notice, however, that this rarely happens, in all our experiments we noticed a maximum of 4 networks of this kind. due to time and hardware constraints, our simulation considers 100,000 nodes, and the results need to be scaled up by a factor of 57 to match the brazilian population statistics. this factor was empirically found by approximating the model results in the number of reported cases in brazil. it is important to stress that for better statistics it should be considered the largest possible number of nodes to represent a population, i.e. the ideal case would be n = total country/city population. however, the computational cost of the simulation grows directly proportional to the number of nodes and edges of the network, and considering the critical situation of the moment at hand, 100,000 nodes are our limit to promptly present results of the epidemic dynamics. in the experiments when varying the social distancing, the same network is considered in each iteration, i.e. comparisons of including/excluding layers are made in the same random network. we considered the epidemic began on february 26, which is the day the first confirmed case was officially reported. it is important to emphasize that we made various optimistic assumptions throughout the model construction and simulation, such as to consider that people are behaving with more caution by reducing direct contact, wearing masks, and doing proper home/hospital isolation when infected. it is also important to notice that we are not considering the number of available icu/regular hospitalization beds for the death count, i.e. all the critical and severe cases are effectively treated. it is not trivial to estimate the direct impact of these numbers on the epidemic, however, this is an essential factor that directly impacts the number of deaths. here we focus on the impacts of different actions on the overall epidemic picture, such as the increase and reduction of cases, deaths, and occupied beds in hospitals. the social network starts normally, with all its layers and the original infection probabilities. the infection starts at a node with the closest degree to the average network degree and propagates at iterations of 1 day (up to 300 days). we consider an optimistic scenario, in which people are aware of the virus since the beginning, thus the initial infection probability is β = 0.3. this represents a natural social distancing, a reduction of direct contacts that could cause infection (hugs, kisses, and handshakes), and also precautions when sneezing, coughing, etc. we empirically found that this initial value of β yields results with a higher correlation to the brazilian pandemic. a moderated quarantine is applied after 27 days, representing the isolation measures applied on march 24 by most brazilian states, such as são paulo [1]. to simulate this quarantine we remove the layers of religious activities and schools and reduce the contacts on transports and work down to 30% of its initial value, i.e. β = 0.09. the remaining activities on these layers represent services that could not be stopped, such as essential services, activities that are kept taking higher precautionary measures, and also those who disrespect the quarantine. we compare the output of the model in the first 83 days with real data available from the brazilian epidemic (up to may 18) [24, 34, 42, 43] . the model achieves a significant overall similarity within its standard deviation. the greatest difference in the number of diagnosed cases at the last 10 days may be related to the increase in the number of tests being performed in brazil, or yet, the constant decrease of isolation levels in the country (below 50% for most days of the past month) [22] . we considered here a fixed isolation level around what was observed in the first days after the government decrees in brazil, but data in ref. shows that these levels are constantly changing. therefore, the number of diagnosed cases and deaths for the remaining simulation may be greater than the reported on this paper (see the "keep isolation" scenario in the next section). concerning the daily death toll, the average number of the proposed model is greater than the official numbers. this is somehow expected, considering that the underdetection rates may be greater in contrast to the fewer number of tests being performed. to better understand this, we analyzed the number of death in brazil from january 1 to april 30, comparing cases between 2019 and 2020, the results are shown in figure 5 . it is possible to observe a clear increasing pattern after february 26, which is the day of the first officially confirmed case of covid-19 in brazil. this indicates that the real death toll for the disease may be significantly greater than the official numbers. [24] , and world health organization (who) [42] . the dotted lines represent the standard deviation, in the case of the real data the curve is the average over a 5-day window, and the solid lines the real raw data. the greatest average number of deaths produced by the proposed model may be related to underdetection (see figure 5 ). [2] . then the total death difference is compared to the covid-19 records of the who [42] and the brazilian government [2] data. the largest difference that appears right after the first confirmed case may indicate a significant underdetection of covid-19 cases. after the initial epidemic phase, we consider 4 possible actions that can be taken after 90 days (may 26): a) do nothing more, maintaining the current isolation levels; b) stop isolation, returning activities to normal (initial network layers and weights); c) return only work activities, restoring the initial probability of the layer; or d) increase isolation, stopping the remaining activities in the work and transports layers (home and random remains). firstly, we analyze the impacts on the number of daily new cases and deaths, results are shown in figure 6 . as previously mentioned, at the start of the covid-19 pandemic, brazil was performing a fewer number of tests by an order of magnitude, in comparison to other countries with similar epidemic numbers, therefore we considered as diagnosed only the severe and critical cases, which are pronounced subjects for testing, and 20% of the mild cases. the total infection ratio is discussed later. considering keeping the current isolation levels, the peak of daily new cases occurs around 100 days after the first case (june 5), with around 11,000 confirmed cases. after 202 days (september 15), the average daily cases is around 500, and it goes below 100 daily cases after around 237 days (october 19). the peak of daily new deaths occurs around 118 days (june 23), with an average of 1900 deaths, and goes below 100 new occurrences after around 210 days (september 24). it is important to stress that this is a hypothetical scenario where the isolation level remains the same from day 27 to 300, which is hardly true in the real world where it is constantly changing [22] . the total numbers after the last day (300) account for 946,830 (±10, 507) diagnosed cases and 149,438 (±3, 124) deaths. when we consider the return of all activities after 90 days, the number of cases and deaths grows significantly in an exponential fashion. the peak occurs at 108 days (june 13) with an average of 40,937 (± 11,010) new cases, and at 122 days (june 27) with an average of 6,484 (± 1,739) new deaths. although the peak of cases/deaths and the decrease of the numbers occur early, in this case, the final result is critically worse, with a total of 1,340,367 (± 18,513) diagnosed cases and 212,105 (± 4,359) deaths. here it is important to notice that we considered that all the activities return after 90 days and remain fully operational until the last day (300). moreover, we do not account for the overloading of hospitals, which directly impacts the final death count. therefore, the number of deaths may be considerably higher. another possible scenario is the return of only the work layer, keeping reduced transports and no schools and religious activities, however, the pattern is similar to returning all activities, considering the growth time, peak, and decay time. the final numbers in this case are 1,253,119 (± 26,009) diagnosed cases and 197,756 (± 5,693) deaths. if the isolation is strictly increased after 90 days (lockdown), the infection and death counts drop significantly in comparison to the other approaches. moreover, the recovering time is much faster, as daily new cases stop earlier than the other scenarios. the peak of daily new cases happens around day 93 (june 1), and of daily new deaths around day 106 (june 11). the total numbers of diagnosed cases and deaths after day 300 are, respectively, 552,855 (± 195,802) and 87,059 (± 30,871). considering the hospitalization time described in the scheme of figure 3 it is possible to estimate the number of occupied beds for regular hospitalization (severe cases) and icu/ventilation (critical cases). we also show the difference between the cumulative growth of diagnosed and undiagnosed cases and recovered cases. the same approach as the previous experiment is considered (except for "return work") with 3 possible actions after 90 days (may 26), results are shown in figure 7 . the overall pattern of results is similar to the previously observed for the number of diagnosed cases and deaths. it is possible to notice that the number of undiagnosed cases is much higher than the diagnosed cases. this reflects the number of asymptomatic cases and the lack of tests for mild cases. in the worst scenario, which means ending the isolation, the total infected number may go above 5 million cases. the recovered rate is directly proportional to the infected rate, as one needs to be infected to either die or become resistant to the disease. if the infected rate is high, so is the recovered rate, e.g. the scenarios of keeping or ending isolation, and a high recovered rate also helps in mitigating the epidemic propagation (natural immunization). however, increasing isolation decreases the propagation much faster than natural immunization, with a considerably smaller death toll. it is also possible to observe the differences at the start of effective recovering, i.e. when the recovered rate surpasses the infected rates, this is due to the early increase in isolation levels. the peak of hospitalization occupancy occurs around a week before the death peaks, in any scenario. in this case, icus are very important because critical patients are treated there, which represents the cases of higher death rates. within the "end isolation" setting, patients may occupy up to an average of 215,285 (± 48,682) regular beds and 109,520 (± 24,647) icu beds. these numbers are by far greater than entire brazil's capacity, as publicly-available and private icu beds sum up to 45,848 [3] . even considering the better scenario, i.e. the lower bound of the standard deviation, the number of occupied icu beds may reach around 86,000, which is also critical for brazil's capacity (almost 2 times it's capacity). in this setting of "end isolation", the healthcare system would surely collapse. when the isolation levels are kept, the numbers are significantly lower. however, the occupancy of 66,110 (± 16,759) regular beds and 33,470 (± 7,926) icu beds is still critical for the brazilian health system. considering the creation of new provisional icu units and good patient logistics, the situation may still remain under control during the peak of hospitalization occupancy. however, the results show that the hospital occupancy is prolonged considerably in this scenario, and they may stay functioning around their maximum capacity for up to a month (with an average of occupied icu beds above 30,000). when increasing the isolation the peak of occupied beds is smaller, with an average of 63,226 (± 20,682) regular beds and 31,816 (± 10,592) icu beds. moreover, the shape of the curve throughout the days is different and the final numbers are considerably smaller. the peak also occurs around a week earlier and then decreases much faster. this scenario would be preferable as it has much more chances of not overloading the brazilian healthcare system, relieving the hospital occupancy considerably faster and, therefore, contributing to the reduction of the number of deaths. this work presents a new approach for the modeling of the covid-19 epidemic dynamics based on multi-layer complex networks. each node represents a person, and edges are social interactions divided into 6 layers: home, work, transports, schools, religions, and random relations. each layer has its own characteristics based on how people usually interact in that activity. the propagation is performed using an agent-based technique, a modification of the sir model, where weights represent the infection probability that varies depending on the layers and the groups the node interacts, scaled by a β term that controls the chances of infection. the network structure is built based on demographic statistics of a given country, region, or city, and the propagation simulation is performed at time iterations, that represent days. here, we studied in depth the case of the brazilian epidemic considering its population properties and also specific events, such as when the first isolation measures were taken, and the impacts of future actions. brazil is a large and populated country with a wide variety of geographical location types, climates, and it also has a lengthy border with other countries to the west. it is a challenging setting for any epidemiological study. here we consider an average over all the country population, as we adjust the model output to match some statistics of the epidemic official reports. brazil is performing fewer tests in comparison to other countries at the same epidemic scale, however, it is known that testing for infection is always limited, either due to the low number of tests or to the velocity of infections which the testing procedure cannot keep up to. we then considered that only hospitalization cases and 20% of the mild cases are diagnosed. asymptomatic cases are not diagnosed and keep acting normally in the network, considering the active layers. regarding the isolation of infected nodes, we take some optimistic assumptions: mild cases (even those not diagnosed) are aware of its symptoms and isolate themselves at home. severe and critical cases are eventually hospitalized, and then fully isolated from the network (removal of all its edges). under the described scenario, the network starts with all its layers and β = 0.3, representing that people are aware of the virus since the beginning (even before isolation measures). after 27 days of the first confirmed case, the first isolation measures are taken where schools and religious activities are stopped and work and transports keep functioning at 30% of the initial scale (achieved further reducing the β term). different actions are then considered after 90 days of the first case: keep the current isolation levels, increase isolation, end isolation returning all activities to 100%, or returning only the work activities. the results show that keeping approximately the current isolation levels results in a prolonged propagation, as we are near the estimated peak (around june 5) with an average of 11,000 daily new cases and 1900 daily new deaths, and an average of 946,830 diagnosed cases (up to 3,6 million infected) and 149,438 deaths until the end of the year. in this scenario, hospitals may exceed its maximum capacity around june 11, but the efficient implementation of new icu beds and good logistic management of patients may still keep the situation under control. however, this is a very optimistic assumption, considering that our definition of "keep isolation" considers social isolation above 50% as registered at the beginning of the brazilian quarantine [22] . the social isolation levels in brazil are constantly decreasing even when we are still in a state of moderated quarantine, and it is possible to observe average isolation below 50% in most days of the past month (middle of april to middle of may 2020). moreover, the results show that this prolonged scenario may cause hospitals to keep functioning at maximum capacity for up to a month. when analyzing other possible scenarios the situation may be considerably different. relaxing isolation measures from now on causes an abrupt increase in the daily growth of cases and deaths, up to 5 times higher in comparison to the current isolation levels. even if only work activities return while schools, religion, and transport activities remain inactive/reduced, the impact is very similar to returning all the activities, with a possible number of above 1,34 million diagnosed cases (up to 5,2 million infected), and around 212,105 deaths until the end of the year. this is, again, a very optimistic assumption as we do not consider the hospital overflow to calculate the death toll. considering this aspect, icu beds may be fully occupied in early june, and around the middle of the month their demand may reach up to 134,000 beds, which is around 3 times higher than the entire country's capacity. the other alternative, which is the increase of isolation levels (lockdown), appears to be the only alternative to stop the healthcare system from entering a very critical situation. in this scenario, the growth in the number of daily cases and deaths would be mitigated, and faster. as we are near the peak of new cases at current isolation levels, estimated to be between the beginning and middle of june, increasing the isolation levels does not cause a significant impact on when the peak occurs or its magnitude. however, the disease spreading and the occurrences of new cases decrease much faster in this scenario in comparison to any other scenario studied here, with a difference of months. moreover, the final numbers are considerably smaller, with an average of 552,855 diagnosed cases (up to 2.1 million infected) 87,059 deaths until the end of the year. although the proposed method includes various demographic information for the network construction, and an improved sir approach to covid-19, it still does not cover all factors that impact the epidemic propagation. as future works, one may consider more information such as the correlation between the age distribution within the social organization and the clinical spectrum of the 4 infection types (e.g. severe and critical cases are mostly composed of risk groups). another possible improvement consists of increasing n (number of nodes of the networks), e.g. using a value near the real population of the studied society, which we avoided here due to hardware and time constraints (graph processing is costly). another important point regarding the obtained results is related to the "keep isolation" scenario, which may be underestimated as we take various optimistic assumptions and also consider a fixed isolation level based on previously observed data, while most recent data shows that these levels are decreasing [22] . therefore, during the network evolution, a possible improvement is the use of dynamic isolation levels to better represent reality. it is also possible to consider various scenarios for future actions, such as 2 or more measures of increasing/reducing isolation. this may allow the discovering of new epidemic waves if social activities return too soon after the isolation period, such as what happened in 1918 with the spanish flu. portal da transparência -painel covid registral amib. brazilian intensive care medicine association: updated data on icu beds in brazil, 2020, visited on 2020-05-08 infectious diseases of humans: dynamics and control incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china presumed asymptomatic carrier transmission of covid-19 the mathematical theory of infectious diseases and its applications emergence of scaling in random 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model covid-19 coronavirus data model studies on the covid-19 pandemic in sweden the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china clinical characteristics of asymptomatic and symptomatic patients with mild covid-19 collective dynamics of 'small-world'networks world health organization -coronavirus disease 2019 (covid-19): situation report, 73 world health organization -modes of transmission of virus causing covid-19: implications for ipc precaution recommendations world health organization coronavirus disease (covid-19) dashboard clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study key: cord-022176-hprwqi4n authors: löscher, thomas; prüfer-krämer, luise title: emerging and re-emerging infectious diseases date: 2009-07-28 journal: modern infectious disease epidemiology doi: 10.1007/978-0-387-93835-6_3 sha: doc_id: 22176 cord_uid: hprwqi4n emerging infectious diseases (eids) are characterized by a new or an increased occurrence within the last few decades. they include the following categories emerging diagnosis of infectious diseases: old diseases that are newly classified as infectious diseases because of the discovery of a responsible infectious agent. europe including great britain as well as in india, china, and japan. emerging vector-borne disease events concentrated in densely populated subtropical and tropical regions mostly in india, indonesia, china, sub-saharan africa, and central america (see figs. 3.3, 3.4 , and 3.5). the identification of new infectious agents in old diseases with unknown etiology is still the basis in many epidemiological studies. such newly detected bacteria and viruses in the last few decades are listed in table 3 .1. since the detection of helicobacter pylori in 1983, this infection has been identified as the causative agent in 90% of b-gastritis cases. the risk of duodenal ulcer is increased by 4-25-fold in patients with helicobacter-associated gastritis. who declared h. pylori as a carcinogen of first order because of its potential to enhance the risk of stomach carcinoma and malt lymphoma in long-term infection. in highprevalence regions for h. pylori, the frequency of stomach carcinoma is significantly higher compared to low-endemic areas (correa et al. 1990 ). the identification of h. pylori facilitates curative treatment of most associated diseases in individuals. but the most important epidemiological effect on associated diseases is attributed to increased hygienic standards in industrialized countries with a substantial reduction of h. pylori prevalences in younger age cohorts. transmission of h. pylori occurs mainly in childhood. in western developed countries the overall prevalence is around 30%, higher in older age groups due to a cohort effect, and this increases with low socioeconomic status (rothenbacher et al. 1989 ). in countries with low hygienic standards the prevalences are still high in younger age groups and reach 90% in developing countries. in developed countries, migrant subpopulations from less-developed regions show significantly higher prevalences in comparison to the nonmigrant population (mégraud 1993 ). since the early 20th century, a characteristic expanding skin lesion, erythema migrans (em), and an arthritis associated with previous tick bites were known. borrelia for many decades. increased outdoor activities facilitated contacts between humans and ticks in the 1970s and the 1980s and increased transmission of borrelia to humans at the northeastern coast of north america, leading to the discovery of borrelia burgdorferi in 1981 by willy burgdorfer. three different stages of the disease that describe the stage of infection and the involvement of different organ systems are known: stage 1, early localized infection; stage 2, early but disseminated infection; and stage 3, late stage with persistent infection. lyme disease is endemic at the east coast and in minnesota in the united states, in eastern and central europe, and russia. seroprevalence rates that reflect about 50% of nonclinical infections vary between 2 and 18% in the general population in germany (hassler et al. 1992; weiland et al. 1992) . in high-risk groups like forest workers in germany the prevalences reach 25-29% (robert koch institute 2001a). in ticks (ixodes) the prevalences are between 2 and 30% depending on the geographical area and the testing method used [immunofluorescence test, ift and polymerase chain reaction (pcr)]. in most studies the main risk factors of infection are age (children: 4-9 years, adults 35-60 years), outdoor activities, skin contacts with bushes and grass, and the presence of ticks in domestic animals (robert koch institute 2001b) . the probability of infection (seroconversion) after a tick bite in germany is 3-6% and the probability of a clinical disease is 0.3-1.4%. the probability that the bite of an infectious tick leads to infection in the host is 20-30%. this depends on the time duration that the tick is feeding on the human body. since the detection of the etiologic infectious agent and the subsequent development of laboratory diagnostic tests in the 1980s, the number of reported cases of lyme disease has increased from 0 to 16,000 per year, indicating that it is an "emerging diagnosis." the reported numbers vary depending on the reproduction of the hosting rodents for ticks as well as the contacts between humans and nature (spach et al. 1993) . ticks may live for several years and their survival, reproduction rate, and activity are directly affected by changes in seasonal climate through induced changes in vegetation zones and biodiversity, hence causing local alterations of the tick's habitat and in the occurrence of animals that are carriers of different pathogens (like small rodents). several studies in europe have shown that in recent decades the tick ixodes ricinus, transmitting lyme borreliosis and tick-borne encephalitis (tbe), has spread into higher latitudes (e.g., sweden) and altitudes (e.g., czech republic, austria), and has become more abundant in many places. such variations have been shown to be associated with recent variations in climate. as a result, new risk areas of both diseases have recently been reported from the czech republic. climate change in europe seems likely to facilitate the spread of lyme borreliosis and tbe into higher latitudes and altitudes, and to contribute to extended and more intense transmission seasons. currently, the most effective adaptive strategies available are tbe vaccination of risk populations and preventive information to the general public (danielova et al. 2004; lindgren et al. 2006; materna et al. 2005 ). an effective vaccine was licensed for b. burgdorferi in 1999. in europe, where different variants of borrelia are present (mostly b. afzelii and b. garinii), this vaccine is not protective. trivalent vaccines for europe are in clinical trials. in recent years, norovirus infections are increasingly recognized as the cause of large outbreaks of diarrheal diseases in the general population, school classes, nursing homes, hospitals, and cruise ships in western countries with peaks in colder seasons (winter epidemics) (centers of disease control 2006; verhoef et al. 2008; robert koch institute 2008a) . this is a typical example for emerging diagnosis due to increasing availability of routine pcr testing for these viruses in stool samples. noroviruses (family caliciviridae) are a group of related, single-stranded rna viruses first described in an outbreak of gastroenteritis in a school at norwalk, ohio, in 1968. five genogroups are known. immunity seems to be strain specific and lasts only for limited periods, so individuals are likely to get the infection repeatedly throughout their life. it is estimated that noroviruses are the cause of about 50% of all food-borne outbreaks of gastroenteritis. for several years there has been an ongoing epidemic in several european countries due to drift variants of a new genotype (gg ii.4jamboree) previously unknown to this nonimmune population (robert koch institute 2008a). as a result of an analysis of 232 outbreaks in the united states between 1997 and 2000, direct contamination of food by a food handler was the most common cause (57%), person-to-person transmission was less prevalent (16%), and even less frequently waterborne transmission could be proved (3%) (centers for disease control 2006). vomiting is a frequent symptom of norovirus enteritis and may result in infectious droplets or aerosols causing airborne or contact transmission. this may explain the difficulty to stop outbreaks in hospitals, nursing homes, and similar settings despite precautions to prevent fecal-oral transmission. also on cruise ships, person-to-person transmission is most likely in those closed settings, and drinking tap water is a risk factor as well (verhoef et al. 2008 ). searching for an agent which causes large outbreaks of enterically transmitted non-a hepatitis in asia and other parts of the world, the hepatitis e virus (hev) was first described in 1983 and cloned and sequenced in 1990 (reyes et al. 1990 ). meanwhile, hev has been shown to be a zoonotic virus circulating in pigs and other animals. it is implicated in about 50% of sporadic cases of acute hepatitis in developing countries and associated with a high case fatality rate in the third trimester of pregnancy (10-25%). hev is a major cause of large epidemics in asia, and to a lesser extent in africa and latin america, typically promoted through postmonsoon flooding with contamination of drinking water by human and animal feces. recent data show hev also to circulate in european countries and to be associated with severe and fatal disease not only during pregnancy but also in the elderly and in patients with chronic liver conditions. in patients with solid organ transplants, hev may even cause chronic hepatitis and liver cirrhosis (kamar et al. 2008) . a recombinant hev vaccine candidate has demonstrated a high protection rate of approximately 95% during clinical trials in nepal (shrestha et al. 2007 ). for 30 years, specific human papillomaviruses have been linked to certain human cancers and have been identified as causative agents of malignant proliferations. in the 1980s the detection of papillomavirus dna from cervical carcinoma biopsies were published, showing that hpv types 16 and 18 are the most frequent (dürst et al. 1983; boshart et al. 1984) . the relation of hpv infections and cancer is further discussed in chapter 23. definition: only infections that are newly discovered in humans are listed in this chapter: hiv, new variant of creutzfeldt-jakob disease (vcjd), hemorrhagic uremic syndrome (hus) caused by enterohemorrhagic escherichia coli, viral hemorrhagic fevers like hanta, lassa, ebola, and marburg fever, nipah virus encephalitis, monkeypox, human ehrlichiosis, severe acute respiratory syndrome (coronavirus infection, sars), and avian influenza (h5n1) (see fig. 3 .1 and table 3 .2). these infections mostly have their origin in zoonotic wildlife (e.g., avian influenza, monkeypox, hantavirus, nipah virus, and filoviruses) or livestock (e.g., vcjd). factors promoting the spread of these infections in humans are contacts with wildlife, mass food production of animal origin, and globalization (migration, transportation of goods and vectors) (see fig. 3 .2). in addition, new strains or variants of well-known pathogens have emerged showing increased or altered virulence such as clostridium difficile ribotype 027 or staphylococcus aureus strains expressing the panton-valentine leukocidin (see also chapter 22). the epidemiology of hiv is treated in chapter 18 and that of avian influenza and new influenza h1n1 in chapter 16. in the year 1995, 3 years after the peak of the bse epidemic in the united kingdom, with an annual incidence rate in cows of 6.636 per million bovines aged over 24 months, the first mortalities in humans with a new variant of creutzfeldt-jakob disease were observed in the united kingdom. until 2007, smaller incidence rates of bse cases had been reported by 21 other european countries in indigenous bovines and up to more than 43,000 per million in 2004 in ireland. from 1999, bse started to increase in switzerland and portugal, from 2004 in spain and in recent years has spread to eastern european countries (organisation mondiale de la santé animale 2008). the infectious agent is a self-replicating protein, a "prion." the source of infection for cows is infectious animal flour. the transmission to humans occurs through oral intake of cow products, most likely undercooked meat and nerval tissues as well as transplants of cornea, dura mater, contaminated surgical instruments, or the treatment with hypophyseal hormones extracted from animal tissues. after a statuary ban on the feeding of protein derived from ruminants to any ruminant and the export ban of all cow products from england, the epidemic of bse in cows and the occurrence of human infections decreased in the united kingdom since 2004. by june 2008 the total number of deaths in definite/probable cases of vcjd in the united kingdom was 163 (the national creutzfeldt-jakob disease surveillance unit 2008). only a few numbers of vcjd were reported from other european countries and the united states (who 2008). nipah virus encephalitis was first observed in 1997/98 in malaysia. the disease was transmitted by pigs to laborers in slaughterhouses and showed a lethality of 40%. the infectious agent was detected in 1999 (chua et al. 2000; lam and chua 2002) . since then, several outbreaks of nipah virus infections have been observed in asian countries: singapore in 1999, india 2001 , and bangladesh since 2003 (who 2004a harit et al. 2006) . the virus has been isolated repeatedly from various species of fruit bats, which seem to be the natural reservoir (yob et al. 2001 ). west nile is a mosquito-borne flavivirus that was first isolated from a woman with a febrile illness in uganda in 1937. from the 1950s, west nile fever endemicity and epidemics started being reported from africa and the middle east. severe neurological symptoms were thought to be rare. more recent epidemics in northern africa, eastern europe, and russia suggested a higher prevalence of meningoencephalitis with case fatality rates of 4-13%. in 1999, west nile virus was identified as the cause of an epidemic of encephalitis at the east coast of the united states (nash et al. 1999) . a seroepidemiological household-based survey showed that the first outbreak consisted of about 8,000 infections of which about 1,700 developed fever and less than 1% experienced neurological disease ). since then, epidemics occur during summer months in north america each year, with an estimated 35,000 febrile illnesses and over 1,200 encephalitis or meningitis cases in the united states in 2007 (centers for disease control 2008). age above 50 years is the main risk factor for developing severe disease. the virus is transmitted mainly by culex mosquitoes, but also by sandflies, ceratopogonids, and ticks, with birds as reservoir hosts and incidental hosts such as cats, dogs, and horses. efforts are made to reduce the transmitting mosquito population and to prevent mosquito bites through personal protection as well as to prevent transmission through blood donations by screening (centers of disease control 2008). the first case of sars occurred in guangdong (china) in november of 2002, leading to an outbreak with 7082 cases in china and hong kong (8096 cases worldwide) until july 2003. the case fatality rate was 9.6%. a new coronavirus (sars-cov) was identified as the causative agent (drosten et al. 2003) , being transmitted first by infected semidomesticated animals such as the palm civet and subsequently from human to human. some cases were exported to other countries, causing smaller outbreaks there, canada being the most affected country outside asia with 251 cases, before control of transmission was effective. eight thousand and ninety-six cases were reported worldwide, until july 2003, then further transmission stopped (besides one more case of laboratory transmission in 2004), indicating an efficient international cooperation in disease control (who 2004b) . recently, sars-cov has been found in horseshoe bats, which seem to be the natural reservoir of the virus. about 150,000-200,000 cases of hemorrhagic fever with renal syndrome (hfrs) caused by hantaviruses are reported annually worldwide, with more than half in china, many from russia and korea, and numerous cases from japan, finland, sweden, bulgaria, greece, hungary, france, and the balkan with different death rates depending on the responsible virus, ranging from 0.1% in puumala to 5-10% in hantaan infections (schmaljohn and hjelle 1997) . hantaviruses are transmitted from rodent to rodent through body fluids and excreta. only occasionally do humans get infected. different types of hantaviruses are circulating in europe and the eastern hemisphere, predominantly puumala virus, dobrava virus, and tula virus, adapted to different mouse species. depending on the virus type the case fatality rate is between 1 and 50%. as an example, the annual rate of reported cases in germany was about 100 cases per year from 2001 onward. this started to change in 2005 with 448 reported cases and rose dramatically to 1687 cases in 2007. that year, hantavirus infections were among the five most reported viral infections in germany. reasons for the rise in human infections were an increase in the hosting rodent population due to a very mild winter 2006/2007 and an early start of warm temperatures in spring which led to favorable nutritional situations for the mice influencing their population dynamics. in addition, favorable climatic conditions enhanced the outdoor behaviors of humans facilitating transmission in rural areas (robert koch institute 2008b; hofmann et al. 2008) . since 1993, a previously unknown group of hantaviruses (sin nombre, new york, black creek canal, bayou-in the united states and canada; andes, in south america) emerged in the americas as a cause of hantavirus pulmonary syndrome (hps), an acute respiratory disease with high case fatality rates (approx. 35%), causing a new, significant public health concern. a total of 465 cases had been reported until march 2007 in 32 states, most of them in the western part of the united states (centers for disease control 2007). lassa virus was detected for the first time in 1969 during an outbreak affecting nurses in a missionary hospital in lassa, nigeria. however, the disease had previously been described in the 1950s. lassa virus is enzootic in a common peridomestic rodent in west africa, the multimammate rat mastomys natalensis, which is chronically infected and sheds the virus in urine and saliva. human infection through direct or indirect contact with rats or their excretions is rather common in some west african countries and estimates from seroepidemiological and clinical studies suggest that there are several hundred thousand cases annually. however, only a minority of infections seems to progress to severe hemorrhagic disease with a case fatality rate of 5-30% in hospitalized cases. the virus can be transmitted by close person-to-person contact and nosocomial spread has been observed under poor hygienic conditions. marburg and ebola viruses, which were first detected during outbreaks in 1967 and 1975, respectively, have so far been observed only during several limited outbreaks and a few isolated cases in certain countries of sub-saharan africa. however, very high case fatality rates (25-90%), the occurrence of outbreaks that were difficult to control in resource-poor settings, and the obscure origin of these viruses have attracted considerable public interest worldwide. recently, evidence was found for both marburg and ebola viruses to occur in certain species of bats that probably constitute the natural reservoir of these filoviruses (towner et al. 2007 ). although the disease burden of these viral hemorrhagic fevers is low, they gained considerable international attention due to -their high case fatality rates, -the risk of person-to-person transmission, -several imported cases to industrialized countries, and -fears of abuse of these agents for bioterrorism. as a consequence, considerable resources have been invested, even in nonendemic countries, in the setting up of task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing. this highly virulent strain of c. difficile expresses both cytotoxins a and b and, in addition, the binary toxin cdt, an adp-ribosyltransferase. due to a deletion in the regulatory tcdc gene, the synthesis rates of toxin a and b are increased by 16-and 23-fold, respectively. this strain was detected in 2000 for the first time in pittsburgh, usa. since then it has spread to canada, and in 2003 it reached europe causing multiple outbreaks in hospitals and nursing homes (warny et al. 2005) . c. difficile 027-associated colitis has shown high case fatality rates (10-22%) and an increased relapse rate. containment of outbreaks in hospitals and other institutions necessitates isolation of patients or cohorts and strict hygienic measures. during recent decades, a large variety of well known infectious diseases has shown regional or global re-emergence with considerable public health relevance (table 3. 3). globally, tuberculosis is probably the most important re-emerging infectious disease. in developing countries, tb infection still is extremely common and, in the wake of the hiv pandemic, the percentage of those developing overt disease has increased dramatically. worldwide, tb is the most common opportunistic infection in patients with aids. the significance of tb and hiv/tb coinfection is reviewed in chapters 16 and 18. the re-emergence of some infectious diseases is closely related to the lack or the breakdown of basic infrastructures as seen in periurban slums and in refugee camps in developing countries, or as a consequence of war, breakdown of the civil society, or natural or man-made disasters. cholera is a formidable example for both re-emergence and epidemic spread under those conditions. another important group of re-emerging infectious diseases is caused by various vector-borne infections, such as malaria, dengue fever, and yellow fever. these major vector-borne diseases are treated in more detail in chapter 21. in addition, there are a variety of re-emerging infections transmitted by arthropod vectors such as various arboviral diseases and some protozoal diseases other than malaria (i.e., leishmaniasis, human african trypanosomiasis). the reasons for the emergence of several vector-borne diseases are rather variable and may range from climatic factors (e.g., global warming, rainfall), lack or breakdown of control, to changes in agriculture and farming and in human behavior (e.g., outdoor activities). these factors are usually quite specific for each of these diseases and largely depend on the specific ecology of the agent, its vectors, and reservoirs. cholera, an acute diarrheal infection transmitted by fecally contaminated water and food, had been endemic for centuries in the ganges and brahmaputra deltas in the 19th century before it started to spread to the rest of the world. since 1817, six pandemics caused by the classical biotype of vibrio cholerae were recorded that killed millions of people across europe, africa, and the americas. it has been a major driving force for the improvement of sanitation and safe water supply. the seventh pandemic was caused by the el tor biotype, first isolated from pilgrims at the el tor quarantine station in sinai in 1906. it started in 1961 in south asia, reached africa in 1971, and is still ongoing. after more than hundred years, cholera spread to the americas in 1991, and beginning in peru, a large epidemic hit numerous latin american countries with 1.4 million cases and more than 10,000 fatalities reported within 6 years. out of the 139 serogroups of v. cholerae, only o1 and o139 can cause epidemics. the serogroup o139, first identified in bangladesh in 1992, possesses the same virulence factors as o1 and creates a similar clinical picture. currently, the presence of o139 has been detected only in southeast and east asia, but it is still unclear whether v. cholerae o139 will extend to other regions. since 2005, the re-emergence of cholera has been noted in parallel with the everincreasing size of vulnerable populations living in unsanitary conditions. cholera remains a global threat to public health and one of the key indicators of social development. while the disease is no longer an issue in countries where minimum hygiene standards are met, it remains a threat in almost every developing country. the number of cholera cases reported to the who during 2006 rose dramatically, reaching the level of the late 1990s. a total of 236,896 cases were notified from 52 countries, including 6,311 deaths, an overall increase of 79% compared with the number of cases reported in 2005. this increased number of cases is the result of several major outbreaks that occurred in countries where cases had not been reported for several years such as sudan and angola. it is estimated that only a small proportion of cases -less than 10% -are reported. the true burden of disease is therefore grossly underestimated. the absence or the shortage of safe water and sufficient sanitation combined with a generally poor environmental status are the main causes of spread of the disease. typical at-risk areas include periurban slums where basic infrastructure is not available, as well as camps for internally displaced people or refugees where minimum requirements of clean water and sanitation are not met. however, it is important to stress that the belief that cholera epidemics are caused by dead bodies after disasters, whether natural or manmade, is false. on the other hand, the consequences of a disaster-such as disruption of water and sanitation systems or massive displacement of population to inadequate and overcrowded camps-will increase the risk of transmission. chikungunya virus, an arbovirus belonging to the alphavirus group, is transmitted by various mosquitoes. the virus was first isolated in tanzania in 1952 and since then has caused smaller epidemics in sub-saharan africa and parts of asia with low public health impact. in 2005, the largest epidemic ever recorded started in east africa, spread to réunion and some other islands of the indian ocean, and then spread further to asia, with more than 1.5 million cases in india alone so far. characteristics of the disease are high fever and a debilitating polyarthritis, mainly of the small joints that can persist for months in some patients. now, for the first time, severe and fatal cases have been observed that may be due to certain mutations of the epidemic strain (parola et al. 2006) . the asian tiger mosquito aedes albopictus has proved to be an extremely effective vector in recent epidemics causing high transmission rates in big cities and leading to epidemics with high public health impact. this southeast asian mosquito species has been shipped by transport of used tires and plants harboring water contaminated with larvae to other continents and, since 1990, ae. albopictus has successfully spread in italy and other parts of southern europe. in august 2007, an outbreak of chikungunya fever occurred in northern italy with more than 200 confirmed cases. the index case was a visitor from india who fell ill while visiting relatives in one of the villages and further transmission was facilitated by an abundant mosquito population during that time, as a consequence of seasonal synchronicity (rezza et al. 2007 ). ross river virus (rrv) is another arbovirus of the alphavirus group that causes an acute disease with or without fever and/or rash. most patients experience arthritis or arthralgia primarily affecting the wrist, knee, ankle, and small joints of the extremities (epidemic polyarthritis). about one-quarter of patients have rheumatic symptoms that persist for up to a year. the disease can cause incapacity and inability to work for months. it is the most common arboviral disease in australia with an average of almost 5,000 notified cases per year. rrv is transmitted by various mosquito species and circulates in a primary mosquito-mammal cycle involving kangaroos, wallabies, bats, and rodents. a human-mosquito cycle may be present in explosive outbreaks which occur irregularly during the summer months in australia and parts of oceania. heavy rainfalls as well as increasing travel and outdoor activities are considered as important factors contributing to the emergence of rrv epidemics. this flavivirus is transmitted by certain culex mosquitoes and is a leading cause of viral encephalitis in asia with 30,000-50,000 clinical cases reported annually. it occurs from the islands of the western pacific in the east to the pakistani border in the west, and from korea in the north to papua new guinea in the south. only 1 in 50-200 infections will lead to encephalitis, which is, however, often severe with fatality rates of 5-30% and with a high incidence of neurological sequelae. despite the availability of effective vaccines, je causes large epidemics and has spread to new areas during recent decades (e.g., india, sri lanka, pakistan, torres strait islands, and isolated cases in northern australia). je is particularly common in areas where flooded rice fields attract water fowl and other birds as the natural reservoir and provide abundant breeding sites for mosquitoes such as culex tritaeniorhynchus, which transmit the virus to humans. pigs act as important amplifying hosts, and therefore je distribution is very significantly linked to irrigated rice production combined with pig rearing. because of the critical role of pigs, je presence in muslim countries is low. crimean-congo virus is a bunyavirus causing an acute febrile disease often with extensive hepatitis resulting in jaundice in some cases. about one-quarter of patients present hemorrhages that can be severe. fatality rates of 7.5-50% have been reported in hospitalized patients. cchf is transmitted by hyalomma ticks to a wide range of domestic and wild animals including birds. human infection is acquired by tick bites or crushing infected ticks, and also by contact with blood or tissue from infected animals that usually do not become ill but do develop viremia. in addition, nosocomial transmission is possible and is usually related to extensive blood exposure or needle sticks. human cases have been reported from more than 30 countries in africa, asia, southeastern europe, and the middle east. in recent years, an increase in the number of cases during tick seasons has been observed in several countries such as russia, south africa, kosovo, and greece. in turkey, where before 2002 no human cchf cases had been observed, a total of 2,508 confirmed cases, including 133 deaths, were reported between 2002 and june 2008. the emergence of cchf has been associated with factors such as climatic features (temperature, humidity, etc.), changes of vector population, geographical conditions, flora, wildlife, and the animal husbandry sector. rvf is a mosquito-borne bunyavirus infection occurring in many parts of sub-saharan africa. it infects primarily sheep, cattle, and goats, and is maintained in nature by transovarial transmission in floodwater aedes mosquitoes. it has been shown that infected eggs remain dormant in the dambos (i.e., depressions) of east africa and hatch after heavy rains and initiate mosquito-livestock-mosquito transmission giving rise to large epizootics. remote sensing via satellite can predict the likelihood of rvf transmission by detecting both the ecological changes associated with heavy rainfall and the depressions from which the floodwater mosquitoes emerge. transmission to humans is also possible from direct and aerosol exposure to blood and amniotic fluids of livestock. most human infections manifest themselves as uncomplicated febrile illness, but severe hemorrhagic disease, encephalitis, or retinal vasculitis is possible. in 1977, rvf has been transported, probably by infected camels to egypt, where it caused major epidemics with several hundred thousand infections of humans. it has been suggested that introduction of rvf may be a risk to other potentially receptive areas such as parts of asia and the americas. floods occurring during the el niño phenomenon of 1997 in east africa subsequently gave rise to large epidemics and further spread to the arabian peninsula. most recent epidemics occurred in 2006 and 2007 following heavy rainfalls in kenya, somalia, and sudan, causing several hundred deaths. besides mosquito control, epidemics are best prevented by vaccination of livestock. leishmaniasis, a protozoal transmitted by sandflies, has shown a sharp increase in the number of recorded cases and spread to new endemic regions over the last decade. presently, 88 countries are affected with an estimated 12 million cases worldwide. there are about 1.5 million new cases of cutaneous and mucocutaneous leishmaniasis, a nonfatal but debilitating disease with 90% of cases occurring in afghanistan, brazil, bolivia, iran, peru, saudi arabia, and syria. the incidence of visceral leishmaniasis (vl), a disease with a high fatality rate when untreated, is estimated at around 500,000 per year. the situation is further aggravated by emerging drug resistance (table 3 .4) and the deadly synergy of vl/hiv coinfection. epidemics usually affect the poorest part of the population and have occurred recently in bangladesh, brazil, india, nepal, and sudan. for many years, the public health impact of the leishmaniases has been grossly underestimated. they seriously hamper socioeconomic progress and epidemics have significantly delayed the implementation of numerous development programs. the spread of leishmaniasis is associated with factors favoring the vector such as deforestation, building of dams, new irrigation schemes, and climate changes, but also with urbanization, migration of nonimmune people to endemic areas, poverty, malnutrition, and the breakdown of public health. antimicrobial resistance of epidemiological relevance has emerged as a major problem in the treatment of many infectious diseases (table 3 .4). resistance is no longer a problem that predominantly affects the chemotherapy of bacterial infections. it became increasingly important in parasitic and fungal diseases, and despite the short history of antiviral chemotherapy, it already plays a prominent role in the treatment of hiv infection and other viral diseases. resistance is also a problem in some of the emerging infections and will further complicate their treatment and control. resistance of bacterial pathogens has become a common feature in nosocomial infections, especially in the icu and in surgical wards. currently, the number one problem in most hospitals is s. aureus resistant to methicillin (mrsa, see chapter 22). however, common problems of resistance also extend to other major bacterial pathogens such as enterococci, various gram-negative enteric bacilli, and pseudomonas species. resistance has developed not only to standard antibiotics (e.g., penicillins, cephalosporins, aminoglycosides, macrolides, or quinolones) but also to second-line antibiotics including carbapenems, glycopeptides, and newer quinolones. however, there is considerable geographic variation. in 2006, the european antimicrobial resistance surveillance system (earss), a network of national surveillance systems, reported vancomycin-resistant rates among enterococci ranging from none in iceland, norway, romania, bulgaria, denmark, and hungary to 42% of enterococcus faecium strains in greece (earss 2006) . a surveillance study conducted in the united states hospitals from 1995 to 2002 showed that 9% of nosocomial bloodstream infections were caused by enterococci and that 2% of e. faecalis isolates and 60% of e. faecium isolates were vancomycin resistant (wisplinghof et al. 2004) . rates and spectrum of antibacterial resistance of e. coli and other gram-negative enteric bacilli may differ considerably from one hospital to the other. in some important pathogens of hospital-related infections such as klebsiella, enterobacter, and pseudomonas species, resistance to almost all available antimicrobials has been observed. this may complicate the choice of an effective initial chemotherapy considerably. therefore, each hospital has to monitor the epidemiological situation of resistance regularly, at least for the most important bacteria causing nosocomial infections, such as staphylococci, enterococci, gram-negative enteric bacilli, and pseudomonas. even in community-acquired infections, there has been a considerable increase in resistance problems. at present, approximately 15% of pneumococcal isolates in the united states are resistant to penicillin, and 20% exhibit intermediate resistance. the rate of resistance is lower in countries that, by tradition, are conservative in their antibiotic use (e.g., netherlands, germany) and higher in countries where use is more liberal (e.g., france). in hong kong and korea, resistance rates approach 80%. in addition, about one-quarter of all pneumococcal isolates in the united states are resistant to macrolides. this rate is even higher in strains highly resistant to penicillin, and increasingly there is multiresistance against other antibiotics such as cephalosporins. the prevalence of meningococci with reduced susceptibility to penicillin has been increasing, and high-level resistance has been reported in some countries (e.g., spain, united kingdom). although high-dose penicillin is effective in infections with strains of intermediate resistance, most national and international guidelines recommend broad-spectrum cephalosporins such as ceftriaxone as first-line drugs. however, in most developing countries, penicillin and chloramphenicol are the only affordable drugs. in recent years, certain strains of community-acquired s. aureus with resistance to methicillin (cmrsa) have been observed which produce a toxin (panton-valentine leukocidin) that is cytolytic to pmns, macrophages, and monocytes, and which are an emerging cause of community-acquired cases and outbreaks of necrotic lesions involving the skin or the mucosa, and in some patients also of necrotic hemorrhagic pneumonia with a high case fatality (vandenesch et al. 2003) . development of resistance is mainly determined by two factors: -the genetic potential of a certain pathogen, i.e., mobile elements such as plasmids, transposons, or bacteriophages, genes coding for resistance, and mutation rate. -the selection pressure caused by the therapeutic or the para-therapeutic application of antimicrobial drugs. in the hospital these factors are supported by -microbial strains that are highly adapted to this environment (e.g., rapid colonization of patients, resistance to disinfectants), -an increasing percentage of patients who are highly susceptible to infections due to old age, multimorbidity, immunosuppression, extended surgery, and invasive procedures, and -the frequent use of broad-spectrum antibiotics or combinations of antimicrobial drugs. another source of resistant bacteria has been identified in mass animal production and the use of antimicrobials as growth promoters (e.g., the glycopeptide avoparcin, the streptogramin virginiamycin) or as mass treatment in the therapy or the prevention of infections. the inadequate use of antimicrobial drugs is also an important factor responsible for the development of resistance in community-acquired infections. this is especially true in developing countries where only a limited spectrum of antibiotics is available, where shortage of drugs often leads to treatment that is underdosed or too short, and where uncontrolled sale and use of antibiotics is commonplace. as a consequence, resistance of gonococci is extremely frequent in southeast asia, and resistance of salmonella typhi, shigella, and campylobacter to standard antibiotics is common. some of the still effective second-line antibiotics have to be given parenterally or are not available because they are too costly. a typical example of the consequences of insufficient chemotherapy due to lack of compliance and/or unavailability of drugs is the alarming increase in multiresistance and extreme resistance in tb (see chapter 16). resistance is also a problem in parasitic diseases such as malaria (see chapter 21), leishmaniasis, or african trypanosomiasis. plasmodium falciparum developed resistance against all major antimalarial drugs as soon as they were used on a broad scale. resistance had contributed significantly to the increase in malaria-associated morbidity and mortality observed in many endemic areas (wongsichranalai et al. 2002) . a recent report on failures of the new artemisinin combination treatment for p. falciparum malaria at the thai-cambodian border supports fears of the development of resistance to this most promising class of drug at present (dondrop et al. 2009 ). resistance against antiviral drugs has developed almost from the beginning of antiviral chemotherapy (table 3 .4). in the treatment of hiv infection, the risk of development of resistance has been drastically reduced by the combination of several drugs with different mechanisms of action (see chapter 18). however, drug resistance remains the achilles' heel of the highly active antiretroviral therapy (haart) and may be at a considerable risk of expanding haart to the developing world. today, we have to realize that as we develop antimicrobial drugs, microbes will develop strategies of counterattack. antimicrobial resistance occurs at an alarming rate among all classes of pathogens. even in rich countries it causes real clinical problems in managing infections that were easily treatable just a few years ago. in life-threatening infections such as sepsis, nosocomial infections, or falciparum malaria, there is a substantial risk that the initial chemotherapy might not be effective. in addition, the delay caused by inadequate treatment might favor transmission to other people and support the spread of resistant pathogens (e.g., multiresistant tb). last but not the least, surveillance and control and the necessity to use expensive second-line drugs or combinations of antimicrobials are enormous cost factors. for developing countries this is a major limitation in the treatment and control of infections caused by resistant agents. so, in many ways, emerging resistance contributes to the emergence of infectious diseases. despite the availability of effective strategies for treatment and prevention, infectious diseases have remained a major cause of morbidity and mortality worldwide. however, the problems associated with infections are due to considerable changes. in industrialized countries the mortality caused by infectious diseases has decreased tremendously during more than 100 years. however, during recent years, both mortality and morbidity associated with infections are increasing again. ironically, this is closely associated with the advances in medicine which have contributed to profound changes in the spectrum of both patients and their infections. advanced age, underlying conditions, and an altered immune response are common features in the seriously infected hospital patient today. immunosuppressive therapy is frequently used to treat neoplastic and inflammatory diseases or to prevent the rejection of transplants. some infections, most notably hiv/aids, cause immunosuppression by itself. in the compromised patient, infections are generally more severe or may be caused by opportunistic pathogens that will not harm the immunocompetent host. antimicrobial treatment is often less effective in these patients and tends to be further complicated by antimicrobial resistance which may manifest itself or develop at a higher frequency in the immunocompromised patient. an increasing percentage of infections are hospital acquired or otherwise health care associated. it is estimated that nosocomial infections affect 1.7 million patients and contribute to approximately 100,000 deaths in us hospitals annually (klevens et al. 2007 ). considering the rising number of elderly and immunocompromised patients, a further increase in severe infections can be predicted. in developing countries, the significance of infectious diseases has remained high for ages and despite the advances in medicine. until now, infections are by far the leading cause of both disability-adjusted life years and life years lost. the reasons are obvious and mostly related to poverty and lack of development causing poor and unhealthy living conditions, inadequate health systems, and lack of resources for prevention and treatment. this is, of course, just an integral part of the general socioeconomic problems of developing countries. however, poor health conditions per se are an important obstacle to development, and infections such as hiv/aids in sub-saharan africa can be a major cause of lack of development, increasing poverty, and political instability. generally, the situation of many developing countries has not improved during the last two decades, and the gap between the first and the third world has increased. however, most of the mortality and morbidity associated with infectious diseases is avoidable. as laid down in the millennium goals, a major task of the world community will be to counteract the imbalance between the industrialized and the developing countries and to find strategies to ensure participation in the progress of modern medicine for all. developing countries also carry the main burden of diseases caused by newly emerging and re-emerging infections (table 3. 2 and 3.3) . however, the consequences of economical and political crises on emerging infectious diseases are obvious in industrialized countries also-such as the return of diphtheria or the increase in tb and multiresistant tb after the breakdown of the former soviet union. today, all countries worldwide are affected by emerging infections as well as by emerging antimicrobial resistance. in the age of globalization, travel and transport of people, animals, and goods of all kinds have increased tremendously. as a consequence, infectious agents may travel over long distances and at high speed. this is clearly evident with influenza pandemics or outbreaks such as the sars epidemic or with imported cases of viral hemorrhagic fever transmissible from person to person. the spread of antimicrobial resistance or the re-emergence of tb seems to be less spectacular, but the consequences may be at least as important in the long run. management and control of emerging and re-emerging infectious diseases can be very different from disease to disease and has to allow for all relevant factors of the populations at risk and of the specific disease including the ecology of the agent, its vectors, and reservoirs. however, some basic principles apply to all situations: -surveillance -information and communication -preemptive planning and preparedness -provision and implementation of • adequate treatment • adequate control and prevention -international cooperation active and passive surveillance systems with rapid reporting and analysis of data are essential for the early detection of outbreaks, changes in epidemiology, and other events of public health concern (see chapters 8 and 9). however, many resourcepoor countries do not have functional surveillance systems. in addition, reporting of infectious diseases may be neglected or delayed because of fears of stigma, international sanctions including trade and travel restrictions, or interference with tourism. classical examples are plague and cholera, but also recent examples such as the bse/vcjd crisis in the united kingdom or sars originating from china showed undue delays between first occurrence of cases and information to the public. although, in outbreaks of new and unknown diseases it may be difficult, or even impossible, to predict or assess the magnitude of the problem and the potential consequences, timely and adequate information and communication is not only obligatory, according to international regulations, but also the best strategy to avoid rumors, misbeliefs, panic, or disregard. in recent years, many countries have installed national plans of action for important epidemiological scenarios and outbreaks such as pandemic influenza, bioterrorism, import of viral hemorrhagic fevers transmissible from person to person, sars, and comparable diseases or outbreaks. all member states of the world health assembly that have so far not been able to install functional surveillance and/or pre-emptive planning are obliged to do so within a maximum of 5 years after their ratification of the new international health regulations (who 2005) . preparedness not only means surveillance and planning but also has to include the provision of facilities to adequately treat and, if necessary, to isolate patients with infectious diseases of public health importance and relevant epidemic potential and/or at risk of transmission to other persons including health-care workers. task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing have been set up in several countries. however, all health facilities of a certain level such as general hospitals should be prepared by their organization and structure to treat patients with infections of public health relevance such as multiresistant tb under appropriate isolation and barrier nursing conditions. this also applies to hospitals in resource-poor countries. adequate training of health-care workers and strict management have been effective to control outbreaks of highly contagious infections within rural african hospitals lacking sophisticated technical equipments (cdc 1998) . strategies for control and prevention may be quite different for various emerging infections. effective vaccinations are available only for some infections and are usually lacking for newly emerging infections (table 3 .5). for the majority of emerging infections, control and prevention have to rely on information, education and exposure prophylaxis, interruption of transmission by vector control and control of reservoir hosts (e.g., rodents), and case finding with early diagnosis and treatment. for diseases and outbreaks caused by infections of public health relevance that are transmissible from person to person, containment procedures including isolation and treatment of patients under condition of barrier nursing as well as tracking and surveillance of contacts are warranted by national and international health regulations. here, international cooperation is essential to successfully contain outbreaks and epidemics such as the sars epidemic in 2003. despite dramatic progress in their treatment and prevention, infectious diseases are still of enormous global significance with tremendous economic and political implications. emerging and re-emerging infectious diseases as well as emerging antimicrobial resistance are major challenges to all countries worldwide. for the management of current and future problems, it will be most important to counteract the imbalance between the industrialized world, new economies, and developing countries, and to adequately and timely react to new threats on a global scale. a new type of papillomavirus dna, its presence in genital cancer and in cell lines derived from genital cancer world health organization: infection control for viral haemorrhagic fevers in the african health care setting nipah virus: a recently emergent deadly paramyxovirus helicobacter and gastric carcinoma. serum antibody prevalence in populations with contrasting cancer risks effects of climate change on the incidence of tick-borne encephalitis in the czech republic in the past two decades artemisinin resistance in plasmodium falciparum malaria identification of a novel coronavirus in patients with severe acute respiratory syndrome a papillomavirus dann from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions susceptibility results for e. faecium isolates lyme-borreliose in einem europäischen endemiegebiet: antikörperprävalenz und klinisches spektrum hantavirus outbreak global trends in emerging infectious diseases hepatitis e virus and chronic hepatitis in organtransplant recipients estimating health care-associated infections and deaths in u.s. hospitals nipah virus encephalitis outbreak in malaysia lyme borreliosis in europe: influences of climate and climate change, epidemiology, ecology and adaptation measures. who regional office for europe altitudinal distribution limit of the tick ixodes ricinus shifted considerably towards higher altitudes in central europe: results of three years monitoring in the krkonose mts epidemiology of helicobacter pylori infection 1999: results of a household-based seroepidemiological survey outbreak of west nile virus infection novel chikungunya virus variant in travelers returning from indian ocean islands isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis infection with chikungunya virus in italy: an outbreak in a temperate region waldarbeiter-studie berlin-brandenburg 2000 zu zeckenübertragenen und andere zoonosen risikofaktoren für lyme-borreliose: ergebnisse einer studie in einem brandenburger landkreis übertrifft die infektionszahlen der vorjahre zahl der hantavirus-erkrankungen erreichte 2007 in deutschland einen neuen höchststand prevalence and determinants of helicobacter pylori infection in preschool children: a population-based study from germany hantaviruses: a global disease problem safety and efficacy of a recombinant hepatitis e vaccine tick-borne diseases in the united states the national creutzfeld-jakob disease surveillance unit (ncjdsu) marburg virus infection detected in a common african bat community-acquired methicillin-resistant staphylococcus aureus carrying panton-valentine leukocidin genes: worldwide emergence multiple exposures during a norovirus outbreak on a river-cruise sailing through europe toxin production by an emerging strain of clostridium difficile associated with outbreaks of severe disease in north america and europe prevalence of borrelia burgdorferi antibodies in hamburg blood donors nipah virus outbreaks in bangladesh revision of the international health regulations nosocomial bloodstream infections in us hospitals: analysis of 24 179 cases from a prospective nationwide surveillance study large outbreak of norovirus: the baker who should have known better epidemiology of drugresistant malaria nipah virus infection in bats (order chiroptera) in peninsular malaysia key: cord-018116-99z6ykb2 authors: healing, tim title: surveillance and control of communicable disease in conflicts and disasters date: 2009 journal: conflict and catastrophe medicine doi: 10.1007/978-1-84800-352-1_13 sha: doc_id: 18116 cord_uid: 99z6ykb2 nan tim healing • to describe the principles of health surveillance in conflict and disaster situations • to assist in organizing a health surveillance system in conflict and disaster situations • to describe the principles of control of communicable diseases in conflict and disaster situations • to assist in organizing a response to outbreaks and epidemics • to introduce the challenges associated with health surveillance and communicable diseases in conflict and disaster situations there are five fundamental principles for the control of communicable disease in emergencies: • rapid assessment -identify and quantify the main disease threats to the population and determine the population's health status • prevention -provision of basic health care, shelter, food, water, and sanitation • surveillance -monitor disease trends and detect outbreaks • outbreak control -control outbreaks of disease. involves proper preparedness and rapid response (confirmation, investigation, implementation of controls) • disease management -prompt diagnosis and effective treatment rapid assessment has been dealt with elsewhere in this book as have the prevention aspects of disease control (adequate shelter, clean water, sanitation, and food, together with basic individual health care). this chapter therefore covers surveillance, outbreak/epidemic control, and public health aspects of disease management. the topics are dealt with in general terms. more details can be found in references. disasters, particularly conflicts, by damaging or destroying the infrastructures of societies (health, sanitation, food supply) and by causing displacement of populations, generally lead to increased rates of disease. outbreaks and epidemics are not inevitable in these situations and are relatively rare after rapid-onset natural disasters, but there is a severe increase in the risk of epidemics during and after complex emergencies involving conflict, large-scale population displacement with many persons in camps and food shortages. in most wars more people die from illness than from trauma. preventing such problems, or at least limiting their effects, falls on those responsible for the health care of the population affected by the emergency. they must be able to • assess the health status of the population affected and identify the main health priorities • monitor the development and determine the severity of any health emergency that develops (including monitoring the incidence of and case fatality rates from diseases, receiving early warning of epidemics and monitoring responses) at first sight, undertaking public health activities in emergencies, especially in conflicts, may seem to be difficult or impossible. the destructive nature of warfare may prevent or inhibit the provision of adequate food and shelter, of clean water and sanitation and vaccination programs. despite the difficulties that warfare imposes, it is generally possible to undertake at least limited public health programs, including disease surveillance and control activities. in other types of disaster public health activities may be expected to be less affected by the security situation than in a war (although aid workers may be at risk if populations are severely deprived of resources such as food, shelter, water, or cash), and with limited access and damage to communication systems and other parts of the infrastructure assessment, surveillance and control activities can be severely restricted. for example, following the pakistan earthquake late in 2005 access was severely restricted for some time and the urgent need to treat the injured and provide food and shelter meant that the limited transport available was heavily committed. the surveillance and control of communicable disease require data which can be collected in one of three ways: 1. surveillance systems -covering all or at least a significant proportion of the population 2. surveys -in which data are collected from a small sample of the affected population considered to be representative of the whole 3. outbreak investigations -in-depth investigations designed to identify the cause of deaths or diseases and identify control measures although the latter two can provide valuable information for disease control and form part of the surveillance process, proper control of disease requires regular monitoring of the overall disease situation, which in turn requires the establishment of a properly designed health surveillance system. it is important therefore that responsibility for surveillance activities is defined at the beginning of planning for an aid mission. generally speaking, a team will be required, including a team leader (often an aid agency health coordinator), who should ideally have surveillance experience, clinical workers, a water and sanitation specialist, and representatives of the local health services and communities. the team may also need clerical, logistic, information technology and communications specialists. the world health organization defines health surveillance as "the ongoing systematic collection, analysis and interpretation of data in order to plan, implement and evaluate public health interventions." data for surveillance must be accurate, timely, relevant, representative, and easily analyzed, and the results must be disseminated in a timely manner to all who need to receive them. in addition the data collected, the methods used for collection and the output must be acceptable to those surveyed (health-care professionals and the population). in emergencies the time that can be given to surveillance by medical personnel is likely to be limited and surveillance activities will be far from the minds of most of those involved. therefore the methods used need to be rapid, practical, and consistent, and while the greatest possible accuracy must be achieved, "the best must not be the enemy of the good." it is necessary to strike a balance between collecting large amounts of information ("what we would like to know") and collecting too little which can lead to an ineffective response. those responsible for establishing surveillance programs must therefore try to determine what is really needed ("what we need to know"). it is better to err on the side of too much than of too little. ideally any existing surveillance system should be used. there is no point in establishing a system if one already exists, unless the existing one is inadequate or inappropriate or has broken down irretrievably. surveillance systems for use in conflict and disaster situations should therefore adhere as far as possible to the criteria given in table 13 .1. notes on these criteria: complexity and inflexibility are incompatible with surveillance systems generally and particularly when operating in emergencies where collection of data may be difficult and where situations can change very fast. defining what you "need to know" will allow you to set up the appropriate data collection methods (questionnaires, sites, etc.) and to design the system so that it can obtain and handle the information required. information that is accurate but out of date is useless for immediate disease control purposes and of little value for forward planning. communications therefore form an integral part of any surveillance system. do not try to overreach when setting up a system. for example, expatriate staff may best be used to recruit local staff for the system and in supervisory activities rather than in collecting data. this criterion is certainly a goal to aim for as sustainability must be the target for all aid work. however, there may be situations where an emergency system is needed rapidly and where it cannot readily be integrated into existing systems or be developed as a new long-term system. 6. based on standardized sampling methods the sampling system must use the same data collection methods throughout if data are to be comparable. ideally this should be methods that are internationally agreed and approved. agreement should be sought for the methods from the other agencies on the ground to ensure consistency. without case definitions that are agreed by all parties the likelihood of success of a surveillance system is very low. this is especially so when laboratory support is minimal or absent since clinical case definitions have to be drawn very tightly if different diseases are not to be confused. routine surveillance requires more than material from ad hoc sources. sites such as medical centers (in towns, villages, or refugee camps), hospitals, and/or public health units should be recruited. the more comprehensive the coverage of the system, the more likely is it that the data will be accurate and complete and that problems will not be missed. such coverage can be problematic. the coverage of the different systems that can be used is discussed below. the data collected and the methods used should ideally fit in with systems that are operating or have previously operated in the area. following from criterion 10, if systems are already in existence or in abeyance but revivable then this should be done so as to ensure compliance by local health-care services and continuity of data collection and analysis. existing records are of considerable value for predictive purposes. knowledge of past problems makes it possible to anticipate future trends and problems and allows for early planning decisions. if several health agencies are operating it is essential to ensure collaboration among them in surveillance activities to avoid confusion and duplication of effort. 14. involve collaboration with local services so as to avoid duplication as above, early involvement of local health and surveillance services will reduce workloads and avoid duplication of effort. if those from whom the data are collected, those who are collecting the data, and those who will receive the results are unhappy with the system, the system is unlikely to operate effectively. these criteria can be used to evaluate a plan for a surveillance system and also, with some additions, to evaluate an existing system. however, failure to fulfil all these criteria need not rule out a system. in many emergencies it can be difficult to meet such a wide range of "best case" criteria, and the question that must be asked is whether the proposed system is capable of fulfilling its purposecan it provide sufficiently accurate essential information to those who need it when they need it? the emphasis of an emergency surveillance program may need to be altered as the situation changes especially if a particular item emerges as being of key importance. those running the surveillance program should use the data gathered and a continuous assessment of the general running of the system, to alter the program as required (preferably after consultation with relevant stakeholders). when designing health surveillance systems, it is essential to do the following: the population under surveillance may be relatively small and well defined (such as the population of a refugee camp) or a much less defined group such as mobile groups of refugees or idps or the population of a village, town, or region, the size of whose population may be unknown or may be fluctuating because of a disaster. establishment of denominators may therefore be difficult. even refugees or idp camps may present a challenge as, while the size of the population may appear to be (or actually be) stable, its makeup may vary over time because of movements in and out. if the age or sex makeup of the camp alters, the pattern of disease may also alter. both the number of cases detected and the rate of factors such as morbidity or mortality per unit of population are important values needed to inform emergency programs. those responsible for all aspects of health care need to know what numbers of cases are involved so as to ensure adequate provision of services (amounts of medicines, numbers of hospital beds, etc.). however, simple numbers are of little value in assessing trends and patterns since increases or decreases in numbers of cases (or numbers of deaths) may reflect changes in population size (resulting, for example, from population displacement) rather than a trend due to (for example) a particular disease. in addition, several rates (such as the crude mortality rate) are key indicators in defining health emergencies (see below). knowing the demography of the affected population is therefore important and all agencies working in an emergency should agree on and use the same population figures. the essential demographic data needed include the following: • total population size • population structure -overall sex ratio and the sex ratio in defined age groups -population under 5 years old, with age breakdown (0-4 years) -this group has special needs and is usually a key factor in planning the emergency response -age pyramid -ethnic composition and place of origin -number of vulnerable persons (e.g., pregnant and lactating women, members of female-headed households, unaccompanied children, destitute elderly, disabled and wounded persons) at the outset it is therefore important to establish methods to obtain demographic data. often the best that can be managed initially is a rough estimate, but this can usually be refined later. it is helpful to use several methods and cross-check the figures to obtain the best estimate. surrogates of the whole population (such as those attending a clinic) may be the best that can be achieved early on. the ease with which such data can be obtained usually depends on the size and scale of the population under consideration. the demography of a well-run refugee camp is quite easy to obtain but that of a larger area may be much more difficult. a lack of knowledge of the size of a displaced group can be confounded by a lack of knowledge of the size of the resident population. in many countries with poor infrastructures, accurate census data are not available. in some instances tax records may be helpful if these can be obtained. it should be noted that demographic data, especially if they involve refugees and idps, can be politically sensitive and interested parties may place undue weight on any figures that are given. ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. a surveillance system in a refugee or idp camp is effectively a miniature comprehensive system as it is possible to cover the whole population. there are situations where comprehensive surveillance is not possible and these often arise in disasters. damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. as far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. the calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. if there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. surveillance must provide information on key health indicators, which should include the following: the selection of information sought in these categories must be done carefully. it is neither possible nor desirable to monitor everything, especially in the early stages of a disaster response. at that stage (the acute phase) the priority of surveillance is the detection of factors that can have the greatest and most rapid effect on the population. in terms of communicable disease this means diseases that affect large numbers of people and have epidemic potential. in most instances this also means diseases for which effective rapid control measures exist. while gathering data on other largescale disease problems should not be excluded, the main surveillance and control efforts should be aimed where they can do the most immediate good. in the very early stages, only clinical information may be available since laboratory diagnostic services will probably be damaged or simply unavailable. however, this need not be a problem if the medical response is also geared to a syndromic approach. as the situation stabilizes, laboratory support becomes available, and longer term control measures can be supported, the surveillance can become more refined and additional diseases (for example, those which can cause severe morbidity and mortality in the longer term -such as tuberculosis, hiv or aids, and stds) can be added to the list. the main morbidity figures that are routinely sought are as follows: • incidence -the number of new cases of a particular disease reported over a defined period • attack rate (used in outbreaks -usually expressed as percentage) (also called incidence proportion or cumulative incidence) -number of new cases within a specified time period/size of the population initially at risk (×100). (e.g., if 30 per 1,000 persons develop a condition over 2 weeks, the ar/ip/ci is 30/1,000 [3.0%]) • incidence rate -number of new cases per unit of person-time at risk. in the above example, the ir is 15/1,000 person-weeks. (this statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) • prevalence -the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (nb: prevalence "rate" is the number of cases of a disease at a particular time/population at risk) there are a number of ways of estimating morbidity. health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. other ways of gathering morbidity data include the following: • surveys -in which data are collected from a small sample of the emergencyaffected population deemed to be representative of the whole (or from a particular group for a specific purpose) • outbreak investigations -which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures as with disease, changes in numbers of deaths may reflect changes in population size. determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. all deaths occurring in the community must therefore be recorded. the following indicators can provide the essential information to define the health situation in a population: • crude mortality rate (cmr) is the most important indicator as it indicates the severity of the problem, and changes in cmr show how a medical emergency is developing. cmr is usually expressed as number of deaths per 10,000 persons per day. if the cmr rises above 1/10,000 per day (>2/10,000 per day for young children) an acute emergency is developing and the emergency phase lasts until the daily cmr falls to 1/10,000 per day or below. • age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). in children this is usually given as the number of deaths in children younger and older than 5 years/1,000 children of each age/day). nb: if population data for the under 5s are not available, an estimate of 17% of the total population may be used. • maternal mortality rate. maternal mortality is a sensitive indicator of the effectiveness of health-care systems. a maternal death is usually defined as the death of a woman while pregnant or within 42 days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. the 42-day cut-off is recommended by who but some authorities use a time of up to a year. maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × 1,000 (or ×100,000) • cause-specific death rates (case fatality rates -usually given as a percentage). proportion of cases of a specified condition which are fatal within a specified time. case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × 100 the following indicators must be measured: • prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children 6-59 months of age (or 60-110 cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) • prevalence of severe acute malnutrition in children 6-59 months of age (or 60-110 cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) • • estimate number of children needing to be cared for in selective feeding programs • estimate number of additional calories per day provided by selective feeding programs immunization programs are a vital part of the public health measures undertaken following disasters. for example, measles vaccination is one of the most important health activities in such situations. the need for campaigns may be assessed on the basis of national vaccination records if they exist. in the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). the effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. in less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. depending on the circumstances it may be necessary to monitor these elements in the affected population. indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. activities in related sectors such as water and sanitation, shelter and security may also be included. the major sources of health data will be hospitals and clinics (both national and those established by aid agencies), individual medical practitioners, and other health-care workers. specialized agencies should be able to provide data on particular needs (e.g., food, water, sanitation, and shelter). case definitions are an essential part of surveillance. if the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless -changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. this is especially important when laboratory confirmation is not possible. it is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. case definitions must be prepared for each health event or disease or syndrome. if available, the case definitions used by the host country's moh should be used to ensure continuity of data. several different sets of case definitions already exist, either in generalized form (for example, those produced by the centers for disease control in atlanta) or sets prepared for specific emergencies (e.g., the who communicable disease toolkit for the iraq crisis in 2003). standard case definitions may have to be adapted according to the local situation. it should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. when case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see table 13 .2). although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. samples can subsequently be sent to a referral laboratory for confirmation. once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. visits to surveillance sites and discussions with staff involved will help define the recording and data transmission systems required. the great advances in information technology that have been made in recent years have greatly facilitated the collection, recording, transmission, and analysis of surveillance data, but care must be taken that the systems put in place are appropriate. in areas where electricity supplies are problematical and communications poor it may be better to use a paper recording system and verbal data transmission by radio than a computerized system. data verification is essential for the credibility of a surveillance system. those responsible for surveillance systems must ensure good adherence to case definitions if a symptom-based system is in operation and that laboratory quality control systems operate where appropriate. regular assessments of record keeping and the accuracy of data transfer are required. triangulation of results from several sources can sometimes help to detect anomalies. frequency of reporting will usually depend on the severity of the health situation. in general, daily reporting during the acute phase of an emergency will be needed, although in an acute medical emergency (such as a severe cholera outbreak) even more frequent reporting may be necessary, especially if the situation is fluctuating rapidly. the frequency may reduce to (say) weekly as the situation resolves. who is to analyze the data and how it is to be analyzed must be established at the outset. in a relatively defined area such as a camp, a data analysis session may be the last of the daily activities of the person responsible for surveillance. if record keeping and analysis protocols have been carefully worked out initially this task is not necessarily a large additional burden. surveillance systems that cover larger areas and bigger and more diffuse populations usually rely on a central data collection point where designated staff analyze the data. use of such a system requires good data transmission systems. output is as important as input. collecting data without dissemination of results is a sterile exercise and tends rapidly to demotivate those who are collecting the data. there are some important points to consider: • the results of surveillance must be presented in a readily comprehensible form. • surveillance reports should be produced regularly and widely distributed to aid agencies, and to national and international governments and organizations. this will help those involved to understand the overall picture, rather than just that in the area where they are working, and will allow them to take informed decisions about future actions. surveillance systems should be evaluated constantly to ensure that they are working properly, that the data are representative, analysis is appropriate and accurate, and that results are being disseminated to where they are needed. the public health aspects of communicable disease control can be broadly divided into preventive activities (such as vector control and vaccination programs) and the investigation and control of outbreaks and epidemics. experience from many emergencies and disasters has made it possible to identify a number of syndromes or diseases that are most likely to occur in such situations (table 13 .3). this makes it possible to plan activities and interventions on the basis of likely occurrences, even before those involved are present at the scene of the disaster, and to make initial purchases and establish stockpiles of appropriate medicines and equipment. "prevention is better than cure" and proper attention to preventive measures from the earliest stage of the response to the disaster will greatly reduce the risks to the health of the population from infectious disease. a key method of preventing communicable disease is the provision of shelter, adequate amounts of clean water, sufficient safe food, and proper sanitation (latrines and facilities for personal hygiene, clothes washing, and drying). arthropod vectors (mosquitoes, ticks) can be controlled by appropriate spraying programs and also by habitat management (e.g., the removal of places where water can accumulate and mosquitoes breed). provision of bed nets, particularly nets impregnated with insecticide, is effective for reducing infection with agents such as malaria and leishmania. control of rodents, by proper control of rubbish, by rodent proofing food stores, by attention to domestic hygiene and by use of rodenticides, will reduce the risks of transmission of rodent-borne diseases such as plague and lassa fever. medical waste includes laboratory samples, needles and syringes, body tissues, and materials stained with body fluids. this requires careful handling, especially the sharps, as infectious agents such as those causing hepatitis b and c, hiv and aids, and viral hemorrhagic fevers can be transmitted by these materials. used sharps should be disposed of into suitable containers (proper sharps boxes are ideal but old metal containers such as coffee or milk powder tins are adequate). medical waste should ideally be burned in an incinerator. this should be close to the clinic or hospital but downwind of the prevailing wind. a 200-l oil drum can be used for this purpose with a metal grate half way up and a hole at the bottom to allow in air and for the removal of ash. larger-scale and more permanent incinerators can be constructed if necessary. burning pits can be used in emergency. if burning is not possible items should be buried at least 1.5 m deep. this is more suitable than burning for large items of human tissue such as amputated legs. ensure there is no risk of groundwater contamination. a few others, such as malaria and other vector-borne diseases (e.g., typhus and leishmaniasis), are also likely to occur but are region specific. tb and hiv or aids can also cause major problems in the longer term this is a complex process involving not just considerations of infection risk but also legal, sociocultural, and psychological factors. there are a number of specialist publications which can be of help. after almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. however, in sudden impact disasters (such as the indian ocean tsunami in 2004), the pattern and incidence of disease found in the dead will generally reflect those in the living. the situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. in fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. the diseased living are far more dangerous. the decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. those most at risk are those handling the deceased, not the community. the most likely risks to them are as follows: mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. the dead must always be treated with dignity and respect. as far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. if the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. when an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). in general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). bodies should be buried at least 1.5 m deep or, if more shallowly, should have earth piled at least 1 m above the ground level and 0.5 m to each side of the grave (to prevent access by scavengers and burrowing insects). disinfectants such as chloride of lime should not be used. new burial sites should be at least 250 m from drinking water sources and at least 0.7 m above the saturated zone. vaccination programs are an essential part of disease prevention. information about existing vaccination programs must be obtained during the assessment process and this should include information from external assessors (e.g., who, unicef, ngos) as to the effectiveness of the vaccination programs that have been undertaken in the past. it cannot be assumed that simply because children have received vaccines that these vaccines were effective. measles kills large number of children in developing countries and is one of the greatest causes of morbidity and mortality in children in refugee and idp camps. mass vaccination of children between the ages of 6 months and 15 years should be an absolute priority during the first week of activity in humanitarian situations and can be conducted with the distribution of vitamin a. a system for maintaining measles immunization must be established once the target population has been covered adequately in the initial campaign. this is necessary to ensure that children who may have been missed in the original campaign, children reaching the age of 6 months, and children first vaccinated at the age of 6-9 months who must receive a second dose at 9 months of age are all covered. some of the children vaccinated during such a mass campaign may have been vaccinated before. this does not matter and a second dose will have no adverse effect. it is essential to ensure full coverage against measles in the population. other epi vaccinations for children are not generally included in the emergency phase because they can only prevent a minor proportion of the overall morbidity and mortality at that stage. however, should specific outbreaks occur then the appropriate vaccine should be considered as a control measure. vaccination programs require the following: • appropriate types of vaccines. • appropriate amounts of these vaccines. • equipment (needles, syringes, sterilization equipment, sharps disposal). emergency immunization kits, including cold chain equipment, are available from a number of sources, including unicef and some ngos (e.g. msf). • logistics (transport, cold chain). • staff: a vaccination team may be quite large. it must include the following personnel: -a supervisor. -logistics staff. -staff to prepare and administer vaccines. -record keepers. -security staff (to maintain order and control crowds) may also be needed. maintenance of the cold chain is particularly important. this is the system of transporting and storing vaccines within a suitable temperature range from the point of manufacture to the point of administration. the effectiveness of vaccines can be reduced or lost if they are allowed to get too cold, too hot, or are exposed to direct sunlight or fluorescent light. careful note should be taken of the conditions needed to transport different vaccines because these can vary. the essential cold chain equipment needed to transport and store vaccines within a consistent safe temperature range includes the following: • dedicated refrigerators for storing vaccines and freezers for ice packs (fridges and freezers powered by gas or kerosene are available as alternatives to electric machines, and solar-powered fridge/freezer combinations specially designed for vaccine storage are also available) • a suitable thermometer and a chart for recording daily temperature readings if possible, vaccines should be stored in their original packaging because removing the packaging exposes them to room temperature and light. check the temperature to ensure the vaccines have not been exposed to temperatures outside the normal storage ranges for those vaccines (see table 13 .4). max. storage time at the different levels: primary, 6 months; region, 3 months; district, 1 month; health center, 1 month; health post, daily usemax. 1 month diluents must never be frozen. freeze-dried vaccines supplied packed with diluent must be stored between +2 and +8°c. diluents supplied separately should be kept between +2 and +8°c vaccines must be kept at the correct temperature since all are sensitive to heat and cold to some extent. all freeze-dried vaccines become much more heat-sensitive after they have been reconstituted. vaccines sensitive to cold will lose potency if exposed to temperatures lower than optimal for their storage, particularly if they are frozen. some vaccines (bcg, measles, mr, mmr, and rubella vaccines) are also sensitive to strong light and must always be protected against sunlight or fluorescent (neon) light. these vaccines are usually supplied in dark brown glass vials, which give them some protection against light damage, but they must still be covered and protected from strong light at all times. only vaccine stocks that are fit for use should be kept in the vaccine cold chain. expired or heat-damaged vials should be removed from cold storage. if unusable vaccines need to be kept for a period before disposal (e.g., until completion of accounting or auditing procedures) they should be kept outside the cold chain, separated from all usable stocks and carefully labelled to avoid mistaken use. diluents for vaccines are less sensitive to storage temperatures than are the vaccines with which they are used (although they must be kept cool), but may be kept in the cold chain between +2 and +8°c if space permits. however, diluent vials must never be frozen (kept in a freezer or in contact with any frozen surface) as the vial may crack and become contaminated. when vaccines are reconstituted, the diluent should be at same temperature as the vaccine, so sufficient diluent for daily needs should be kept in the cold chain at the point of vaccine use (health center or vaccination post). at other levels of the cold chain (central, provincial, or district stores) it is only necessary to keep any diluent in the cold chain if it is planned to use it within the next 24 h. freeze-dried vaccines and their diluents should always be distributed together in matching quantities. although the diluents do not need to be kept in the cold chain (unless needed for reconstituting vaccines within the next 24 h), they must travel with the vaccine at all times, and must always be of the correct type, and from the same manufacturer as the vaccine that they are accompanying. each vaccine requires a specific diluent, and therefore, diluents are not interchangeable (for example, diluent made for measles vaccine must not be used for reconstituting bcg, yellow fever, or any other type of vaccine). likewise, diluent made by one manufacturer for use with a certain vaccine cannot be used for reconstituting the same type of vaccine produced by another manufacturer. some combination vaccines comprise a freeze-dried component (such as hib) which is designed to be reconstituted by a liquid vaccine (such as dtp or dtp-hepb liquid vaccine) instead of a normal diluent. for such combination vaccines, it is again vital that only vaccines manufactured and licensed for this purpose are combined. note also that for combination vaccines where the diluent is itself a vaccine, all components must now be kept in the cold chain between +2 and +8°c at all times. as for all other freeze-dried vaccines, it is also essential that the "diluent" travels with the vaccine at all times. the effectiveness of a vaccination program will need to be assessed. the program can be evaluated both by routinely collected data and, if necessary, by a survey of vaccination coverage. routine data on coverage is obtained by comparing the numbers vaccinated with the estimated size of the target population (and clearly depends on accurate assessment of the latter). a coverage survey requires the use of a statistical technique called a two-stage cluster survey details of which can be found in the appropriate who/epi documents. information about the effectiveness of the campaign should be obtained from routine surveillance of communicable disease. if, for example, large number of measles cases continue to occur, or there is an outbreak, then data on coverage should be reexamined. if this is shown to be good (over 90%) then the efficacy of the vaccine must be suspected. if the field efficacy is below the theoretical value 85% (for measles vaccine -data on efficacy of other vaccines can be obtained online) then possible causes of a breakdown in the vaccination program must be investigated (failure of the cold chain, poorly respected vaccination schedule). methods for measuring vaccine efficacy can be found in the who/epi literature. mass chemoprophylaxis for bacterial infections such as cholera and meningitis is not usually recommended except on a small scale (for example, the use of rifampicin may be considered to prevent the spread of meningococcal meningitis among immediate contacts of a case), but the difficulties of overseeing such activities and the risks of the development of antibiotic resistance outweigh any benefits that might be gained. the use of chemoprophylaxis for malaria must be undertaken with care. it may be indicated for vulnerable groups of refugees/idps (for example, children and pregnant women) arriving in an endemic area, particularly if they come from a nonmalarious area, but care must be taken to provide drugs to which the local strains of malaria are sensitive. the spread of resistance means that many of the standard drugs are ineffective and the replacements are both costly and may have unwanted side effects. public health education and information activities play a vital role in disease prevention. vaccination programs will not work unless there is acceptance by the public of the necessity for such programs. individuals must be informed as to why these programs are necessary and also where and when they need to take their children for vaccination. such activities are also essential to inform people about particular health programs (for example, feeding programs or vector control programs) and about the steps they can take to protect their health and that of their families (e.g., good hygiene). information can be propagated in many ways: staff who are trained in this type of activity therefore play a key role in disease prevention. heath education also requires transport and equipment (such as video or film projectors, screens, generators, blackboards, etc.). details of the treatment of individuals for various infectious diseases and the facilities needed are covered elsewhere in this book and in many textbooks covering disasters and disease response. in terms of the population aspects of the treatment of disease, important requirements are to ensure that there are • appropriate laboratories (microbiological, parasitological, hematological, biochemical) available to confirm diagnoses and monitor treatment. • adequate supplies of appropriate antimicrobial agents available and the facilities to transport these, store, and distribute them under appropriate conditions (e.g., controlled temperature), together with relevant instruction for use. the provision of laboratory facilities in emergencies is usually limited to basic tests such as those for malaria. more advanced tests, including identification of microorganisms and the determination of antimicrobial sensitivities, require more sophisticated facilities. these may be available in the affected country but are unlikely to be operating in the disaster-affected area. it is more likely that specimens will have to be transported to laboratories abroad. collection of specimens requires appropriate equipment. this will include items such as swabs, transport media, needles, syringes, or vacum sampling systems for blood sampling, different blood collection bottles (with and without anticoagulants) and other sterile specimen tubes, and containers for faeces and urine. transporting specimens must be done safely, and packing specimens for shipment requiring specially trained personnel. treatment of disease requires good supplies of appropriate antimicrobial agents. it is important to ensure that the agents chosen are suitable for use in the area. it is common for doctors in affected areas to ask for the latest therapeutic agents. however, these agents, although effective, are often expensive and not part of the normal treatment programs in the region. the local doctors may not therefore be familiar with the use of these agents, nor may laboratories be capable of monitoring their use. it is better to use funds, which are often limited, to supply larger amounts of older (generic) agents. one caveat is the possibility that regular use may have allowed resistance to certain agents to develop in a country. data on this may be available from local surveillance records. antimicrobials should always be supplied with relevant guidelines in a language that can be understood locally. if local laboratories are unable to test microbes for resistance to antimicrobials, isolates or specimens should be sent as soon as possible to appropriate reference laboratories for testing. outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. an epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. an outbreak is an epidemic limited to a small area (a town, village, or camp). the term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. the areas where vaccination campaigns are a priority need to be identified and campaigns started. the term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. this will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. infections where a single case represents a potential outbreak include the following: infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: • human african trypanosomiasis • visceral leishmaniasis a surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. however, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. in addition to the establishment of surveillance, outbreak preparation involves the following: • preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. • ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. • stockpiling essential supplies. this includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. • identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. • identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. ensure that vaccination supplies (needles, syringes, etc.) are adequate. make sure the cold chain can be maintained. • identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. if the number of reported cases is rising, is this in excess of the expected number? ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. in the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including moh, who, other un organizations, ngos, etc. in the case of an epidemic the moh will probably take the lead or may ask who or another un agency to do so. the team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. this may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the moh or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. this team should meet at least once a day to review the situation and define the necessary responses. it has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. the team should also act as the point of contact for the media. a media liaison officer should be appointed and all media contact should be through this individual. this will allow team members to refer media representatives to a central point and reduce interference with their activities. it will also ensure that a consistent message based on the most complete data is given to the media. the appropriate national authorities should be informed of the outbreak. in addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the revised international health regulations (2005) to report outbreaks of certain diseases. these include four diseases regarded as public-health emergencies of international concern: • smallpox • polio (wild-type) in some cases, member states must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and west nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, rift valley fever, and meningococcal disease). once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: • produce a case definition for the outbreak. this is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. • collect and analyze descriptive data by time, person, and place (time and date of onset, individual characteristics of those affected -age, sex, occupation, etc., location of cases). plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). • determine the population that is at risk. • determine the number of cases and the size of the affected population. calculate the attack rate. • formulate hypotheses for the pathogen about the possible source and routes of transmission. • conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). • report results and make recommendations for action. the two main statistical tools used to investigate outbreaks are as follows: • case-control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) • cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms however the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. • implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) • prevent infection (e.g., by vaccination programs) • prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) • evaluate the outbreak detection and response -were they appropriate, timely, and effective? • change/modify policies and preparedness to deal with outbreaks if required • what activities are needed to prevent similar outbreaks in the future (e.g., improved vaccination programs, new water treatment facilities, public health education, etc.)? • produce and disseminate an outbreak report. the report should include details of the outbreak, including the following: -cause -duration, location, and persons involved -cumulative attack rate (number of cases/exposed population) -incidence rate -case fatality rate -vaccine efficacy (if relevant) (no. of unvaccinated ill − no. of vaccinated ill/no. of unvaccinated ill) -proportion of vaccine-preventable cases (no. of vaccine-preventable cases/no. of cases) -recommendations this is an easy-to-use tool which is of great value for handling epidemiological data and for organizing study designs and results, which can be downloaded free of charge from the internet. it is produced by the centers for disease control (atlanta) and is a series of microcomputer programs which can be used both for surveillance and for outbreak investigation and includes features used by epidemiologists in statistical programs, such as sas or spss, and database programs such as dbase. public health action in emergencies caused by epidemics. geneva: who, 1986. cdc atlanta. case definitions for infectious conditions under public health surveillance updated guidelines for evaluating public health surveillance systems epidemiology for the uninitiated communicable disease control in emergencies -a field manual last jm (ed). dictionary of epidemiology medicins sans frontieres. refugee health -an approach to emergency situations geneva: international committee of the red cross sphere project. humanitarian charter and minimum standards in disaster response. geneva: the sphere project key: cord-103291-nqn1qzcu authors: chapman, lloyd a. c.; spencer, simon e. f.; pollington, timothy m.; jewell, chris p.; mondal, dinesh; alvar, jorge; hollingsworth, t. deirdre; cameron, mary m.; bern, caryn; medley, graham f. title: inferring transmission trees to guide targeting of interventions against visceral leishmaniasis and post-kala-azar dermal leishmaniasis date: 2020-02-25 journal: nan doi: 10.1101/2020.02.24.20023325 sha: doc_id: 103291 cord_uid: nqn1qzcu understanding of spatiotemporal transmission of infectious diseases has improved significantly in recent years. advances in bayesian inference methods for individual-level geo-located epidemiological data have enabled reconstruction of transmission trees and quantification of disease spread in space and time, while accounting for uncertainty in missing data. however, these methods have rarely been applied to endemic diseases or ones in which asymptomatic infection plays a role, for which novel estimation methods are required. here, we develop such methods to analyse longitudinal incidence data on visceral leishmaniasis (vl), and its sequela, post-kala-azar dermal leishmaniasis (pkdl), in a highly endemic community in bangladesh. incorporating recent data on infectiousness of vl and pkdl, we show that while vl cases drive transmission when incidence is high, the contribution of pkdl increases significantly as vl incidence declines (reaching 55% in this setting). transmission is highly focal: >85% of mean distances from inferred infectors to their secondary vl cases were <300m, and estimated average times from infector onset to secondary case infection were <4 months for 90% of vl infectors, but up to 2.75yrs for pkdl infectors. estimated numbers of secondary vl cases per vl and pkdl case varied from 0-6 and were strongly correlated with the infector's duration of symptoms. counterfactual simulations suggest that prevention of pkdl could have reduced vl incidence by up to a quarter. these results highlight the need for prompt detection and treatment of pkdl to achieve vl elimination in the indian subcontinent and provide quantitative estimates to guide spatiotemporally-targeted interventions against vl. . pkdl has therefore been recognised as a major 49 potential threat to the vl elimination programme in the isc 50 (10), which has led to increased active pkdl case detection. 51 nevertheless, the contribution of pkdl to transmission in 52 field settings still urgently needs to be quantified. 53 although the incidence of asymptomatic infection is 4 to 54 17 times higher than that of symptomatic infection in the 55 isc (21), the extent to which asymptomatic individuals con(fig. 1a) . the data from this study are fully de-105 scribed elsewhere (8, 31) . briefly, month of onset of symptoms, 106 treatment, relapse, and relapse treatment were recorded for 107 vl cases and pkdl cases with onset between 2002 and 2010 108 (retrospectively for cases with onset before 2007), and year of 109 onset was recorded for vl cases with onset before 2002. there 110 were 1018 vl cases and 190 pkdl cases with onset between 111 january 2002 and december 2010 in the study area, and 413 112 vl cases with onset prior to january 2002. 113 over the whole study area, vl incidence followed an epi-114 demic wave, increasing from approximately 40 cases/10,000/yr 115 in 2002 to ≥90 cases/10,000/yr in 2005 before declining to 116 <5 cases/10,000/yr in 2010 (fig. 1b) . pkdl incidence fol-117 lowed a similar pattern but lagging vl incidence by roughly 118 2yrs, peaking at 30 cases/10,000/yr in 2007. however, vl 119 and pkdl incidence varied considerably across paras (aver-120 age para-level incidences: vl 18-124 cases/10,000/yr, pkdl 121 0-31 cases/10,000/yr, table s5 ) and time (range of annual 122 para-level incidences: vl 0-414 cases/10,000/yr, pkdl 0-120 123 cases/10,000/yr, fig. s15 ). ú ci = credible interval, calculated as the 95% highest posterior density interval † risk of subsequent vl/asymptomatic infection if susceptible ‡ based on assumed infectiousness § in the absence of background transmission and relative to living directly outside the case household. based on the relative infectiousness of vl and the di erent 151 types of pkdl from the xenodiagnostic data, in the absence 152 of any other sources of transmission, the estimated probability 153 of being infected and developing vl if living in the same 154 household as a single symptomatic individual for 1 month 155 following their onset was 0.018 (95% ci: 0.013, 0.024) for vl 156 and ranged from 0.009 to 0.023 (95% cis: (0.007,0.013)-(0.018, 157 0.031)) for macular/papular pkdl to nodular pkdl. living 158 in the same household as a single asymptomatic individual, 159 the monthly risk of vl was only 0.00037 (95% ci: 0.00027, 160 0.00049), if asymptomatic individuals are 2% as infectious as 161 vl cases. 162 the risk of infection if living in the same household as an 163 infectious individual was estimated to be more than 10 times 164 higher than that if living directly outside the household of an 165 infectious individual (hazard ratio = 12.0), with a 95% ci 166 well above 1 (8.3, 16.7). the estimated spatial kernel (fig. 167 s16 ) around each infectious individual shows a relatively rapid 168 decay in risk with distance outside their household, the risk of 169 infection halving over a distance of 84m (95% ci: 71, 99m). mission. we assess the contribution of di erent infectious 172 groups to transmission in terms of their relative contribu-173 tion to the transmission experienced by susceptible individuals 174 ( fig. 2a and fig. s17 ). the contribution of vl cases was 175 fairly stable at around 75% from 2002 to the end of 2004 176 before decreasing steadily to 0 at the end of the epidemic, 177 while the contribution of pkdl cases increased from 0 in 178 2002 to ≥73% in 2010 (95% ci: 63, 80%) (fig. s17) . only a 179 small proportion of the total infection pressure on susceptible 180 individuals, varying between 9% and 14% over the course of 181 the epidemic, was estimated to have come from asymptomatic 182 and pre-symptomatic individuals. reconstructing the transmission tree. by sampling 1,000 193 transmission trees from the joint posterior distribution of 194 the transmission parameters and the unobserved data (as de-195 scribed in materials and methods), we can build a picture of 196 the most likely source of infection for each case and how infec-197 tion spread in space and time. fig. 3 shows the transmission 198 tree at di erent points in time in part of the south-east cluster 199 of villages. early in the epidemic and at its peak (figures 3a 200 and 3b), most new infections were due to vl cases. towards 201 the end of the epidemic, some infections were most likely due 202 to pkdl cases and there was some saturation of infection 203 around vl cases (fig. 3c) . the inferred patterns of trans-204 mission suggest that disease did not spread radially outward 205 from index cases over time, but instead made a combination 206 of short and long jumps around cases with long durations of 207 symptoms and households with multiple cases. . arrows show the most likely source of infection for each case infected up to that point in time over 1,000 sampled transmission trees, and are coloured by the type of infection source and shaded according to the proportion of trees in which that individual was the most likely infector (darker shading indicating a higher proportion). asymptomatic infections are not shown for clarity. s/a = susceptible or asymptomatic, e = pre-symptomatic, i = vl, r = recovered, d = dormantly infected, p = pkdl (see si text). gps locations of individuals are jittered slightly so that individuals from the same household are more visible. an animated version showing all months is provided in si movie 1. there is considerable heterogeneity in the estimated contriby each vl/pkdl case is typically less than 1 (fig. s19a ). the times after onset of symptoms in the infector at which 336 secondary vl cases become infected are typically longer for 337 pkdl infectors than for vl infectors (fig. 4b) detected after a longer delay than subsequent cases and there 356 will be some delay in mounting a reactive intervention, such 357 as active case detection and/or targeted irs around the index 358 case(s), interventions will need to be applied in a large radius 359 (up to 500m) around index cases to be confident of capturing 360 all secondary cases and limiting transmission. our results demonstrate the importance of accounting for 362 spatial clustering of infection and disease when modelling 363 vl transmission. previous vl transmission dynamic models 364 (23, 41-43) have significantly overestimated the relative con-365 tribution of asymptomatic infection to transmission (as up 366 to 80%), despite assuming asymptomatic individuals are only 367 1-3% as infectious as vl cases, by treating the population 368 as homogeneously mixing, such that all asymptomatic indi-369 viduals can infect all susceptible individuals via sandflies. in 370 reality, asymptomatic individuals do not mix homogeneously 371 with susceptible individuals as they are generally clustered 372 together around or near to vl cases (25, 28), who are much 373 more infectious and therefore more likely to infect suscepti-374 ble individuals around them, even if they are outnumbered 375 by asymptomatic individuals. asymptomatic infection also 376 leads to immunity, and therefore local depletion of suscep-377 tible individuals around infectious individuals. hence, for 378 the same relative infectiousness, the contribution of asymp-379 tomatic individuals to transmission is much lower when spatial 380 heterogeneity is taken into account. nonetheless, our results suggest that asymptomatic indi-382 viduals do contribute a small amount to transmission and 383 that they can "bridge" gaps between vl cases in transmission 384 chains, as the best-fitting model has non-zero asymptomatic 385 relative infectiousness. superficially, this appears to conflict 386 with preliminary results of xenodiagnosis studies in which 387 asymptomatic individuals have failed to infect sandflies ac-388 cording to microscopy (44). however, historical (12, 45) and 389 experimental (46) data show that provision of a second blood 390 meal and optimal timing of sand fly examination are criti-391 cal to maximizing sensitivity of xenodiagnosis. these data 392 suggest that recent xenodiagnosis studies (11, 44), in which 393 dissection occurred within 5 days of a single blood meal, may 394 underestimate the potential infectiousness of symptomatic 395 and asymptomatic infected individuals. occurrence of vl in 396 isolated regions where there are asymptomatically infected 397 individuals, but virtually no reported vl cases (27, 47), also 398 seems to suggest that asymptomatic individuals can generate 399 vl cases. however, it is possible that some individuals who de-400 veloped vl during the study went undiagnosed and untreated, 401 and that we have inferred transmissions from asymptomatic 402 individuals in locations where cases were missed. we will in-403 vestigate the potential role of under-reporting in future work. 404 the analysis presented here is not without limitations. as 405 can be seen from the model simulations (fig. s20) , the model is 406 not able to capture the full spatiotemporal heterogeneity in the 407 observed vl incidence when fitted to the data from the whole 408 study area, as it underestimates the number of cases in higher-409 incidence paras (e.g. paras 1, 4 and 12). there are various 410 possible reasons why the incidence in these paras might have 411 been higher, including higher sandfly density, lower initial lev-412 els of immunity, variation in infectiousness between cases and 413 within individuals over time, dose-dependence in transmission 414 (whereby flies infected by vl cases are more likely to create 415 vl cases than flies infected by asymptomatic individuals (22)), 416 where k(d) = e ≠d/is the spatial kernel function that determines 7 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . bangladesh (protocol #2007-003) and the centers for disease con • recovered (i.e. treated for primary vl, vl relapse or pkdl, or self-resolved from pkdl, or recovered from asymptomatic upon infection, individuals either develop pre-symptomatic infection with probability pi or asymptomatic infection with probability 1 ≠ pi (see table s2 for values of fixed parameters used in the model 2 of 37 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. whereis the rate constant for spatial transmission between infected and susceptible individuals; k(dij) is the spatial kernel function that scales the transmission rate by the distance dij between individuals i and j; " (ø 0) is a rate constant for additional within-household transmission; 1ij is an indicator function for individuals living in the same household, i.e. between an exponentially decaying spatial kernel and a cauchy-type kernel in our previous study (1) (the exponential kernel 73 gave a marginally better fit), we use the exponential kernel here: and asymptomatic individuals, we take the relative infectiousness of pre-symptomatic individuals, h0, to be the same as that of 93 asymptomatic individuals (i.e. h0 = h4). one-hundred and thirty-eight of the 190 pkdl cases underwent one or more examinations by a trained physician to 95 determine the type and extent of their lesions (table s1 ). data from a recent xenodiagnosis study in bangladesh (20) pre-symptomatic -thus, individual j's infectiousness at time t is given by [3] 113 incubation period. following previous work (1), we model the incubation period as negative binomially distributed nb(r, p) 114 with fixed shape parameter r = 3 and 'success' probability parameter p, and support starting at 1 (such that the minimum 115 incubation period is 1 month): we estimate p in the mcmc algorithm for inferring the model parameters and missing data (see below). vl onset-to-treatment time distribution. several vl cases with onset before 2002 have missing symptom onset and/or treatment times (only their onset year is recorded), and may therefore have been infectious at the start of the study period. in order to be able to infer the onset-to-treatment times of these cases, ot õ j = min(r õ j , d õ j ) ≠ i õ j (j = 1, . . . , ni 0 ), in the mcmc algorithm 121 (see below) we model the onset-to-treatment time distribution as a negative binomial distribution nb(r1, p1) and fit to the 122 onset-to-treatment times of all vl cases for whom both onset and treatment times were recorded ( figure s2a ): 124 to obtain r1 = 1.34 and p1 = 0.38 (corresponding to a mean onset-to-treatment time of 3.2 months). assume that all cases not recorded as having immediate recurrence of symptoms su ered treatment relapse and that the time to relapse follows a geometric distribution geom(p4) with pmf: where fitting to the recorded gaps gives p4 = 0.13 (corresponding to a mean time to relapse of 7.9 months). relapse cases are 156 assumed to be uninfectious from their treatment month to their relapse time and their duration of symptoms upon relapse 157 is assumed to follow the same distribution as the onset-to-treatment time for a first vl episode (eq. (5)). we assume all 158 relapse cases were treated for relapse before the end of the study, since the latest treatment time for primary vl in a case that 168 169 while the probabilities of pre-symptomatic or asymptomatic infection in month t given susceptibility up to month t ≠ 1 are, respectively: [12] model for initial status of non-symptomatic individuals. as there was transmission and vl in the population before the start the probabilities of each non-symptomatic individual initially present (i.e. with vj = 0) being susceptible, asymptomatically infected, or recovered from asymptomatic infection at time t = 0 can then be found by calculating the probability of avoiding infection in every month from their birth to the start of the study, summing over the probabilities of being infected in one of the months between their birth and the start of the study and recovering after the start of the study, and summing over the 6 of 37 probabilities of being infected in a month before the start of the study and recovering before the start of the study, respectively: ps 0 (aj) := p(aj > 0, rj > 0) = e ≠⁄ 0 a j [13] pa 0 (aj) := p(aj ae 0, where aj is the age of individual j in months at t = 0. since we assume that non-symptomatic individuals who are born, or 183 who immigrate into the study area, after the start of the study (with vj > 0) are susceptible, for notational convenience we 184 define the probabilities for these individuals as ps 0 (aj) = 1, pa 0 (aj) = pr 0 (aj) = 0. we estimate the historical asymptomatic infection rate, ⁄0, by fitting the model to age-prevalence data on leishmanin skin 186 test (lst) positivity amongst non-symptomatic individuals from a cross-sectional survey of three of the study paras conducted 187 in 2002 (28) (see figure s4 ). we assume that entering state r corresponds to becoming lst-positive, as lst positivity is 188 a marker for durable, protective cell-mediated immunity against vl (28, 29), and estimate ⁄0 by maximising the binomial with these definitions, the complete data likelihood for the augmented data z = (y, x) given the model parameters ◊ = (-, -, ', ", p) is composed of the products of the probabilities of all the di erent individual-level events over all months: the joint posterior distribution of the model parameters ◊ = (-, -, ', ", p) and the missing data x given the observed data y [17] 223 we do this using a metropolis-within-gibbs mcmc data augmentation algorithm in which we iterate between sampling from 224 the conditional posterior distribution of the parameters given the observed data and the current value of the missing data, t=v j qj(t) + qj(t + 1) is a normalising constant to account for the fact that we know that j was not preand ", which are non-negative, since there is little information available with which to construct informative priors (table s3) . the mean of the prior distribution foris chosen as 100m based on our previous findings (1) . a beta distribution, beta(a, b) , 266 is chosen as a conjugate prior for the incubation period parameter p, since it is a probability (p oe where -= (-, -, ', "), so p can be updated e ciently in the mcmc by drawing from this full conditional distribution rather than using a random walk metropolis-hastings update. 1 ≠ aj,0) for rj,0 = t + 1. by repeating the following steps. note that throughout the following we suppress notation of conditional dependencies in the 320 likelihood terms where they are obvious to maintain legibility. the algorithm also accounts for the fact that some individuals 321 were born or migrated or died during the study when updating the unknown pre-symptomatic infection times and asymptomatic 322 infection and recovery times (using the birth/migration/death times as bounds on the proposed unobserved times), but we 323 omit these details from the following description for simplicity. (b) accept the infection time move with probability where (c) i. if aj = 0: if rj = 0, if rj > 0. ii. if aj = t + 1: a. if a õ j = t + 1, then r õ j = t + 1, so accept immediately as the likelihood does not change. step 4(c)ic, except with . 365 iii. if aj oe [1, t ]: a. if a õ j = 0, follow step 4(c)ia, but with q replaced by step 4(c)ib except with step 4(c)ic but with 6. update missing treatment times of vl cases during the study: 382 update the treatment time of the vl case whose treatment time is missing but whose onset time is known, conditional 383 on the treatment time being before their pkdl onset: 384 (a) propose a new treatment time as update the onset and treatment times of all cases who potentially had active vl at the start of the study (t = 1) who n(0, 4) ) " = 0 p1) . update the treatment times of cases who potentially had active vl at the start of the study whose treatment times were 390 not recorded but whose onset times are known, one by one. for each case j: p1) . 9. update whole relapse period of cases missing both relapse and relapse treatment times: 392 update the relapse and relapse treatment times of all vl cases who su ered relapse during the study who are missing step 4 in the above algorithm may appear complicated, but essentially consists of proposing a new asymptomatic infection is the empirical covariance of the last k ≠ f (k) + 1 samples offrom the chain, with the mean of the last k ≠ f (k) + 1 samples; 0 is the initial guess for the covariance matrix, and k0 determines the rate at 442 which the influence of 0 on k+1 decreases (the weight of 0 halves after the first 2k0 iterations). we use k0 = 1000 here. if f (k) = f (k ≠ 1) (i.e. if k is odd with f (k) chosen as above), an additional observation is added to the estimate of the 444 covariance matrix if k is even), the new observation replaces the oldest it has been shown that n (k , 2.38 2 /n -), where is the covariance matrix of the posterior distribution, is the optimal 453 proposal distribution for rapid convergence and e cient mixing of the mcmc chain for symmetric product-form posterior 454 distributions as nae oe, and leads to an acceptance rate of 23.4% (38, 39) . this corresponds to a scaling of c k = 1 in eq. (23). however, we are in a context with a large amount of missing data, which is strongly correlated with some of the transmission 456 parameters (see parameter estimates below), so the posterior distribution is not symmetric, and this scaling is not optimal. 457 we therefore follow (36) and scale c k adaptively as the algorithm progresses to target an acceptance rate of approximately 458 23.4% for updates to -. we do this by rescaling c k by a factor of x k > 1 every time an acceptance occurs and by a factor of 459 x ‹/(‹≠1) k < 1 every time a rejection occurs such that the acceptance rate ‹ approaches 23.4% in the long run, if proposal is rejected. in order to satisfy the 'diminishing adaptation' condition (40), which is necessary to ensure the markov chain is ergodic and converges to the correct posterior distribution, it is required that c k tends to a constant as k ae oe. so that the adaptation 463 diminishes as k increases, we use the sequence 465 where m0 is the number of iterations over which the scaling factor x k decreases from 2 to 1.5. here, we use m0 = 100. model comparison 467 we compare the goodness of fit of models with di erent asymptomatic and pre-symptomatic relative infectiousness (between 0% and 2% of that of vl cases), with and without additional within-household transmission, to test di erent assumptions about how infectious asymptomatic and pre-symptomatic individuals are, using dic (41). dic measures the trade-o between model fit and complexity and lower values indicate better fit. since some variables were not observed, we use a version of dic appropriate for missing data from (42), which is based on the complete data likelihood l(◊; z) = p(y, x|◊). this is equivalent to the standard version of dic for fully observed data except that it is averaged over the missing data: where d(◊) is the deviance (the measure of model fit), given (up to an additive constant dependent only on the data) by . [26] the relative contribution of state x to the infection pressure on the ith vl case at their infection time, i.e. the probability 483 that i's infection source is x (fig. 2b in the main text), is: , x oe {a, e, i, p}. [27] the probability that the ith vl case is infected from the background transmission is ' ⁄i(ei ≠ 1) . reconstructing the epidemic reconstructing the transmission tree. we reconstruct the transmission tree following the 'sequential approach' described 488 in (44). we draw n samples (◊ k , x k ) (k = 1, . . . , n) from the joint posterior distribution from the mcmc, calculate the 489 probability that infectee i was infected by individual j conditional on their infection time ei and uncertainty in the parameter values and missing data (over the posterior distribution). we use n = 1000 here. calculating transmission distances and times. the mean infector-to-vl-infectee distance and mean infector-onset-to-vlinfectee-infection time for each vl and pkdl infector ( figures 4a and 4b in the main text) are calculated from the sample of n transmission trees by averaging the distances and times from each infector to their vl infectees within each tree, and then averaging these quantities over all the trees in which that vl/pkdl case is an infector: where · [33] the absolute contribution of each infectious state to the e ective reproduction number at time t is: where x oe {a, i} denotes the infectious state, and, as described above, in the main text we split the numbers of secondary 517 infections (rj) arising from vl and pkdl for cases that had both. to assess the fit of the model and simulate hypothetical interventions against pkdl, we create a stochastic simulation version of 520 the individual-level spatiotemporal transmission model described above. we follow standard stochastic simulation methodology 521 for discrete-time individual-level transmission models (47), converting infection event rates into probabilities in order to 522 determine who gets infected in each month. we assume that an individual's progression through di erent infection states 523 following infection occurs independently of the rest of the epidemic (i.e. is either governed by internal biological processes or 524 random external processes of detection), which enables the simulation of an individual's full infection history from the point of 525 infection. so that we can simulate durations of pkdl infectiousness, we fit a negative binomial distribution nb(r5, p5) to the observed 527 pkdl onset-to-treatment times and onset-to-resolution times for self-resolving pkdl cases in the data: given these pieces of information, the simulation algorithm proceeds as follows: negative binomially distributed (48). the pmf of a size-biased negative binomial random variable x ú corresponding to 542 x ≥ nb is: and assign a pkdl infectious by drawing from cat({h1, h2, h3, hu}, p) . 562 ii. else the individual recovers without developing pkdl, so draw a recovery time: figure 575 s5 and table s4 respectively. based on the deviance distributions and dic values, the best-fitting model is the model with 576 additional within-household transmission and the highest level of relative pre-symptomatic and asymptomatic infectiousness 577 (both 2% as infectious as vl). hence, we focus on the output of this model in the main text and below. and ") and incubation period distribution parameter p for the di erent models are shown in table s4 . the parameter estimates 580 are very similar across the di erent models and vary in the way expected -the spatial transmission rate constantand 581 background transmission rate ' are lower for models with additional within-household transmission (" > 0) and decrease with 582 increasing relative asymptomatic infectiousness h4, and the mode foris slightly larger for models with " > 0 (since a flatter 583 kernel shape compensates for the extra within-household transmission). the posterior distributions for the incubation period 584 distribution parameter p correspond to a mean incubation period of 5.7-6.9 months (95% hpdis (4.8,6.6)-(6.0,7.8) months). the log-likelihood trace and posterior distributions for the parameters for the best-fitting model are shown in figure s6 . the parameters are clearly well defined by the data, as the posterior distributions di er significantly from the weak prior 587 distributions. the corresponding autocorrelation plots are shown in figure s7 . the high degree of autocorrelation evident for all the 589 parameters is due to strong correlation between the transmission parameters and the missing data, in particular between the 590 spatial transmission rate constantand the asymptomatic infection times. figure s8 shows thatis strongly negatively 591 correlated with the mean asymptomatic infection timeā. this is expected since a higher overall transmission rate leads to show that there is some negative correlation betweenand ', -and ', and " and p. these correlations are not surprising: the 603 more transmission that is explained by proximity to infectious individuals (the higher -), the less needs to be explained by the 604 background transmission (the lower '); the flatter the spatial kernel (the larger -), the fewer infections need to be explained by 605 the background transmission; and the more infections are accounted for by transmission within the same household (the higher 606 "), the longer the incubation period (the lower p) needs to be (due to long times between onsets of cases in the same household). the acceptance rate for the transmission parameter updates (step 1 in the mcmc algorithm) was 23. demonstrate that the data augmentation algorithm works as expected. figure s10 shows the incidence curve of vl and pkdl 615 cases for the whole study area and the inferred incidence curve of asymptomatic infections (averaged over the mcmc chain). the number of asymptomatic infections increases and decreases with the number of vl cases as expected given the assumption 617 that the incidence ratio of asymptomatic to symptomatic infection is fixed. the posterior probabilities that individuals were asymptomatically infected during the study (shown in figure s11 , with is higher). this is as expected given the structure of the model (the decrease in the risk of infection with distance from an 622 infectious individual encoded in the spatial kernel) and the estimates of the transmission parameters. the examples shown in figure s12 demonstrate that non-symptomatic individuals' asymptomatic "infection" time posterior distributions (red). note that asymptomatic "infection" in months 0 and t + 1 = 109, represent asymptomatic infection before the study and no asymptomatic infection before the end of the study, respectively. (a) individual who migrated into a house with an active vl case from outside the study area in month 53 and therefore had a high initial probability of asymptomatic infection, followed by further peaks in asymptomatic infection risk in months 64 and 69 with the pkdl and vl onsets of two other household members in months 63 and 68 respectively. (b) individual born in month 2 with a high probability of having avoided asymptomatic infection for the duration of the study. (c) individual who was 23-years-old at the start of the study with a moderately high risk of having been asymptomatically infected before the study and a small peak in asymptomatic infection risk when a fellow household member had vl onset in month 45. the role of case proximity in transmission of visceral leishmaniasis in a highly endemic village in 649 visceral leishmaniasis in the indian subcontinent: modelling epidemiology and control feasibility of eliminating visceral leishmaniasis from the indian subcontinent: explorations with a set 653 of deterministic age-structured transmission models elimination of visceral leishmaniasis in the indian subcontinent: a comparison of predictions from 655 three transmission models risk factors for kala-azar in bangladesh increasing failure of miltefosine in the treatment of kala-azar in nepal and the potential role of parasite 658 drug resistance, reinfection, or noncompliance single locus genotyping to track leishmania donovani in the indian 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quantification of the natural history of visceral leishmaniasis and consequences for control. parasites 700 & vectors characterization and identification of suspected counterfeit miltefosine 702 capsules loss of leishmanin skin test antigen sensitivity and potency in a longitudinal study of visceral leishmaniasis 704 in bangladesh age trends in asymptomatic and symptomatic leishmania donovani infection in the indian 706 subcontinent: a review of data from diagnostic and epidemiological studies estimating parameters in stochastic compartmental models using markov chain methods bayesian inference for partially observed stochastic epidemics bayesian analysis for emerging infectious diseases linking time-varying symptomatology and intensity of 715 infectiousness to patterns of norovirus transmission coupling and ergodicity of adaptive markov chain monte carlo algorithms accelerating adaptation in the adaptive metropolis hastings random walk algorithm weak convergence and optimal scaling of random walk metropolis algorithms. the 722 annals appl optimal scaling for various metropolis-hastings algorithms examples of adaptive mcmc deviance information criteria for missing data models the deviance information criterion: 12 years on (with discussion) methods to infer transmission risk factors in complex outbreak data di erent epidemic curves for severe acute respiratory syndrome reveal similar impacts of control 733 measures a bayesian approach to quantifying the e ects of mass poultry vaccination upon the spatial and 735 temporal dynamics of h5n1 in northern vietnam dynamics of the 2001 uk foot and mouth epidemic: stochastic dispersal in a heterogeneous 737 landscape the mathworks, inc julia: a fresh approach to numerical computing julia v1.0.5. the julia project 3 37 2 3 nw 998 830 18 3 24 4 4 nw 2185 1768 197 35 124 22 5 nw 934 774 15 2 22 3 6 nw 1640 1363 94 22 77 18 7 se 604 493 42 7 95 16 8 se 585 496 8 0 18 0 9 se 969 809 33 6 45 8 10 se 701 595 17 3 32 6 11 se 1300 1080 28 9 29 9 12 se 2807 2388 102 25 47 12 13 se 933 816 36 7 49 10 14 se 446 391 23 3 65 9 15 se 660 574 15 3 29 6 16 nw 905 762 26 9 38 13 17 nw 2080 1764 75 13 47 8 18 nw 3212 2653 61 11 26 5 19 nw 774 647 62 18 107 31 total 24781 20798 1018 190 54 key: cord-102850-0kiypige authors: huang, c.-c.; lai, j.; cho, d.-y.; yu, j. title: a machine learning study to improve surgical case duration prediction date: 2020-06-12 journal: nan doi: 10.1101/2020.06.10.20127910 sha: doc_id: 102850 cord_uid: 0kiypige predictive accuracy of surgical case duration plays a critical role in reducing cost of operation room (or) utilization. the most common approaches used by hospitals rely on historic averages based on a specific surgeon or a specific procedure type obtained from the electronic medical record (emr) scheduling systems. however, low predictive accuracy of emr leads to negative impacts on patients and hospitals, such as rescheduling of surgeries and cancellation. in this study, we aim to improve prediction of operation case duration with advanced machine learning (ml) algorithms. we obtained a large data set containing 170,748 operation cases (from jan 2017 to dec 2019) from a hospital. the data covered a broad variety of details on patients, operations, specialties and surgical teams. meanwhile, a more recent data with 8,672 cases (from mar to apr 2020) was also available to be used for external evaluation. we computed historic averages from emr for surgeonor procedure-specific and they were used as baseline models for comparison. subsequently, we developed our models using linear regression, random forest and extreme gradient boosting (xgb) algorithms. all models were evaluated with r-squre (r^2), mean absolute error (mae), and percentage overage (case duration > prediction + 10 % & 15 mins), underage (case duration < prediction 10 % & 15 mins) and within (otherwise). the xgb model was superior to the other models by having higher r^2 (85 %) and percentage within (48 %) as well as lower mae (30.2 mins). the total prediction errors computed for all the models showed that the xgb model had the lowest inaccurate percent (23.7 %). as a whole, this study applied ml techniques in the field of or scheduling to reduce medical and financial burden for healthcare management. it revealed the importance of operation and surgeon factors in operation case duration prediction. this study also demonstrated the importance of performing an external evaluation to better validate performance of ml models. it becomes more and more important for clinics and hospitals in managing resources for 2 critical cares during the covid-19 pandemic. statistics show that approximately 60 % 3 of patients admitted to the hospital will need to be treated in the operation room 4 (or) [11] , and the average cost of or is up to 2,190 dollars per hour in the united 5 states [1, 6] . hence, the or is considered as one of the highest hospital revenue 6 generators and accounts for as much as 42 % of a hospital's revenue [6, 10] . based on 7 these statistics, a good or schedule and management is not only critical to patients 8 who are in need of elective, urgent and emergent operations, but is also important for 9 surgical teams to be prepared. owing to the importance of or, improvement of or 10 efficiency has high priority so that the cost and time spent on or is minimized while the 11 utilization of or is maximized to increase surgical case number and patient access [15] . 12 in a healthcare system, numerous factors are involved in affecting or efficiency, for 13 example patient expectation and satisfaction, interactions between different professional 14 specialties, unpredictability during operations, surgical case scheduling and etc [20] . 15 although the process of or is complex and involves multiple parties, one way to 16 enhance or efficiency is by increasing the accuracy of predicted surgical case duration. 17 over-or under-utilization of or time often leads to undesirable consequences such as 18 idle time, overtime, cancellation or rescheduling of surgeries, which may implement 19 negative impact on the patient, staffs and hospital [21] . in contrast, high efficiency in 20 or scheduling not only contribute to better arrangement for the usage of operating 21 room and resources, it can also lead to cost reduction and revenue increment since more 22 surgeries can be performed. 23 currently, most hospitals schedule surgical case duration by employing estimations 24 from surgeon and/or averages of historical case durations, and studies show that both of 25 these methods have limited accuracy [14, 17] . for case length estimated by surgeons, 26 factors including patient conditions, anesthetic issues might not be taken into 27 consideration. moreover, underestimation of case duration often occurs as surgeon 28 estimations were usually made by leaning towards maximizing block scheduling to 29 account for potential cancellations and cost reduction. furthermore, operations with 30 higher uncertainty and unexpected findings during operation add difficulties and 31 challenges into case length estimation [14] . historic averages of case duration for a 32 specific surgeon or a specific type of operation obtained from electronic medical record 33 (emr) scheduling systems have also been used in hospitals. however, these methods 34 have been shown to produce low accuracy due to large variability and lack of same 35 combination in the preoperative data available on the case that is being performed [25] . 36 in order to improve the predictability, researchers utilized linear statistical models, 37 such as regression, or simulation for surgical duration prediction and evaluation of the 38 importance of input variables [8, 12, 13] . however, a common shortcoming of these 39 studies is that relatively lesser input variables or features were used in their models due 40 to the limitation of statistical techniques in handling too many input variables. similarly, we combined categories for primary surgeon's id, specialty, anesthesia type 101 and room number which had case numbers less than 50 into the category of 'others'. in addition, since operation case duration can be related to the performance of 103 surgeons and surgeons' performance is affected by their working time, we also analysed 104 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 12, 2020. . figure 1 . the workflow of model training for this study. the data set used for model training fall within the time range of jan 1, 2017 to dec 31, 2019. from this data set, about 17 % of the cases were excluded based on these criteria: patients with two or more surgical procedures performed at the same time, emergent and urgent cases, surgeons with age under 28, patients with age younger than 20, pregnant patients, procedure duration longer than 10 hours or less than 10 minutes and cases with missing value. the total number of cases included in the data set for model building was 142,448. this data set was then split into training (80 %) and validation (20 %) subsets for model development. machine learning and linear regression models were developed on the training data set and validated on the validation data set using r-square and mean absolute error. percentage of cases with actual duration differences falling within 10 % and 15 minutes of predicted procedure duration was also computed. eventually, the models were further evaluated on the most recent surgical cases (from mar 1 to apr 30, 2020) which were not included in the original data set for model training. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. 105 surgical minutes performed by the same primary surgeons on the same day as well as 106 within the last 7 days, and the number of urgent and emergent operations prior to the 107 case that was being performed by the same surgeon were included in the analysis. together, 24 predictor variables were included for predictive model building in this 109 study. these predictors can be categorised into 5 groups: patient, surgical team, 110 operation, facility and primary surgeon's prior events (see table 1 ). model development and training 112 we applied multiple ml methods for operation case duration prediction. operation case 113 duration (in minutes) is the total period starting from the time patient entering into the 114 or to the time exiting the or. historic averages of case durations based on 115 surgeon-specific or procedure-specific from emr systems were used as baseline models 116 for comparison in case duration prediction. at the beginning, we performed multivariate 117 linear regression (reg) to predict operation case duration. however, when we looked at 118 the distribution of operation case duration, it was observed to be skewing to the right 119 ( fig. 2) . we performed logarithmic transformation on operation case duration to reduce 120 the skewness. the model built from log transformed multivariate linear regression 121 (logreg) outperformed reg in all evaluation indexes. subsequent ml algorithms were 122 also trained by using the log transformed case duration as the target. the first ml algorithm that we tested is random forest (rf), a tree-based 124 supervised learning algorithm. rf uses bootstrap aggregation or bagging technique for 125 regression by constructing a multitude of decision trees based on training data and 126 outputting the mean predicted value from the individual trees [19] . bagging technique 127 is unlikely to over-fitting, in other words, it reduces the variation without increasing the 128 bias. tree-based techniques were suitable for our data since they include a large number 129 of categorical variables, e.g. icd code and procedure type, most of which were sparse. 130 the number of trees that was set in study is 50. extreme gradient boosting (xgb) 131 algorithm is the other supervised ml algorithm that was tested for comparison to rf. 132 recently, xgb algorithm gains popularity within the data science community due to its 133 ability in overcoming the curse of dimensionality as well as capturing the interaction of 134 variables [18] . xgb is also a decision tree-based algorithm but more computationally efficient for 136 real-time implementation than rf. xgb and rf algorithms are different in the way of 137 how the trees are built. it has been shown that xgb performs better than rf if 138 parameters are tuned carefully, otherwise it would be more likely to over-fitting if the 139 data are noisy [3, 9] . we adopted 5-fold cross validation strategy to tune out the best 140 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . data-splitting strategy was used in the training for all the models to prevent 145 over-fitting consequences. we randomly separated the data into training and testing 146 subsets at a ratio of 4:1. the training data were used to build different predictive 147 models as well as to extract important predictor variables. the testing data were used 148 for internal evaluation of the models.in addition to interval evaluation, external 149 evaluation on all the models were performed using data from mar 1 to apr 30, 2020. surgeon-or procedure-specific calculated from emr were also evaluated on the same 154 internal and external testing sets to ensure fair and uniform comparison across all 155 models. data processing and cleaning as well as model development in this study were 156 performed using r software. the packages "xgboost and "randomforest were used to 157 implement xgb and rf algorithms in r [4, 5] . 164 r 2 is the coefficient of determination, it represents the proportion of the variance for 165 the actual case duration that is explained by predictor variables in our models. mean absolute error (mae) measures the average of errors between the actual case 167 6/15 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. hand, the average model based on a specific procedure had lower percentage underage 188 and overage compared to the surgeon-specific model. these differences were due to an 189 extensive procedure classification in the procedure-specific model. however, the 190 percentage underage was still quite high. since no other information is taken into 191 consideration in the average model, except durations of operation cases happened in the 192 past, prediction bias and low accuracy usually result from the average model. 193 we first fitted the reg model by including all the input variables showed in table 1 . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. when its performance is better on training set but poor on testing set. when we log transformed operation case duration and re-ran a regression model (i.e. 203 logreg), the performance of logreg model improved and outperformed reg model. [12, 23] . again in the logreg model, the results of all the evaluation metrics were 208 close for training, internal and external training sets, so the model was not over-fitting. 209 although performance of the logreg model was not bad, an assumption of linear performance of the xgb model was better than the rf model on training set but did 219 not improve a lot compared to the rf model on internal and external testing sets. since xgb was more computing efficient than rf, the xgb model was chosen to be 221 the best model and was used in subsequent analysis. in addition to the three key metrics, we studied inaccuracy of different models by 223 using external testing set. we calculated the total prediction error (in minutes) and the 224 corresponding inaccurate percentage for all the models. the results are reported in 225 in fig. 3 , we plotted scatter plots of actual versus predicted duration on the external 234 testing set for the average models of surgeon-and procedure-specific, and the xgb 235 model. a straight line indicating the theoretical perfect relationship, i.e. predicted and 236 actual procedure duration are identical, was added as a reference in each scatter plot. the data points of the xgb models were aligned closer to the straight line. therefore, 238 the xgb model showed a higher correlation between predicted and actual duration 239 compared to the other two types of average model. fig. 4 shows the density plot of 240 differences between actual and predicted case durations for the two average models and 241 the xgb models. it clearly demonstrates that the error distribution of xgb model was 242 narrower and closer to 0. as a result, the xgb model is more accurate than the other 243 models in predicting operation case duration. 244 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . . density plot of differences between the actual operation case durations and predicted case durations obtained from the xgb model (light blue color) was narrower and centered more at 0 than density plots of those obtained from the average models (pink and cyan colors). in the average models, previous operation case durations, either averaging for a specific surgeon (cyan color) or specific procedure (pink color), were used as predictions. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. how important the variable is in making a branch of a decision tree to be purer [5, 22] . 248 a higher wfg percentage indicates that the variable is more important. the result of 249 the top 15 important variables are shown in table 4 . one thing worth noting is that 3 250 of the top 4 important variables are attributed to operation information. moreover, 251 three of the features which we computed from surgeons' data (i.e. total surgical minutes 252 performed by the surgeon within the last 7 days and on the same day, and number of accurate prediction of operation case duration is vital in elevating or efficiency and 257 reducing cost. this study not only helps to improve accuracy of or case prediction, it 258 also has novelty in the following aspects. first, the data set used in this study contained 259 more than 140,000 cases and more than 400 different types of surgical procedures which 260 set up a new benchmark for huge amount and large diversity. the maximal number of 261 cases that had been used in other studies were in the range of 40,000 to 60,000 [2, 21] . second, or events was modeled as dependent events instead of independent. to this 263 end, we extracted some additional information from surgeons' data, e.g. previous . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06. 10.20127910 doi: medrxiv preprint april 2020 as external testing data for model evaluation. fourth, though urgent and 268 emergent surgeries were excluded from the data, number of urgent and emergent 269 operations prior to the case that was being performed by the same surgeon was included 270 as an input variable to account for its effect on operation case duration. currently, surgical cases at cmuh are scheduled according to estimates made by 272 primary surgeons. however, surgeon estimates rely heavily on prior experiences of the 273 surgeons and many factors beyond expectation will not be taken into consideration. since there is no formal record on surgeon estimates, we used averages calculated based 275 on a specific surgeon or procedure type on the testing set to be our baseline models. the performance of these two average models, as reported in table 2 , clearly showed 277 that these models were poor in predicting operation case duration. they also tended to 278 under-predict operation case duration according to their scatter plots of actual versus 279 prediction and density plot of differences between actual versus prediction (see fig. 3 280 and 4). when 24 feature variables ( table 1) were included in our model development, 281 r 2 , mae, percentage underage, overage and within improved greatly compared to the 282 baseline models. we applied 15 minutes as tolerance threshold for percentage underage, 283 overage and within because â± 15 minutes is an acceptable periodic range in cmuh to 284 be considered as accurately booking. to avoid having too stringent standard and to 285 better compare our outcomes with other studies [2, 24] , tolerance threshold of 10 % was 286 also applied. by using regression and ml approaches, we were able to decrease the total 288 prediction error (table 3 ) of operation case durations at cmuh. among all the models, 289 performance of the xgb model was considered to be the best because it was more 290 computing efficient and had the lowest inaccuracy. moreover, even though the results of 291 evaluation metrics of the rf model were similar to the xgb model, the xgb model 292 was still able to reduce the total prediction error in minutes from 223,686 to 218,415 293 minutes. in other words, the xgb model was able to save more than 5,000 minutes of 294 idle or delay times than the rf model. since most ors usually have multiple cases 295 scheduled per day, the total prediction error represents the cumulative effect of total 296 or cases in the 2-month period of mar to april 2020. this cumulative effect may 297 eventually reflects a significant financial advantage in scheduling an additional 298 operation case [7] . this would also lead to a significant cost reduction and increment in 299 revenue because ors are utilized appropriately and efficiently. it has been reported in the past studies that primary surgeons contributed the 301 largest variability in operation case duration prediction compared to other factors 302 attributed to patients [2, 16, 23] . these studies provide evidence and rationale that more 303 factors relating to primary surgeon should be added as input variables in the training of 304 ml models. moreover, extensive feature engineering usually improve the quality of ml 305 model which can be independent to the modeling technique itself. as a result, in 306 addition to primary surgeon's identifier, gender and age, we computed previous working 307 time and number of previous surgeries performed by the same primary surgeons within 308 the last 7 days and on the same day. we also counted the number of urgent and 309 emergent operations prior to the case that was being performed by the same primary 310 surgeon. these variables extracted from the data of primary surgeon were significantly 311 (p < 0.05) correlated with operation case duration (see table 5 in appendix). the 312 correlation coefficients of these variables also revealed that an operation case duration 313 performed by a primary surgeon may decrease as he or she becomes more familiar with 314 the surgical procedure but may increase if his or her total surgical minutes are too long. 315 although performing a surgery multiple times on different patients may help a primary 316 surgeon to be more efficient in his or her next operation, long working time may also 317 lead to lethargic and affect the primary surgeon's performance. in the methodology of data processing, for predictor variables which contained a lot 319 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 12, 2020. . of categories, we grouped categories that had cases less than 50 into a categories named 320 'others'. in addition to reducing data dimensionality for categorical features, this may 321 aid in generalization of our model. this indicates that our model will still be able to 322 predict case duration even for operations that are rare. moreover, our model can be 323 applied to new primary surgeons, who are not included in the training set during model 324 development, by setting their id as 'others' for case duration prediction. however, 325 there is still a need to update our model after a while, for example, when the operation 326 cases performed by a new primary surgeon has increased beyond a certain number. in 327 terms of timing, we recommend updating the model annually by using operation cases 328 performed in the most recent 3 years as training data. one limitation in this study is that we selected predictor variables which could only 330 be extracted from preoperative data. our ml model still needs to be improved in order 331 to be able to predict surgical case duration dynamically. for example, blood loss during 332 operation may affect case duration as an unexpected increase in blood loss may cause 333 surgeons to take longer time to complete the surgery. therefore, it would be better if 334 intra-operative data are incorporated during ml model development and prediction 335 made by the ml model can be updated during operation. one common issue in all ml 336 studies in predicting operation case duration, including our study, is that ml models 337 were developed using data from a single site. these ml models have difficulties in 338 generalization, since the surgical team, facilities and patient populations are different 339 across entities. it has to be custom made for a given organization using training data 340 containing its patients, procedures, surgeons, medical staffs, and the facility itself. as a 341 result, the exact same ml model is not meant to and will not perform well when 342 applied to another organization or hospital. the other interesting issue of applying ml 343 or artificial intelligence in operation estimation is that medical technologies evolve fast. 344 hence, how frequent should a ml or artificial intelligence model need to be updated 345 still remains to be answered. the xgb model was superior in predictive performance when comparing to the average, 348 the reg and the logreg models. the total inaccuracy of predicted outcomes of the xgb 349 model was the lowest among the other models developed in this study. although the 350 performance of the rf model was close to the xgb model, the xgb model was more 351 computing efficiency than the rf model in which it took shorter time to complete the 352 training process. the coefficient of determination (r 2 ) was higher while percentages of 353 under-and over-prediction of the xgb model built in this study were also lower than 354 other ml studies [2, 21, 24] . moreover, this model improves the current or scheduling 355 method which is based on estimates made by surgeons at cmuh. 356 we propose extracting additional information from operation and surgeons' data to 357 be used as predictor variables for ml algorithm training since their importance was 358 high in the xgb model. moreover, we validated the model types using an external 359 testing set in additional to the internal testing set split from the original data used in 360 model training. this helped us to validate and test the models in a more stringent and 361 rigorous way. therefore, we suggest external evaluation should be used as a tool to 362 better validate the predictive power of ml models in the future. 363 1 appendix . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 12, 2020. table 5 . correlation coefficient, standard error, t-value and p-value of predictor variables extracted from primary surgeons' data. these information were obtained from the log transformed multivariate regression (logreg) model. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 12, 2020. . https://doi.org/10.1101/2020.06.10.20127910 doi: medrxiv preprint optimization and planning of operating theatre activities: an original definition of pathways and process modeling improving operating room efficiency: machine learning approach to predict case-time duration a comparative analysis of xgboost package 'randomforest' title breiman and cutler's random forests for classification and regression package 'xgboost' type package title extreme gradient boosting understanding costs of care in the operating room decrease in case duration required to complete an additional case during regularly scheduled hours in an operating room suite predicting the unpredictable: a new prediction model for operating room times using individual characteristics and the surgeon's estimate greedy function approximation: a gradient boosting machine factors that influence the expected length of operation: results of a prospective study surgical unit time utilization review: resource utilization and management implications surgical duration estimation via data mining and predictive modeling: a case study use of simulation to assess a statistically driven surgical scheduling system improving predictions of pediatric surgical durations with supervised learning the surgical scheduling problem: current research and future opportunities tree boosting with xgboost -why does xgboost win "every" machine learning competition? tree boosting with xgboost -why does xgboost win "every" machine learning competition? newer classification and regression tree techniques: bagging and random forests for ecological prediction operating room efficiency improved prediction of procedure duration for elective surgery decision tree methods: applications for classification and prediction. shanghai archives of psychiatry surgeon and type of anesthesia predict variability in surgical procedure times a machine learning approach to predicting case duration for robot-assisted surgery relying solely on historical surgical times to estimate accurately future surgical times is unlikely to reduce the average length of time cases finish late the authors would like to thank shu-cheng liu, jhao-yu huang and min-hsuan lu in 365 providing feedback during the progress of this study. key: cord-022034-o27mh4wz authors: olano, juan p.; peters, c.j.; walker, david h. title: distinguishing tropical infectious diseases from bioterrorism date: 2009-05-15 journal: tropical infectious diseases doi: 10.1016/b978-0-443-06668-9.50124-1 sha: doc_id: 22034 cord_uid: o27mh4wz nan bioterrorism can be defined as the intentional use of infectious agents or microbial toxins with the purpose of causing illness and death leading to fear in human populations. the dissemination of infectious agents with the purpose of attacking livestock and agricultural resources has similar motives. many of the agents that could potentially be used in bioterror (bt) attacks are also responsible for naturally occurring infectious diseases in the tropics. as such, naturally occurring outbreaks must be differentiated from bt attacks for public health, forensic, and security reasons. if a bt attack occurs in tropical underdeveloped countries, owing to their weak public health infrastructure, the public health implications would be even more dramatic than in developed countries. an outbreak of smallpox due to a bt attack would probably require vaccination and mandatory quarantine of millions of people in order to control the outbreak and quell global public unrest. this chapter will concentrate on selected infectious agents that have the potential to be used as bioterror agents in human populations. the first step in managing the damage from a covert biological dissemination is recognition of the attack and the organism(s). as in most emerging infections, we predict that in bioterrorist attacks the etiological diagnosis will be made by a clinician or pathologist and the recognition of a bioterrorist event will be through geographical and epidemiological anomalies. we have very limited environmental detection capability at this time, and there are no comprehensive pointof-care diagnostics for most of the high-impact bt agents. some diseases such as inhalational anthrax or smallpox may be relatively readily recognized by an alert clinician because of their very distinctive presentation in many cases. however, the leading edge of a bt epidemic may arrive on a pathologist' s doorstep without prior suspicion. for example, individual cases of pneumonic plague as the earliest harbingers of an attack will presumably present as community-acquired pneumonia and probably die without clinical diagnosis. given the short window available for successful treatment, the recognition of these earliest cases is paramount. sartwell 1 has demonstrated empirically that incubation periods follow a log-normal distribution, which results in "front-loading" of cases ( fig. 119-1 ). delay in recognizing the epidemic through reliance on syndromic surveillance or other surrogates will likely result in most of the cases of diseases such as plague and tularemia being well into their disease course and perhaps unsalvageable. 2 bioterrorist events will enlarge our knowledge of tropical diseases. for example, inhalational anthrax and several viral hemorrhagic fevers (vhf) thought to be transmitted mainly by aerosol 3 are under-represented in naturally occurring case series, and a bt attack would provide an opportunity to answer questions about the underlying host factors and pathogenesis. indeed, the extension of the risk population to include children, the elderly, and the immunosuppressed is likely to provide considerable insight into these oftenunderstudied groups. it is also likely that our lack of information about them will challenge our current diagnostic algorithms. in october 2001, anthrax spores were distributed covertly in the u.s. postal service, leading to 22 cases of human anthrax and billions of dollars spent on controlling the potentially devastating effects of a small inhalational anthrax epidemic. 4, 5 this attack was by no means the first intentional attempt to use infectious agents as weapons of terror. ever since the times of the ancient greeks and romans, humans have tried to inflict damage by the use of contagion on other populations. 6, 7 less than 4% of the people or groups responsible for terrorist attacks on human populations take responsibility for their actions. 8 therefore, the use of biological weapons is ideal to conduct covert attacks. in addition, it has been estimated that to kill the same number of human beings with biological weapons as compared to chemical or nuclear weapons, the cost is far less with biological weapons ($2/human casualty) compared with chemical ($2000/ human casualty) and nuclear ($2,000,000/human casualty) weapons. 6 hypothetical bt attacks would range from an overt attack of a large city with a bomb containing several kilograms of an agent (weaponized bacteria, viruses, or toxins) to discrete or covert intentional release of the infectious agent through a delivery system, such as spray devices, postal service, ventilation ducts, water supplies, and food supply. based on transmissibility, severity of morbidity and mortality, and likelihood of use (availability, stability, weaponization), potential bt agents are divided into three categories (table 119-1) . this chapter will concentrate on selected agents from categories a and b and on the diagnostic challenges posed by illnesses caused by such agents. table 119 -1 are capable of producing illness under natural circumstances. therefore, the first challenge is to identify the infectious agent responsible for a certain disease correctly, followed by a thorough epidemiologic and microbiologic analysis of the epidemic or outbreak. in some circumstances, the identification of a bt attack would be obvious. a case of smallpox in any human population is an international emergency that would trigger a massive response of the public health systems around the world. sophisticated epidemiological investigations would follow in order to characterize the outbreak, identify the source, and possibly label it "intentional." in other cases, the identification of the outbreak as secondary to intentional dissemination of an infectious agent will require the use of sophisticated epidemiological and molecular tools, especially for diseases endemic to the area where the outbreak occurs. the need to use genetic sequences as markers has spawned a new discipline referred to as microbial forensics, sister to phylogenetics and "molecular epidemiology." differentiation between natural infections and a biological warfare attack rests firstly on disease patterns given by several epidemiological clues. they include presence of disease outbreaks of the same illness in noncontiguous areas, disease outbreaks with zoonotic impact, different attack rates in different environments (indoor versus outdoor), presence of large epidemics in small populations, increased number of unexplained deaths, unusually high severity of a disease for a particular pathogen, unusual clinical manifestations owing to route of transmission for a given pathogen, presence of a disease (vector-borne or not) in an area not endemic for that particular disease, multiple epidemics with different diseases in the same population, a case of a disease by an uncommon agent (smallpox, viral hemorrhagic fevers, inhalational anthrax), unusual strains of microorganisms when compared to conventional strains circulating in the same affected areas, and genetically homogenous organisms isolated from different locations. 9, 10 these are a few guidelines that could prove helpful when investigating an outbreak, but it has to be kept in mind that the deduction will not be based on any single finding but rather the pattern seen in its totality. first and foremost, the possibility of an attack must be ever in mind, or differentiation of a covert bt attack and a natural outbreak of an infectious disease may not be made. in fact, the outbreak of salmonellosis in oregon in 1984 was due to a covert attack planned by the rajneeshee leadership and accompanied by distinctive epidemiological clues. it was not labeled as intentional until somebody came forward with the information leading to the responsible group; as in most of medicine, the unsuspected diagnosis is the easiest to miss. 11 an increasing number of public health departments are now acquiring the technology necessary to perform syndromic surveillance. this new method of surveillance is based on syndromic disease rates such as respiratory, gastrointestinal, and neurological syndromes or analysis of other health-related activities such as laboratory test requests and results, purchasing rates for certain pharmaceutical agents, unexplained death rates, and veterinary surveillance. 2, 10, 11 the purpose of syndromic surveillance is to detect a bt attack as early as possible by analyzing the previously mentioned variables by extracting and analyzing data through computer networks. the rationale behind syndromic surveillance is the nonspecific nature of early signs and symptoms of many of the illnesses caused by bt agents. examples of proposed syndromes are as follows: gastroenteritis of any apparent infectious etiology, pneumonia with the sudden death of a previously healthy adult, widened mediastinum in a febrile patient, acute neurologic illness with fever, and advancing cranial nerve impairment with weakness. 12 a key component factors affecting syndromic surveillance include selection of data sources, definition of syndrome categories, selection of statistical detection thresholds, availability of resources for follow-up, recent experiences with false alarms, and criteria for initiating investigations. it must be emphasized that these systems are experimental and not yet of proven value in managing bt attacks. they are expensive, require follow-up confirmation, have unproven sensitivity and specificity, and ultimately depend on the clinician. 2 they may prove to be more useful in managing an event than in expeditiously detecting one. conventional epidemiological investigations are by no means obsolete with the availability of more sophisticated methods to study possible bt attacks. they include the confirmation of an outbreak once it is suspected. confirmation is based in many cases on laboratory analysis of patients' samples or autopsy material. a case definition is constructed to increase objectivity of the data analyzed and to enable determination of the attack rate. other variables are included in the analysis, such as time and place, and an epidemiological curve can be constructed. 10 epidemiological curves are an important tool to analyze epidemics and suggest the mode of transmission and propagation. a point source epidemic curve is classically log-normal in distribution 1 and would suggest a common exposure of a population to an infectious agent. of course, there can be variations depending on the presence of susceptible subpopulations (e.g., children, immunosuppressed, aged) and on varying doses of the agent. propagative curves are more characteristic of highly communicable agents such as smallpox. a short description of selected category a and b agents follows. all these pathogens are addressed as naturally occurring disease agents in other chapters of this book. bacillus anthracis (anthrax) b. anthracis (see chapter 39) is without a doubt the microorganism that has received the most attention as a bt agent due to its high lethality (inhalational form), ease of propagation, and high environmental stability. fortunately, the disease is not transmitted from person to person. however, the first three characteristics make it one of the ideal bioweapons. anthrax presents in humans as four different clinical syndromes, depending on the portal of entry: cutaneous (the most common form of the disease resulting from contact with infectious animal products), gastrointestinal and oral/oropharyngeal (both secondary to ingestion of contaminated meat), and inhalational (woolsorter' s disease), secondary to inhalation of spores from the environment. in the event of a bioterror attack, either overt or covert, the clinical presentation of the patients affected by the attack would be that of inhalational anthrax. this form of anthrax is so rare that a single case of inhalational anthrax should raise immediate suspicion, as dramatically demonstrated during the bt attacks in the fall of 2001. [13] [14] [15] during those attacks, 50% of cases were cutaneous anthrax thought to be secondary to handling of anthraxlaced mail envelopes or environmental surface contamination in the presence of minor cutaneous lesions, providing a portal of entry for the spores. 5 an outbreak of inhalational anthrax also took place in sverdlovsk (former soviet union) as a result of an accidental release into the air of b. anthracis spores from a facility producing anthrax for the bioweapons program in the ussr. 5, [16] [17] [18] inhalational anthrax should be suspected clinically in any individual presenting with fever and a widened mediastinum on chest radiograph (due to hemorrhagic mediastinitis). 19, 20 the incubation period is normally 3 to 5 days, but in some cases it can be as short as 2 days and as long as 60 days depending on inoculum and the time of germination of the spore. 17 based on research performed on rhesus monkeys, the ld 50 is estimated to be 8000 to 10,000 spores. [21] [22] [23] however, as few as 1 to 3 spores may be capable of producing a fatal outcome in approximately 1% of those exposed to these quantities. 24 the initial symptoms are nonspecific and consist of fever, malaise, anorexia, fatigue, and dry cough. these symptoms are followed in 3 to 4 days by an abrupt onset of respiratory insufficiency, stridor, diaphoresis, and cyanosis. the subsequent clinical course is rapid, and patients usually die within 24 to 36 hours after clinical deterioration. mortality is 100% without antibiotic therapy. 20, [25] [26] [27] early diagnosis, aggressive treatment with antimicrobial agents to which the bacteria are susceptible, and aggressive supportive therapy decreased the mortality to 40% in the 2001 attacks. 5 pathologic studies performed on the sverdlovsk victims confirmed some of the findings in animal models of inhalational anthrax, such as hemorrhagic lymphadenitis and mediastinitis. however, many patients also developed hematogenous hemorrhagic pneumonia. pleural effusions were usually large and frequently led to severe lung atelectasis. in about half of cases, hemorrhagic meningitis developed, leading rapidly to central nervous system (cns) manifestations terminating in coma and death. 16, 28, 29 yersinia pestis (plague) y. pestis (see chapter 42) is a gram-negative, aerobic, nonsporulating coccobacillus, member of the enterobacteriaceae with a wide host range, including rodents, felines, and humans. 30 the most important reservoirs are urban rats, and its main vector is the rat flea. in rural epizootics, reservoirs include prairie dogs and squirrels in the united states. 31 y. pestis has been responsible for some of the most devastating pandemics in human history in the preantibiotic era (6th, 14th, and 19th centuries). 32 public health measures have made this disease a rarity in the united states (around 20 cases/year) and around the world, although approximately 1000 cases are reported to the world health organization (who) every year (countries reporting plague include madagascar, tanzania, and peru, among others). clinical presentation in naturally acquired infections takes five forms, namely bubonic, septicemic, pneumonic, cutaneous, and meningeal. the pneumonic form is the most likely presentation in a case of plague due to a bt attack. it is worth mentioning that plague has already been used as a bt agent when japan dropped thousands of y. pestis-infected fleas over china leading to small outbreaks of bubonic plague in continental china during world war ii. 33, 34 the incubation period for pneumonic plague is short, ranging from 2 to 3 days. it is the rarest form in natural infections (1% or less) but has the highest mortality, reaching 100% in untreated patients. the initial presentation is nonspecific and consists of cough, fever, and dyspnea. cough may be productive (bloody, purulent, or watery in the initial phases). this is followed by a rapid clinical course leading to respiratory failure and the patient' s demise if not treated with antibiotics early in the course of the disease. 30, 31, 35 the factors that led to the severe manchurian pneumonic plague outbreaks in the early 20th century are unknown, but weather, hygiene, and crowding were important factors. more recent outbreaks worldwide and particularly in the united states have been much smaller and readily controlled. pneumonic cases are common in the united states, but secondary transmission has been rare in the last 50 years. modeling of pneumonic transmission using eight small outbreaks to derive the parameters find average of secondary cases per primary case (ro) to be approximately 1.3 prior to any control measures. 36 this is one of the most scientifically neglected microorganisms with bt potential. tularemia is a zoonotic infection caused by a strictly aerobic, gram-negative, nonsporulating small coccobacillus. two subspecies are recognized, namely f. tularensis subspecies holarctica (jellison type b) and f. tularensis subspecies tularensis (jellison type a). 37 type a is by far the more virulent and is present only in north america. of the bacteria with potential as bt agents, f. tularensis has by far the widest host range, including wild and domestic animals, humans, fish, reptiles, and birds. vectors are also numerous and include ticks, fleas, mosquitoes, and biting flies. 37, 38 this is an impressive range for any human pathogen. in contrast to other diseases described in this chapter, tularemia does not have the remarkable history that some of the other pathogens have. in europe, tularemia was first described in 1532; in the united states, it was first described in 1911 in california in the aftermath of the san francisco earthquake. 38 in natural infections, the most common source of infection is a tick bite and manipulation of infected animals such as wild rabbits. six different clinical syndromes have been described as follows: ulceroglandular, glandular, oculoglandular, pharyngeal, pneumonic, and typhoidal. marked overlap exists among all these forms, and for practical purposes two syndromes (ulceroglandular and typhoidal) have been proposed. [39] [40] [41] as a bt agent, f. tularensis will most likely cause a disease with a primary pulmonary component with secondary dissemination (typhoidal/systemic). in natural infections, both ulceroglandular and typhoidal forms can have a hematogenous pulmonary component, although it is more common in typhoidal forms. pulmonary features include cough, pleural effusions, and multifocal bronchopneumonic infiltrates. if not treated promptly, patients usually develop adult respiratory distress syndrome leading to respiratory insufficiency and the patient' s demise. case-fatality rate approaches 30% if not treated with appropriate antibiotics. 41 smallpox eradication remains the single most important victory in the war against infectious diseases. smallpox (see chapter 58) is the only disease so far eradicated from the face of the earth due to human intervention. the who declared smallpox eradicated in 1980 after the last case of natural disease was diagnosed in somalia in 1977, 42 and vaccination ceased around the world, rendering humankind vulnerable to reintroduction of the virus. [43] [44] [45] a laboratory accident was responsible for two more cases in 1978 in england. this accident prompted the who to restrict the frozen virus to two places in the world: the cdc in atlanta, georgia, and the institute for polyomyelitis and viral encephalitides in moscow, later moved to npo vector, novosibirisk, russia. however, it is suspected that secret military repositories exist after the fragmentation of the soviet union and the subsequent exodus of scientists involved in its bioweapons program (biopreparat). 46, 47 the agent responsible for this disease is an orthopox virus with no known animal reservoir, but high aerosol infectivity, stability, and mortality. although not a category a agent, monkeypox is responsible for outbreaks in africa and is the only other member of the orthopox genus capable of producing systemic disease in humans. the clinical disease is potentially indistinguishable from smallpox, where mortality rates in tropical africa are around 10% to 15%. in may and june 2003, an outbreak of monkeypox occurred in the united states. 48 thirty-seven infections were laboratory-documented and involved humans exposed to infected prairie dogs that had become infected because of contact with infected gambian rats and dormice, two animal species shipped from africa earlier that year. infected humans included veterinarians, exotic pet dealers, and pet owners. the clinical spectrum in this outbreak ranged from asymptomatic seroconversions to febrile illness with papulovesicular rash. no deaths were associated with this outbreak. however, phylogenetic analysis of the virus placed it in the west africa clade as opposed to the central africa clade which carries the previously mentioned case-fatality rate of 10% to 15%. a single case of smallpox would trigger a massive public health response in order to contain the outbreak. an outbreak in germany in 1970 resulted in 19 cases with 100,000 people vaccinated to contain the infection. in 1972, yugoslavia underwent an epidemic with a total of 175 cases (35 deaths) and a vaccination program that included 20 million people in order to contain the outbreak and obtain international confidence. vaccination with the vaccinia virus (a related orthopox virus) is the most effective way to prevent the disease and can be administered up to 4 days after contact with ill patients. strict quarantine with respiratory isolation for 17 days is also mandatory. the newer generation of antivirals that have been developed after the disease was eradicated has never been tested in human populations, but in vitro data and experiments in animal models of poxvirus disease suggest some antiviral activity for the acyclic nucleoside phosphonates such as cidofovir. 49 the only vaccine available in the united states is dryvax, and sufficient doses have been manufactured to cover the entire u.s. population. however, newer vaccines that may have fewer side effects are being developed. the clinical presentation is characteristic. the incubation period ranges from 10 to 12 days. the initial phase is nonspecific, common to other viral syndromes, and is characterized by abrupt onset of fever, fatigue, malaise, and headaches. during this prodromal phase in 10% of patients with fair complexion, a discrete erythematous rash appears on the face, forearms, and hands. the typical smallpox rash has a centrifugal distribution (that is, more abundant on the face and extremities than on the trunk and abdomen). an enanthem also develops with presence of oral ulcerations by the time the exanthem appears. systemic manisfestations begin to subside once the rash appears and can reappear with superinfection of skin lesions or superimposed bacterial bronchopneumonia. progression of the lesions is synchronous (maculopapules, vesicles, pustules). after pustules rupture, scabs form and detach in 2 to 3 weeks, leaving depigmented, scarred areas. this form of the disease, called variola major, is fatal in up to 30% of unvaccinated patients and 3% of vaccinated individuals. various hemorrhagic forms exist. in some cases, the rash progresses very slowly and hemorrhage develops into the base of the lesions, which remain flat and soft instead of tense, carrying a bad prognosis. in some other cases, the disease is hemorrhagic from the beginning, leading to death 5 to 7 days after the initial symptoms appear (case-fatality rate: 100%). finally, in some cases, a severe and overwhelming illness is followed by dusky skin lesions; these patients have a large quantity of virus and are extremely dangerous epidemiologically. previously vaccinated individuals usually develop a milder disease that consists of a mild pre-eruptive phase followed by few skin lesions that appear more superficial, evolve more rapidly, and are not as synchronous as the classical type. 50 viral hemorrhagic fever (vhf; see chapter 65) is caused by a heterogenous group of rna viruses that belong to several different families. the cdc identified filoviruses (ebola and marburg viruses), arenaviruses (lassa, junin, machupo, guanarito, and sabia), and bunyaviruses (crimean-congo hemorrhagic fever [cchf] and rift valley fever [rvf]). [51] [52] [53] the common denominator in these infections is the increased vascular permeability in the microcirculation leading to hemorrhagic diathesis and systemic manifestations such as pulmonary edema and cerebral edema related to leaky capillaries. 54 these viruses usually have a very narrow geographic range determined by their natural reservoirs and vectors. humans are accidental hosts. these diseases have caught great public attention due to their high mortality. this, combined with their aerosol infectivity, has led to the use of biosafety level 4 laboratories in their study. clinical presentation is usually nonspecific and consists of fever and malaise, followed by signs of increased vascular permeability and circulatory compromise. vhf usually terminates in shock, generalized mucocutaneous hemorrhages, and multiorgan failure. differences exist among the clinical details and pathogenesis of the different viruses (see chapter 65 for an overview and the individual chapters for details). for example, vhf due to filoviruses usually have prominent hemorrhagic manifestations and disseminated intravascular coagulation (dic) as a terminal event. rvf virus leads to liver damage, dic, and hemorrhagic manifestations in approximately 1% of patients with severe disease. cchf also behaves like the filoviral infections with prominent hemorrhagic manifestations. lassa fever has few neurologic or hemorrhagic manifestations. the south american arenaviral hemorrhagic fevers usually have hemorrhagic and neurologic components. toxins in the context of bt agents are substances of biologic origin that are capable of producing human illness. toxins are usually proteins synthesized by living bacteria, fungi, or plants. toxins are generally less dangerous than infectious agents. the most potent biological toxin is that from clostridium botulinum and it is 10-fold or more less lethal than anthrax on a weight basis. other toxins such as ricin are more than a 1000-fold less toxic than botulinum toxin and sarin is 30-fold less toxic than ricin. there are seven similar toxins produced by seven different serotypes of c. botulinum (a to g), all leading to the same clinical manifestations and with the same lethality. the toxins have a molecular weight of approximately 150 kda and block neurotransmission at the presynaptic level in cholinergic neurons including the neuromuscular junction, leading to progressive palsies of cranial nerves and skeletal muscle. botulinal toxins are among the most lethal substances known to mankind with ld 50 of 0.001 î¼g/g of body weight when administered parenterally. 25, 55, 56 the aerosol route decreases its lethality 80 to 100 times. both aerosol attacks and contamination of food supplies are potential bt scenarios. clinical manifestations consist of progressive bulbar and skeletal paralysis in the absence of fever, including diplopia, dysphagia, blurred vision, ptosis, dysarthria, dysphonia, mydriasis, dry mucosae, and descending paralysis. 25, 56 the cause of death in lethal cases is respiratory insufficiency due to paralysis of respiratory muscles. onset of symptoms is variable and depends on the inoculum, ranging from 24 hours to several days after exposure. most cases of naturally occurring intoxication are related to consumption of improperly sterilized canned food or ingestion of preserved fish. rare cases of inhalational botulism were documented in germany in the early 1960s due to accidental laboratory exposure. the rapid absorption through the respiratory tract may offer a different pathogenesis and it is not known if antitoxin is useful in therapy, although animal models show efficacy in prophylaxis. all the agents in category a are generally recognized as serious threats for causing extensive casualties. categories b and c are much more heterogeneous. they were considered to provide significant threat potential but there are continuing reassessments. these conditions are caused by the genus alphavirus, family togaviridae (eastern, western, and venezuelan equine encephalitis [vee] viruses; see chapter 74). natural infections are usually transmitted by mosquitoes, but aerosol transmission is the notorious cause of numerous laboratory infections and is the basis of its historic weaponization. 52, 57 most of these viruses cause systemic illness characterized by fever, myalgias, and prostration. clinically apparent involvement of the central nervous system is present in some cases and varies among the different viruses. eastern equine encephalitis (eee) is by far the most virulent, leading to case-fatality rates of 50% to 75%, and survivors usually have severe neurologic sequelae. 58, 59 vee, in contrast, leads to cns manifestations in no more than 4% of cases and almost all vee infections are symptomatic even in the absence of cns involvement. [60] [61] [62] rickettsia prowazekii (epidemic typhus) and r. rickettsii (rocky mountain spotted fever) typhus (see chapter 51) is another disease that has played a historic role in human populations. [63] [64] [65] [66] millions of people perished in world war i and world war ii due to epidemic, louse-borne typhus. large outbreaks of the disease still occur in tropical regions around the world in areas stricken by war, famine, and poverty. rocky mountain spotted fever (rmsf), on the other hand, is transmitted by tick bites and occurs endemically in south and central america as well as north america. rickettsiae target the microvascular endothelium leading to leaky capillaries systemically. 67 the main causes of morbidity and mortality are noncardiogenic pulmonary edema and cerebral edema leading to diffuse alveolar damage and meningoencephalitis. clinical manifestations are nonspecific and include fever, malaise, headache, myalgias/arthralgias, cough, nausea, vomiting, confusion, stupor, and coma in severe cases. skin rash ranges from maculopapular to petechial, depending on the severity, and is observed in around 90% of patients with rmsf and 2% to 100% of cases of epidemic typhus, depending on the darkness of cutaneous pigmentation. rickettsiae are remarkably underestimated biothreats as they are highly infectious by low-dose aerosol exposure, possess a stable extracellular form, and are resistant to most empirically administered antibiotics, including î²-lactams, aminoglycosides, and macrolides, and are exacerbated by sulfonamides. case-fatality rates can be as high as 40% to 50% without antibiotic therapy and 3% to 5% with adequate antibiotic coverage. lethal cases are usually due to delayed diagnosis. 64, 65, 68 these rickettsiae are highly infectious by aerosol and are potent bt agents. they are often discounted because of their susceptibility to tetracycline and chloramphenicol. however, the severity of the illness, the exhaustion of antibiotics in the face of a mass attack, and the existence of antibiotic-resistant organisms suggest they are still formidable players. this gram-negative, obligately intracellular bacterium has a high degree of infectivity (one organism is capable of causing infection by inhalation) and low lethality. [69] [70] [71] [72] the distribution of q fever is worldwide and results from exposure to animals such as sheep, cattle, goats, cats, rabbits, and others. c. burnetii has spore-like characteristics that can withstand harsh environmental conditions and be transported by wind to other places. in natural infections, 60% of cases are asymptomatic and are diagnosed by seroconversion. in symptomatic cases, the presentation is nonspecific and includes malaise, fever, myalgias, cough, chills, headaches, anorexia, weight loss, and in some cases pleuritic chest pain. hepatomegaly and splenomegaly are sometimes observed, although not frequently. transmission occurs by exposure to infected animal products (meat, milk). less common routes of infection are inhalational and cutaneous. the clinical presentation of brucellosis is highly variable, even after inhalational exposure. the clinical spectrum ranges from asymptomatic seroconversion to severe acute systemic disease. intermediate forms include undulant fever or chronic disease, characterized by presence of brucella in virtually any organ. acute systemic disease is highly incapacitating with high fever, headache, nausea, vomiting, chills, severe sweating, and, in very severe cases, delirium, coma, and death. undulant fever is characterized by relapses of fever, weakness, generalized aching, and headache. chronic infections may have manifestations related to several organ systems such as the gastrointestinal and genitourinary tracts, cns, joints, and bones. [73] [74] [75] developing countries with insufficient water treatment and food security are more vulnerable to enteric bt attack. these agents include shigella dysenteriae, salmonella spp., enterohemorrhagic e. coli, vibrio cholerae, and cryptosporidium parvum. shigella and salmonella have in fact already been used as agents of biorevenge or biopolitics in small-scale attacks: one (shigella) in an office setting by a disgruntled employee and one in oregon by a religious sect that led to nearly 1000 cases of salmonella-related gastroenteritis. 11, 76 these agents are indeed ideal for small-scale attacks since large-scale attacks would require contamination of large water supplies which, because of enormous dilution factors and susceptibility of all these agents (except for c. parvum) to standard chlorinating procedures, would decrease the number of bacteria to below that required to infect large numbers of people. 69 occasional outbreaks of nontyphoidal salmonella and shigella infections occur in the united states. shigella is a highly infectious organism that requires very low numbers (10 2 -10 3 organisms) to provoke clinical disease. the illness caused by shigella and enterohemorrhagic e. coli is explosive and starts with fever, vomiting, severe abdominal cramping, bloody diarrhea, and systemic manifestations such as hypotension, and circulatory collapse if not treated rapidly. both microorganisms produce an exotoxin responsible for most of the systemic manifestations associated. a distinct complication, hemolytic uremic syndrome, occurs in a small percentage of cases, being more common in children younger than 10 years of age, leading to renal failure and hemolysis. salmonella is less infectious and less explosive than shigella, and leads to fever, vomiting, diarrhea, abdominal cramping, and in some cases to typhoidal manifestations. imported cases of v. cholerae have been diagnosed in the united states in the past. however, the disease occurs in southern asia and latin america as large outbreaks. the clinical illness is characterized by explosive watery diarrhea that leads to rapid dehydration and circulatory collapse. c. parvum infections are characterized by watery diarrhea and abdominal cramping for 2 to 3 weeks. the disease is self-limited except in patients with acquired immunodeficiency syndrome (aids) or other conditions of compromise, in whom illness can last for months or years if immune function is not restored. c. parvum is resistant to standard chlorine concentrations in water supplies. 77 the largest outbreak in this country occurred in milwaukee in the early 1990s and was responsible for thousands of cases and increased mortality among those with aids. 69, 78, 79 this section addresses other toxins considered of potential bt use, such as staphylococcal enterotoxin b (seb) and ricin toxin (derived from castor beans, which in turn are the fruit of the ricinus communis plant). the ricin toxin is composed of two glycoproteins of approximately 66,000 kda. 80 the toxin inhibits protein synthesis by blocking elongation factor 2 (ef2) at the ribosomal level. ricin toxin is not a weapon of mass destruction since its lethal dose in humans is much higher than previously believed. however, the use of the toxin in small bt attacks is possible in the tropics because of its ready availability and relatively easy extraction from the beans. clinical presentation depends on the route of administration as does the ld 50 . in cases where large amounts of the toxin are ingested, the manifestations include nausea, vomiting, severe abdominal cramping, rectal hemorrhage, and diarrhea. as the clinical course progresses, anuria, mydriasis, severe headaches, and shock supervene leading to the patient' s demise in 2 to 3 days. clinical manifestations usually appear within 10 hours after ingestion of the toxin. inhalational exposure leads to prominent pulmonary manifestations 8 to 28 hours after exposure and fever, dyspnea, progressive cough, cyanosis, and death. histologically, there is widespread necrosis of pulmonary parenchyma and pulmonary edema. a single case of parenteral intoxication was documented. a defector from bulgaria was injected with a pellet containing ricin from a weapon disguised in an umbrella, resulting in local necrosis, regional lymphadenopathy, gastrointestinal hemorrhage, liver necrosis, nephritis, and dic. 81 staphylococcus aureus enterotoxin b (seb) is a 28-kda, heatstable exotoxin produced by certain strains of s. aureus and is responsible for food poisoning after ingestion of the preformed exotoxin in improperly handled food. in bt scenarios, exposure can occur either by inhalation or ingestion leading to seb food poisoning or seb respiratory syndrome. the toxin is highly incapacitating and not very lethal. the dose that causes symptoms in half of exposed persons and ld 50 differ by a magnitude of 5 log scales for inhalational exposure. 82 thus, it is thought of as an incapacitating agent. incubation time after ingestion is short (4-12 hours) followed by explosive vomiting that persists for several hours. weaponization of the toxin as an aerosol is possible due to its high stability. manifestations after inhalation of the seb are related to the respiratory system and consist of fever, cough, chills, myalgias, chest pain, and pulmonary insufficiency due to alveolar edema. general symptoms and signs are universal and consist of multiorgan failure secondary to a cytokine storm. 25 these toxins are superantigens due to their ability to bind to major histocompatibility complex (mhc) class ii molecules on large numbers of lymphocytes and macrophages, leading to a hyperactivation of the immune system and massive cytokine release including interferon-gamma (ifn-î³), tumor necrosis factor-alpha (tnf-î±), interleukin (il-6), and other mediators such as leukotrienes and histamine. 82 the role of the clinical laboratory in the diagnosis of possible cases related to a bt attack is of utmost importance. 83, 84 on the one hand, standard clinical microbiology laboratories will be receiving specimens for diagnostic purposes, and communication with clinicians regarding their suspicions is critical. certain isolates in the laboratory are not pursued further (bacillus spp. is a classic example) unless specifically requested due to the frequent isolation of contaminants with similar characteristics. in addition, handling of certain specimens will require added biosafety level requirements due to their infectivity (table 119-2) . certain samples will have to be shipped to highly specialized laboratories for initial or further work-up. environmental testing is challenging due to the complexity of the samples to be analyzed. 85, 86 this type of testing takes place in highly specialized laboratories and is not undertaken by the standard clinical microbiology laboratory. the bacterial diseases caused by the bt agents outlined in this chapter, with the exception of c. burnetii and rickettsia spp., can be diagnosed by standard isolation techniques in clinical microbiology laboratories. isolation of rickettsiae and the bt viruses requires specialized laboratories with bsl-3 or bsl-4 biocontainment. 87 serological assays are available for detection of antibodies against all bt agents. however, for many organisms serological assays require the presence of rising antibody titers, and therefore the serologic diagnosis is usually retrospective in nature. for some viral diseases, a reliable diagnosis can be established based on elevation of immunoglobulin m (igm) titers in the acute phase of the disease. with the advent of molecular techniques, rapid and sensitive diagnostic tests are becoming available for bt agents during the acute phase of the disease. [88] [89] [90] this is of utmost importance in a bt event since identification of the first cases would be critical for a rapid and effective public health response. in addition, treatment and prophylactic measures can also be initiated as quickly as possible. molecular diagnostic techniques can be applied to potential bt agents in an additional setting: as part of the epidemiological and forensic investigations that a bt attack would immediately trigger. postmortem diagnosis is also possible by analysis of frozen or paraffin-embedded tissues by immunohistology or nucleic acid-based amplification techniques. rapid diagnosis of the initial case (cases) in a bt event requires a high degree of clinical suspicion from the physicians having contact with such patients in the emergency room or outpatient setting. the clinical laboratories would then play a critical role in detecting the suspected agent and/or referring the appropriate specimens to higher level laboratories for specialized testing (table 119-3) . 83, 85, 91 several of the agents discussed in this chapter are zoonotic diseases. therefore, diagnosis of certain zoonotic diseases in animals may be important in identifying some bt attacks. in such situations, animals could be seen as either direct victims of the attack or as sentinel events in a human outbreak. there are currently efforts to establish a network of laboratories dedicated to diagnosis of veterinary agents. 85 bsl-1 suitable for work involving well-characterized agents not known to cause disease in healthy bacillus subtilis adult humans and of minimal potential hazard to laboratory personnel and the environment. naegleria gruberi canine hepatitis virus bsl-2 suitable for work involving agents of moderate potential hazard to personnel and the measles virus environment. laboratory personnel have specific training in handling pathogenic agents salmonella spp. and are directed by competent scientists; access to the laboratory is limited when work toxoplasma spp. is being conducted; extreme precautions are taken with contaminated sharp items; and hepatitis b virus certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. bsl-3 suitable for work with infectious agents which may cause serious or potentially lethal coxiella burnetii disease as a result of exposure by the inhalation route. in addition to the requirements rickettsia spp. described for work in bsl-2 environment, all procedures are conducted within biological m. tuberculosis safety cabinets, or other physical containment devices, and by personnel wearing alphaviruses appropriate personal protective clothing and equipment. laboratory should be located in a separate building or an isolated zone within a building. laboratories are equipped with double door entry, directional inward flow, and single-pass air. bsl-4 required for work with dangerous and exotic agents that pose a high individual risk of filoviruses aerosol-transmitted laboratory infections and life-threatening disease. members of the arenaviruses laboratory staff have specific and thorough training in handling extremely hazardous infectious agents. they are supervised by competent scientists who are trained and experienced in working with these agents. access to the laboratory is strictly controlled by the laboratory director. the facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. all activities are confined to class iii biological safety cabinets, or class ii biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. the biosafety level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. the diagnosis of inhalational anthrax is based on isolation and identification of b. anthracis from a clinical specimen collected from an ill patient. in cases of inhalational anthrax, samples of sputum, blood, or cerebrospinal fluid (csf) may yield growth of the agent. demonstration of b. anthracis from nasal swabs has more epidemiological and prophylactic implications than clinical importance. standard diagnostic techniques are based on visualization and isolation in the clinical microbiology laboratory and serological demonstration of antibodies against b. anthracis. [92] [93] [94] [95] [96] visualization of b. anthracis from clinical specimens (blood cultures, csf, and cutaneous lesions) by gram stains is not difficult. b. anthracis appears as large gram-positive, spore-forming rods with a bamboo appearance. isolation is achieved by inoculating standard sheep blood agar plates, and colonies appear as small, gray-white, nonhemolytic colonies. a selective medium (polymyxin-lysozyme-edta-thallous acetate agar) is available mostly for environmental samples and inhibits the growth of other bacillus spp., such as b. cereus. growth is rapid (24-48 hours) . 93 confirmatory tests include î³-phage lysis, detection of specific cell wall and capsular antigens, and polymerase chain reaction (pcr) amplification of dna followed by sequencing. 90 serological tests available for clinical diagnosis are based on detection of antibodies directed against protective antigen (pa). cross-reactive antibodies decrease the specificity of this test. assays based on toxin detection are available in specialized centers and are based on capture of anthrax toxins by using antibodies. antibody-coated immunomagnetic beads are then analyzed by electrochemiluminescence technology. the analytical sensitivity of this technique for detection of anthrax toxin is at the picogram to femtogram level (10 â��12 to 10 â��15 ). 97, 98 immunoliposomal technology combined with real-time pcr (for a dna reporter sequence) is also in the early stages of development for several toxins (ricin, cholera, and botulinum) and appears promising with analytical sensitivity in the attomolar to zeptomolar (10 â��18 to 10 â��21 ) range for cholera toxin. 99 the specificity of this assay is given by the toxin-capturing antibody. nucleic acid amplification techniques (pcr) are also available both in standard format and real-time format. extraction of dna from spores is challenging and requires modification of dna extraction protocols in order to facilitate release of dna from spores or induction of germination prior to dna extraction. 90 real-time pcr tests have been developed by applied biosystems (taqman 5' nuclease assay) and roche applied science (lightcycler). [100] [101] [102] the analytical sensitivity of both techniques is extremely high, and testing times have been decreased to 1 to 2 hours. portable pcr instruments are being developed for rapid deployment to the field. 103 examples include the rugged advanced pathogen identification device (rapid), 100 the smartcycler (cepheid, ca), 101 and the miniature analytical thermal cycler instrument (matci) developed by the department of energy' s lawrence livermore national laboratory. 104 this instrument later evolved into the advanced nucleic acid analyzer (anaa) and handheld advanced nucleic acid analyzer (hanaa). 105 molecular subtyping of b. anthracis is also possible by using the 16s ribosomal rna (rrna) subunit gene, multiplelocus vari-able number tandem repeat analysis of eight genetic loci, and amplified fragment length polymorphism (aflp) techniques. 106, 107 environmental testing also plays a role in the investigation of a bt event. in this setting, detection of b. anthracis relies heavily on molecular techniques for confirmation of potentially contaminated samples (e.g., surfaces, air). 108, 109 postmortem diagnosis is also possible by using gram stains on paraffin-based tissues or immunohistochemical procedures using polyclonal or monoclonal antibodies against various anthrax antigens. diagnosis of y. pestis is based on demonstration of the bacillus in blood or sputa from patients. standard diagnostic techniques in the laboratory include visualization of gramnegative coccobacilli, which by giemsa, wright, or wayson stains reveal a "safety pin" appearance. isolation is performed in blood and mcconkey agar plates on which colonies appear as nonlactose fermentors. the organisms are identified preliminarily by direct immunofluorescent assay with y. pestisspecific antibodies, with final identification based on biochemical profiles in clinical microbiology laboratories. 110 molecular diagnostic techniques based on real-time pcr have become available in recent years and involve detection of y. pestis genes such as plasminogen activator (pla), genes coding for the yop proteins and the capsular f1 antigen, and the 23s rrna gene, which allows distinction from other yersinia spp. [111] [112] [113] assays have been developed to detect resistance to particular antibiotics. the importance of these diagnostic techniques in a disease such as plague is evident. the log-normal epidemic curve with a narrow dispersion of the incubation periods (see fig. 119 -1) and the short interval for successful antibiotic therapy mandate recognition of the earliest cases if the bulk of the exposed are to be saved. molecular subtyping of y. pestis is also possible by analyzing polymorphic sites in order to identify the origin of strains in the event of a bt attack. diagnosis is made in the clinical laboratory by demonstration of the microorganisms in secretions (sputa, exudates) by direct immunofluorescence or immunohistochemically in biopsy specimens. isolation in the clinical laboratory may be achieved by using regular blood agar plates, posing a risk to laboratory personnel not employing bsl-3 facilities and procedures. the procedure for isolation of f. tularensis in the laboratory is very similar to that described for y. pestis. final identification in the clinical laboratory is based on the biochemical profile. 114 molecular diagnostic techniques are based on pcr detection of f. tularensis by using primers for different genes such as outer membrane protein (fop) or tul4 and real-time detection systems. 90, 115, 116 smallpox virus diagnosis of variola major is suggested by its clinical presentation and the visualization of guarnieri bodies in skin biopsy samples. preliminary confirmation requires visualization of the typical brick-shaped orthopox virus by electron microscopy, followed by isolation from clinical specimens and accurate molecular identification to differentiate it from the morphologically (and sometimes clinically) similar monkeypox virus. confirmation of this diagnosis is performed only under bsl-4 containment facilities at the cdc. 47 molecular techniques are based on pcr amplification using real-time or standard technology followed by sequencing or use of restriction fragment length polymorphism (rflp) for accurate identification. 117 technologies so far developed for smallpox molecular testing include taqmanand lightcycler-based assays with primers designed for the hemagglutinin gene and a-type inclusion body proteins. [118] [119] [120] [121] sequencing of the smallpox genome has been completed for some asian strains of variola major and one of variola minor. other strains are being sequenced and will provide more information for probe design and treatment targets. 90 diagnosis of these diseases is performed in highly specialized centers in the united states because special isolation procedures and highly contained laboratories are required. initial diagnosis of these diseases is suspected on clinical and epidemiologic grounds. laboratory diagnosis involves isolation, electron microscopy, and serological assays. immunohistochemical detection of hemorrhagic fever viral antigens in paraffin-embedded tissues is also performed in highly specialized centers such as the cdc. [122] [123] [124] [125] [126] molecular diagnostic techniques have also improved dramatically during the last few years. serum or blood is the most common specimen used for reverse transcriptase-pcr amplification of viral nucleic acids. both standard and realtime techniques are available. design of primers for this heterogenous group of rna viruses that are highly variable is one of the limitations. 90 therefore, multiplex pcr techniques are required to detect as many targets as possible in a single assay. 127, 128 real-time pcr based on detection of the target sequence using fluorescent probes therefore limits the number of targets that can be identified because of the limited wavelength range for fluorescent applications (usually only four different wavelengths can be detected at the same time). [128] [129] [130] the use of microchips containing several thousands of oligonucleotides from all viruses known to be pathogenic to humans is an encouraging development. in fact, the rapid identification and characterization of the novel human coronavirus responsible for the sars outbreak in 2003 is an excellent example of the power of hybridization-based microchips. the creation of an automated and easily deployable instrument capable of detecting all possible potential bt agents based on highly sensitive techniques such as electrochemoluminescence (ecl) or pcr would be ideal. the nonspecific nature of presenting symptoms is a major problem with several of the agents. the rapid recruitment of cases into the infected cohort requires that an early diagnosis of the epidemic be established, particularly for organisms such as y. pestis in which there is only a short window for successful treatment. in fact, such projects are already in the making. an example of this system is the automated biological agent testing system (abats) that combines the techniques mentioned previously. 86 the system is the result of integrating several commercially available technologies into a single automated and robotized instrument for detection of viruses, bacteria, and parasites considered potential bt agents. the technologies incorporated into this "super system" include automated specimen preparation (both nucleic acid-based and protein-based such as immunodiagnostics), thermocyclers for pcr detection, chemiluminescent detectors for immunobased assays, sequencers, and software programs for sequence analysis. rickettsia prowazekii (epidemic typhus) and r. rickettsii (rocky mountain spotted fever) diagnosis of these infections in the clinical microbiology laboratory currently rests on the identification of antibodies in serum during the acute and convalescent period in order to demonstrate seroconversion or rising titers. the diagnosis is therefore retrospective. 131, 132 detection of rickettsial dna from blood or skin samples during the acute phase of the disease is possible via pcr assays. however, these assays are not standardized and are not commercially available. primers have been designed for amplification of several rickettsial genes including citrate synthase, 17-kda protein gene, ompa, and ompb. [132] [133] [134] [135] [136] the clinical sensitivity and specificity of standard or real-time pcr techniques have not been determined. most likely real-time pcr is superior due to the higher analytical sensitivity of this technique and low risk of sample contamination with dna amplicons when compared to standard pcr amplification methods. isolation of rickettsiae from clinical specimens is performed in very few specialized laboratories in the nation and requires the use of cell monolayers, embryonated eggs, or animals. detection of rickettsial antigens or whole bacteria in blood specimens is theoretically possible by using ultrasensitive methods, but such assays are currently only in the early phases of development. immunohistochemical detection of rickettsiae in paraffin-embedded tissue has also been applied to tissue samples obtained pre-or postmortem. [137] [138] [139] salmonella spp., shigella dysenteriae, vibrio cholerae, and cryptosporidium parvum (acute enteric syndromes) diagnosis of salmonella, shigella, and vibrio infections is based on isolation of the offending agent on standard microbiological media in the clinical laboratory, followed by specialized confirmatory tests to identify the specific serotype involved. 140 diagnosis of c. parvum is based on visual identification of the protozoan in fecal specimens by using modified trichrome stain. 140 the diagnosis rests on serological demonstration of antibodies by immunofluorescent assay (ifa) or enzyme-linked immunosorbent assay (elisa). antibodies remain elevated for years after the acute infection, and therefore a fourfold rise in titers is the gold standard for diagnosis. pcr detection of c. burnetii dna from blood or tissues also yields a diagnosis of q fever. 88 brucella spp. diagnosis of brucellosis requires a high degree of clinical suspicion due to the protean manifestations related to this disease. laboratory diagnosis is based on isolation of the microorganism from blood, bone marrow, or other tissue samples. isolation is not easy due to the slow-growth of brucella spp. colonies usually appear after 4 to 6 weeks, and therefore communication with the clinical laboratory is important so that appropriate media will be used and the cultures will be held long enough for colonies to be detected. 90 serologic assays for demonstration of rising antibody titers are available, although the diagnosis is retrospective. pcr detection is promising, but it is not standardized. [141] [142] [143] alphaviruses (encephalitic syndromes: venezuelan, eastern, and western equine encephalomyelitis) diagnosis is based on isolation of the virus from serum or brain (postmortem specimens) in a bsl-3 environment. pcr detection of viral sequences is also possible. serologic diagnosis is based on demonstration of antibodies in acute and convalescent sera. [144] [145] [146] botulinum toxins the diagnosis of botulism relies heavily on clinical parameters. an afebrile patient with signs and symptoms of progressive bulbar palsies and descending neuromuscular paralysis is highly suspected of having botulism. demonstration of the toxin in cases of botulism due to ingestion of contaminated food is made from gastric samples, feces, blood, and urine. however, detection of minute amounts of toxin (and contacts with samples from cases may prove fatal due to the toxin' s potency) would be difficult by current immunoassay systems such as elisa platforms. 146 detection techniques based on electrochemiluminescence and immunoliposomes are currently under development. 99, 147 pcr assays can be performed in cases of ingestion of contaminated food in order to detect the genetic material present in c. botulinum. if weaponized toxin is used in the absence of c. botulinum organisms, detection of the genetic material would be difficult and would rely on the presence of residual dna after toxin purification procedures. if inhalational botulism is suspected, respiratory secretions and nasal swabs should be obtained as early as possible. postmortem samples of liver and spleen can be used for detection of botulinum toxins. diagnosis is also based on clinical presentation and requires a high index of suspicion due to the nonspecific nature of the signs and symptoms. laboratory diagnosis rests on detection of the toxin in body fluids by immunoassays (capture elisa and igg elisa). 146 a new generation of tests using more sensitive detection methods is under development (see preceding discussion). diagnosis is also suspected on clinical grounds and confirmed by demonstration of the toxin in nasal swabs early in the disease process, feces, and, in fatal cases, from kidney and lung tissue. serum can be analyzed by elisa, and pcr can be performed for detection of toxin genes of s. aureus if present. 146 the distribution and incubation periods of 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other orthopoxviruses real-time pcr system for detection of orthopoxviruses and simultaneous identification of smallpox virus gene for a-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses the potential of 5â�² nuclease pcr for detecting a single-base polymorphism in orthopoxvirus detection of smallpox virus dna by lightcycler pcr comparative pathology of the diseases caused by hendra and nipah viruses a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever. commission de lutte contre les epidemies a kikwit immunohistochemical and in situ localization of crimean-congo hemorrhagic fever (cchf) virus in human tissues and implications for cchf pathogenesis retrospective diagnosis of hantavirus pulmonary syndrome, 1978-1993: implications for emerging infectious diseases hantavirus pulmonary syndrome. pathogenesis of an emerging 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detection of r. conorii in circulating endothelial cells: a 6-year follow-up differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of pcr-amplified dna of the gene encoding the protein rompa differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of pcr-amplified dna diagnostic tests for rocky mountain spotted fever and other rickettsial diseases immunohistochemical diagnosis of typhus rickettsioses using an anti-lipopolysaccharide monoclonal antibody monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target investigation of foodborne and waterborne disease outbreaks rapid laboratory confirmation of human brucellosis by pcr analysis of a target sequence on the 31-kilodalton brucella antigen dna the 18-kda cytoplasmic protein of brucella species-an antigen useful for diagnosis-is a lumazine synthase characterization of an 18-kilodalton brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections toxins as weapons of mass destruction: a comparison and contrast with biological warfare and chemical warfare agents sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor key: cord-006172-ndmf5ekp authors: akins, paul taylor; belko, john; uyeki, timothy m.; axelrod, yekaterina; lee, kenneth k.; silverthorn, james title: h1n1 encephalitis with malignant edema and review of neurologic complications from influenza date: 2010-09-02 journal: neurocrit care doi: 10.1007/s12028-010-9436-0 sha: doc_id: 6172 cord_uid: ndmf5ekp background: influenza virus infection of the respiratory tract is associated with a range of neurologic complications. the emergence of 2009 pandemic influenza a (h1n1) virus has been linked to neurological complications, including encephalopathy and encephalitis. methods: case report and literature review. results: we reviewed case management of a 20-year old hispanic male who developed febrile upper respiratory tract signs and symptoms followed by a confusional state. he had rapid neurologic decline and his clinical course was complicated by refractory seizures and malignant brain edema. he was managed with oseltamavir and peramavir, corticosteroids, intravenous gamma globulin treatment, anticonvulsants, intracranial pressure management with external ventricular drain placement, hyperosmolar therapy, sedation, and mechanical ventilation. reverse transcriptase polymerase chain reaction analysis of nasal secretions confirmed 2009 h1n1 virus infection; cerebrospinal fluid (csf) was negative for 2009 h1n1 viral rna. follow-up imaging demonstrated improvement in brain edema but restricted diffusion in the basal ganglia. we provide a review of the clinical spectrum of neurologic complications of seasonal influenza and 2009 h1n1, and current approaches towards managing these complications. conclusions: 2009 h1n1-associated acute encephalitis and encephalopathy appear to be variable in severity, including a subset of patients with a malignant clinical course complicated by high morbidity and mortality. since the h1n1 influenza virus has not been detected in the csf or brain tissue in patients with this diagnosis, the emerging view is that the host immune response plays a key role in pathogenesis. the current pandemic of 2009 influenza a (h1n1) (2009 h1n1) virus has presented challenges for clinicians the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. worldwide. neurologic complications of seasonal influenza are likely under-recognized by neurologists and the frequency of acute or post-infectious neurologic complications with 2009 h1n1 virus infection is unknown. it is worth noting the historical relationship between h1n1 and neurology. following the 1918-1919 h1n1 pandemic, an increase was observed in encephalitis lethargica cases [1] . what have neurologists learned about complications of 2009 h1n1 virus infections worldwide? we present a case report of 2009 h1n1-associated encephalopathy and review neurologic complications associated with seasonal influenza and 2009 h1n1 virus infection. the kaiser permanente inpatient neurosurgery service maintains ongoing institutional review board approval for a prospective database registry for clinical research purposes. we identified a case of acute encephalopathy associated with 2009 h1n1 virus infection of the upper respiratory tract referred from an outside kaiser community hospital for management. we conducted a detailed review of the electronic medical records. we also conducted a literature review using pubmed. mesh search terms included influenza, encephalitis, encephalopathy, h1n1, acute necrotizing encephalopathy, and meningitis. a previously healthy 20-year old male college student had 5 days of non-productive cough, rhinorrhea, myalgias, and fever but no headaches or neck stiffness. on the 6th illness day, he presented to the emergency department of a community hospital with lethargy and confusion. he was electively intubated for airway protection. his chest x-ray (cxr) was normal. routine admission laboratory tests including hepatic transaminases were within normal range. a non-contrast head computed tomography (ct) did not reveal any abnormalities (fig. 1, top row) , and he underwent lumbar puncture. cerebrospinal fluid (csf) analysis showed 53 wbc/ll with 91% lymphocytes, 6 rbc/ll, protein 113 mg/dl, and glucose 59 mg/dl. he was diagnosed with meningoencephalitis and started on vancomycin, ceftriaxone, acyclovir, and oseltamivir (150 mg twice daily per nasogastric tube). on the morning of the third-day of hospitalization, he experienced tonic-clonic seizures and remained comatose with extensor posturing afterwards. repeat head ct (fig. 1 , bottom row) demonstrated diffuse brain edema and effaced basal cisterns. he received fosphenytoin, mannitol, and propofol. the treating physicians contacted the neuro-intensive care unit at kaiser sacramento for additional assistance. he was emergently transferred to the kaiser permanente sacramento neuro-intensive care facility (nicu). on arrival, his initial examination demonstrated a glasgow coma scale of 3 (e1v1m1). his repeat cxr did not demonstrate on the bottom row, the small arrow points to effacement of basal cisterns (left) and subcortical brain edema (larger arrows, bottom row, left and right). this subcortical edema is confirmed on mr imaging (fig. 2) any infiltrates or signs of acute respiratory distress syndrome (ards). an external ventricular drain was placed by the neurosurgeon at the bedside. he reported that the csf pressure noted at the time of initial catheter placement was elevated. the first recorded intracranial pressure (icp) was 10 mm hg, and this reading was taken after the expected loss of csf during the procedure. on the second day of nicu hospitalization, his glasgow coma scale (gcs) score was 4 (e1v1m2) and average icp was 7 mm hg. throughout the remainder of the hospitalization, the recorded icp remained below 20 mm hg. initial icp was maintained with external ventricular drainage at 0 cm relative to the external auditory canal and a midazolam infusion (5 mg/h). electroencephalogram (eeg) monitoring demonstrated diffuse, severe slowing in the delta range and no electrographic seizures. on hospital day 3, mri of the brain was obtained (see fig. 2 ). he received 20 days of dual neuraminidase inhibitor treatment (oseltamivir 150 mg twice daily per nasogastric tube, peramavir 600 mg iv daily); intravenous gamma globulin (1 gm/ kg 9 2 days); dexamethasone (10 mg iv load, 6 mg iv every 6 h with taper over 4 weeks); icp monitoring and management; ventilator support; and anticonvulsants (fosphenytoin, levetiracetam). his weekly glasgow scale scores showed delayed improvement (3, e1v1m1, admission): 5 (e2v1m2, week 1), 5 (e2v1m2, week 2), 5 (e2v1m2, week 3), 9 (e3v2m4, week 4). the midazolam infusion was discontinued on hospital day 4, after clinical observation and eeg confirmation that he was not having electrographic seizures. thereafter he received intermittent doses of lorazepam as needed for sedation while on the ventilator. over 3 weeks, neuroimaging demonstrated improvement in his brain edema with restoration of his basal cisterns, and the external ventricular drain was successfully weaned and removed. more rapid weaning of his external ventricular drain was not attempted due to severe neurologic impairments with gcs less than eight and radiographic appearance of diffuse brain edema and effaced basal cisterns. his nicu course was complicated by ventilatorassociated klebsiella pneumoniae and spontaneous pneumomediastinum on day 6 of intensive care. chest ct demonstrated subcutaneous emphysema, mediastinal emphysema, bilateral lower lobe atelectasis, and no pulmonary interstitial emphysema, or pneumothorax. he did not develop adult respiratory distress syndrome or suffer periods of hypoxemia. rt-pcr of an admission nasopharyngeal swab was positive for 2009 h1n1 virus at the california department of public health virology laboratory. rt-pcr analysis of csf samples was negative for influenza a and b viruses, herpes virus type 1, 2, and 6, varicella, enterovirus, and epstein barr virus. nasopharyngeal samples were negative for enterovirus and mycoplasma pcr. bacterial and viral cultures of csf were negative. test results from clinical specimens (blood, endotracheal aspirate, serum, and csf) sent to the california encephalitis project did not reveal an alternative cause. follow-up mri brain imaging (fig. 2b, d) was repeated at 1 month. after 6 weeks, he transitioned to acute rehabilitation, and 1 month later returned home. because he had improved upper extremity use without recovery in his legs, the physiatry staff performed spine mr imaging and no specific cause was identified. at the time of this case report, the patient has returned home with his family. he is talking and interacting with his family normally. he has not returned to college. his gastromy tube has been removed. he has generalized rigidity without tremor or dyskinesia. he is ambulatory but requires a walker due to reduced endurance and leg weakness. fig. 2 magnetic resonance imaging was done at the time of patient transfer a, c to the neuro-intensive care center and at 1 month of treatment b, d with influenza-specific antiviral therapy, corticosteroids, and intravenous gamma globulin therapy. a coronal flair image shows diffuse brain edema with sulcal effacement and symmetric hyperintensities selectively affecting the white matter and sparing cortex and subcortical nuclei such as basal ganglia and thalami. b coronal flair image at 1 month shows resolution of sulcal effacement, marked reduction in white matter hyperintensity, and relative brain atrophy (20 year old patient). c diffusion-weighted imaging on admission showed some increased signal in the periventricular zones that were also bright on t2 and flair sequences consistent with t2 shine-through. d diffusionweighted imaging at 1 month revealed hyperintensity in the caudate and putamen with corresponding decreased signal in adc map and lack of hyperintensities on t2 and flair sequences (see fig 1b) we present a case of a patient with acute encephalitis associated with febrile upper respiratory tract illness due to 2009 h1n1 complicated by seizures and malignant cerebral edema. few adult cases of 2009 h1n1 influenzaassociated acute encephalitis or encephalopathy have been reported to date. descriptions of 2009 h1n1-associated neurologic complications are limited to case reports and small case series, and have been more commonly reported among young children. given the current influenza pandemic, we provide an overview of neurologic complications associated with seasonal influenza and h1n1 (fig. 3 ) and review clinical management and rationale. influenza virus infections can cause human respiratory disease and have been associated with a variety of central nervous system disorders [2] . influenza virus has been rarely detected in csf of patients that developed acute encephalitis/encephalopathy [3] [4] [5] . the systemic inflammatory response syndrome (sirs) to influenza virus infection of the upper respiratory tract is hypothesized to play a prominent role in the more severe stages leading to cytokine dysregulation (''cytokine storm'') in influenzaassociated encephalopathy or encephalitis (iae) patients [6] . elevated cytokines in serum and csf have been reported in patients with seasonal influenza-associated encephalopathy [4, [7] [8] [9] [10] . elevated csf to plasma ratios suggest activation of cytokine production within the cns may have occurred along with the respiratory tract and systemic cytokines [7, 11, 12] . microglia and astrocytes are capable of producing cytokines in the cns [13, 14] . it is known that influenza virus infects and replicates at the nasopharyngeal epithelium leading to extensive damage during infection. below the mucosa, the free nerve endings of the olfactory nerves may also become infected. as seen with herpes simplex viruses, some postulate that influenza virus could penetrate and replicate at the olfactory mucosa and the free nerve endings with resultant axonal transport of virions to the olfactory bulbs, to the olfactory tract, and finally to the brain [15] . there is some literature to support this mechanism when one looks at h5n1, or avian influenza, where mice inoculated intranasally with h5n1 developed cns lesions in the pons, medulla oblongata, and cerebellar nuclei. astrocytes and glial cells were positive for viral antigen but viral replication ceased before 7 days [16, 17] . further study is needed to elucidate the pathogenesis of cns disease complicating influenza a infection. neurologic symptoms associated with influenza can arise at different intervals after the initial influenza illness (fig. 3 , table 1 ). when assessing patients clinically, it is important to determine if the patient has active or recent symptoms (within days) of influenza or if the neurologic symptoms have appeared in a subacute manner. we will first discuss neurologic complications in the setting of recent influenza virus infection and then proceed to complications that present in a delayed manner the development of a confusional state in the setting of influenza illness symptoms and fever raises the possibility of influenza-associated encephalitis or encephalopathy. the degree of encephalopathy varies from a confusional state to obtundation. it is important to recognize that a small portion of cases can rapidly deteriorate to coma and subsequent brain death due to diffuse, malignant cerebral edema. focal and generalized seizures often occur and can be present with either mild or severe cases. the presence of fever and altered mental state should prompt clinicians to pursue csf analysis unless neuroimaging or laboratory studies reveal a contraindication. influenza illness may include upper respiratory symptoms, pneumonia, or diarrhea (more commonly in young children with seasonal influenza). a thorough medical assessment to exclude other causes such as sepsis, metabolic or toxic disorders, structural cns diseases, and other cns infections is warranted. we define encephalitis by the presence of inflammation in the csf or demonstration of viral infection in brain biopsy or autopsy specimens. we define encephalopathy when csf is acellular and brain biopsy or autopsy specimens have failed to demonstrate viral infection. in some cases, this distinction is arbitrary and the case has borderline csf pleocytosis or csf analysis was not performed due to malignant brain edema. a consistent observation is that patients with seasonal influenza-associated encephalopathy rarely ever have evidence of influenza viral rna in csf based on rt-pcr analysis of csf. furthermore, there is no evidence of seasonal influenza virus infection of brain specimens. in one case series, only one out of 18 patients with acute seasonal influenza-associated encephalitis had influenza viral rna detected [5] . terminology for post-infectious encephalitis can be confusing. for example, the international pediatric multiple chronic condition * sometimes classified as adem [18] sclerosis study group [18] listed ten terms that have been used to describe acute disseminated encephalomyelitis (adem). some terms focus on the triggering event, such as post-infectious encephalomyelitis; others on pathologic or pathophysiologic features such as acute demyelinating encephalomyelitis or hyperergic encephalomyelitis. these authors also classify acute hemorrhagic leukoencephalitis, acute necrotizing hemorrhagic leucoencephalitis (also known as acute necrotizing encephalitis, (ane)), and acute hemorrhagic encephalomyelitis as hyperacute forms of adem. these diagnostic terms are of great historical interest. they generally preceded modern neuroimaging and relied more on the clinical and pathologic details. the study group also lumps a diversity of neuroimaging findings under the diagnosis of adem including: ring-enhancing lesions; diffuse and multi-focal regions of t2 hyperintensity with and without associated hemorrhage; multi-focal lesions with associated mass effect (tumefactive lesions); and images with symmetric, bithalamic edema. while we prefer one term (adem) rather than ten terms to describe post-infectious encephalitis, we are concerned that the pathophysiology and outcome of a process leading to the formation of ring-enhancing lesions (demyelinating, for example, acute demyelinating encephalomyelitis) must be radically different than that causing bithalamic edema (necrotizing, for example, ane). in reality, iae presents along a spectrum ranging from milder cases with normal neuroimaging to more malignant cases with abnormal neuroimaging and less favorable outcomes. for the sake of discussion and literature review, we present a simplified classification scheme based on clinical and imaging findings. the iae benign variant can present with fever, confusional state, and seizures but neuroimaging with ct brain or mri brain does not demonstrate any acute abnormalities. csf analysis is within normal limits or has borderline findings. rt-pcr testing for 2009 h1n1 influenza viral rna is positive in upper respiratory secretions but negative when csf is tested [19] [20] [21] . these patients typically recover within 1 week, and most cases have received oseltamavir and anticonvulsants. the initial reports of pediatric cases of 2009 h1n1 encephalopathy in the us were not severe [19] . similarly, other reported adult cases of 2009 h1n1 iae without ards have not been severe with complete recovery [20, 21] . a more recent pediatric case series of 2009 h1n1 iae reported that 2/4 patients had imaging abnormalities and neurologic sequelae [22] , so the treating physicians need to be aware that full recovery is not a certainty. the iae with splenial sign presents with acute febrile respiratory illness and additional neurologic symptoms with a characteristic mri abnormality. we found case reports associated with seasonal influenza but not with h1n1. it has been reported in children, but rarely in adults [23] [24] [25] [26] [27] [28] . encephalopathy is always present and can be severe. seizures are often present. mri imaging demonstrates increased t2 and flair signal and restricted diffusion in the splenium of the corpus callosum. this finding is reversible. the mri finding is not specific and has been reported with other infections, high-altitude brain edema, and certain metabolic states such as hypernatremia [29] . csf analysis is unremarkable. these patients have been treated with oseltamavir and anticonvulsants, and typically recover within 1 month. the iae with posterior reversible leucoencephalopathy syndrome (pres) presents as moderate to severe febrile encephalopathy. this subtype has been reported with seasonal influenza but not specifically with h1n1. the mri imaging appears radiographically identical to pres caused by more typical causes such as pregnancy or malignant hypertension [30, 31] . vascular caliber changes have been observed in these cases; this is non-specific and can be related to infectious vasculitis or pres. given the diverse causes of pres including malignant hypertension, pregnancy, metabolic disorders, and certain medications such as chemotherapeutics and immunosuppressants; it is often difficult to distinguish the pathophysiology of iae in this clinical setting. therapy is focused upon antiviral treatment, corticosteroid administration, and supportive care. iae with malignant brain edema is one of the most challenging subtypes to diagnose and treat. both seasonal influenza and h1n1 can be complicated by severe forms of acute encephalopathy and malignant brain edema [32] [33] [34] [35] . survival in some cases has been achieved with aggressive neuro-intensive case management with other therapies, including administration of antivirals, corticosteroids, immunoglobulin (2 gm/kg in adult patients), hyperosmolar therapy, plasmapheresis, and hypothermia in some cases. one of the goals of treatment is to reduce viral expression with early antiviral treatment and thereby to reduce stimulation of the host inflammatory response. our case presentation illustrates the rapid time course for this complication (see fig. 1 ) and neurocritical care treatment approaches. because of diffuse brain edema, a broad treatment approach using hyperosmolar therapy, intubation, fever control, and sedation were important. to the best of our knowledge, this is the only case description of iae in which an external ventricular drain was utilized, probably because it is difficult to place a catheter into the small, compressed ventricles of patients with diffuse brain edema associated with influenza. another adult case of h1n1 encephalitis has been reported with radiographic findings similar to ours. fugate et al. [35] described an adult with h1n1 influenza-associated acute hemorrhagic leukoencephalitis. like our patient, their case also showed confluent areas of increased t2 signal in the periventricular white matter and centrum semiovale. because of the additional finding of microhemorrhages demonstrated on gradient echo mri sequences, they diagnosed acute hemorrhagic leukoencephalitis or hurst disease. their patient also had restricted diffusion in the basal ganglia (see fig. 2 ). because their patient had severe adult respiratory distress syndrome (ards) with oxygen saturation readings in the range of 70-80%, the authors attributed the basal ganglia findings to hypoxic brain injury. our patient did not have advanced pulmonary disease, hypoxia, or hypotension. care should be taken to distinguish iae with malignant edema from reyes' syndrome in which patients may present with lethargy, confusion, seizures, or coma accompanied by brain edema. reyes' syndrome most commonly occurs in children but has been reported in adults following influenza and aspirin ingestion [36] . it can be distinguished based on the accompanying hepatic abnormalities, hyperammonemia, and hypoglycemia. caution should be taken with any neurosurgical procedures in reyes' syndrome due to increased risk of perioperative bleeding. one of the most devastating complications of seasonal and pandemic influenza is ane [37] [38] [39] . patients develop rapid neurologic deterioration to coma. seizures are often present. initial brain ct may show decreased density in the thalami, and mri of brain demonstrates the characteristic bilateral thalami lesions. this finding may be initially mistaken for ischemic strokes (top-of-basilar syndrome) or venous infarction secondary to thrombosed internal cerebral veins, vein of galen, or straight sinus. it is interesting that there have been case reports for recurrent ane and also familial ane. this suggests that there may be a genetic susceptibility and a gene associated with familial seasonal influenza ane cases has been reported (nuclear pore gene, ranbp2; [40] ). this condition is often fatal or accompanied by permanent neurologic sequelae in surviving cases. it is intriguing that the neuroanatomical changes found in the thalami, midbrain, and cerebellum on neuroimaging correlated with the clinical symptoms reported for encephalitis lethargica, specifically ''sleeping sickness'', ophthalmoparesis, quadriparesis, and delayed parkinsonism (see below). it is conceivable that survivors with less fulminant involvement could manifest a clinical syndrome with symptoms and signs that localize to brainstem structures. a pediatric case of 2009 h1n1-associated ane with bilateral thalamic imaging findings without associated malignant brain edema has been published [41] , but detailed clinical follow-up was not reported. during the subacute period, additional classic neurologic syndromes associated with influenza have been described. post-influenzal cerebellitis is quite uncommon and has been reported rarely in adults [42] [43] [44] . this syndrome was diagnosed in a 31-year old woman who developed ataxia, dysarthria, and truncal titubation 1 month after influenza b virus infection, with neurologic symptoms that resolved gradually after an additional month. ct and mri brain imaging were unrevealing. csf studies detected evidence of the persistence of the np gene of influenza b virus in the csf from samples taken 7 and 9 weeks after the onset of initial influenza illness. a 25-year old woman gradually developed gait and speech problems after influenza a illness that was treated with oseltamavir. csf showed pleocytosis. the cerebellar cortex had increased t2 signal which resolved over an 80 day period. she received pulse intravenous corticosteroid therapy. her symptoms resolved [42] . plasmapheresis [45] and ivig [46] have also been used for this condition. some cases of cerebellitis following viral and mycoplasma illness have developed fulminant cerebellar swelling with secondary brainstem compression, obstructive hydrocephalus, with fatal outcome [47] . interventions with posterior fossa decompression and external ventricular drain placement may lead to a favorable outcome in a child with this severe condition. antibodies to the glutamate receptor have been reported in patients with post-infectious influenza viral cerebellitis [44] . guillain-barre syndrome (gbs) is a subacute, immunemediated disease predominantly affecting the peripheral nervous system. the diagnosis and treatment are wellknown to most neurologists and this condition has been extensively described and reviewed. gbs has been rarely reported in association with seasonal influenza virus infection [48] , but it should be noted that influenza testing is rarely pursued in gbs cases and may be unrevealing. treatment for influenza-related gbs is identical to treatment for other gbs due to other associated causes. monitoring for respiratory compromise due to neuromuscular weakness with timely respiratory support if needed is critical. plasmapheresis or gammaglobulin treatments are also helpful. the precise pathophysiology is uncertain, but molecular mimicry of the infectious agent is presumed to stimulate autoimmune responses. this has been demonstrated to occur in campylobacter jejuni-associated gbs [49] . influenza-associated myositis has been reported with seasonal influenza [50] and h1n1 variant [51] . myalgias are a common symptom of influenza, but some patients develop frank weakness and have elevated serum levels of creatine phosphokinase (cpk). it is more common in children but has been seen in all age groups. the calf muscles are most suspectible, and patients may walk with a stiff gait or toe walk. onset is usually within the first week of infection and spontaneous improvement typically occurs within 2 weeks in most cases. rarely, severe cases can result in myoglobinuria-associated renal failure and compartment syndromes requiring fasciotomies. influenza can also selectively attack specific muscle groups such as the heart (myocarditis). muscle biopsy shows necrosis, regenerating fibers, and occasionally inflammation. post-viral parkinsonism has been reported after an assortment of infections including influenza virus [52] . an outbreak of these cases was temporally noted following the great influenza (h1n1) pandemic of 1918-1919 [53] . patients with this condition respond poorly to medical therapy, and it has an unfavorable prognosis. encephalitis lethargica is also known as von economo encephalitis and sleeping sickness [53] . a wave of such cases was reported following the 1918-1919 influenza a (h1n1) virus pandemic. the cardinal features of this condition are altered consciousness with prolonged somnolence and ophthalmoplegia. after intervals of months to years, survivors are at risk of developing parkinsonism. pathological findings include nerve cell destruction primarily in the midbrain, subthalamus, and hypothalamus [53, 54] . using modern laboratory techniques, formalin-preserved autopsy brain specimens of encephalitis lethargica cases analysed for influenza viral rna were negative [54] . scientists have proposed a ''hit-and-run'' model of early viral-mediated injury with late sequelae [54] . the neurologist oliver sacks [55] drew attention to this mysterious disorder and the discovery of l-dopa, in his book, awakenings later converted to a feature-length movie. the delayed appearance of restricted diffusion in the basal ganglia in our patient and others [35] is concerning for this condition (fig. 2) . we do not know if this indicates that our patient with 2009 h1n1 is at risk of developing postviral parkinsonism, but long-term clinical follow-up will be important. a delayed diffusion neuroimaging abnormality was also reported in the dentate nucleus of a patient with seasonal influenza encephalopathy/splenial sign [42] . we present a case of acute encephalitis associated with 2009 pandemic influenza a (h1n1) virus infection, complicated by malignant brain edema. the emerging hypothesis about acute neurologic complications of seasonal influenza is that the immune response triggered by influenza virus infection of the respiratory tract plays a prominent role in the pathogenesis of neurological manifestations. this hypothesis regarding the development of acute encephalopathy and brain edema is analogous to current theories about the role of the immune system and cytokines in the development of ards with 2009 h1n1 virus infection. we have also provided an overview of the spectrum of acute and post-infectious neurologic complications reported in association with seasonal and pandemic influenza virus infection of the upper respiratory tract. neurologists should be aware of the potential for a wide range of neurologic complications in association with the current 2009 h1n1 pandemic and seasonal influenza. 1918 influenza, encephalitis lethargica, 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consecutive cerebellitis produced by glutamate receptor delta2 autoantibody plasmapheresis improves outcome in postinfectious cerebellitis induced by epstein-barr virus brain spect imaging and treatment with ivig in acute post-infectious cerebellar ataxia: case report acute near-fatal parainfectious cerebellar swelling with favourable outcome guillain barre syndrome and influenza virus infection carbohydrate mimicry between human ganglioside gm1 and campylobacter jejuni lipooligosaccharide causes guillain-barre syndrome benign acute childhood myositis: laboratory and clinical features melting muscles: novel h1n1 influenza a associated rhabdomyolysis viral parkinsonism lack of detection of influenza genes in archived formalin-fixed, paraffin waxembedded brain samples of encephalitis lethargica patients from 1916 to 1920 new york: random house, inc key: cord-001512-u3u2k8hj authors: ding, hua; chen, yin; yu, zhao; horby, peter w; wang, fenjuan; hu, jingfeng; yang, xuhui; mao, haiyan; qin, shuwen; chai, chengliang; liu, shelan; chen, enfu; yu, hongjie title: a family cluster of three confirmed cases infected with avian influenza a (h7n9) virus in zhejiang province of china date: 2014-12-31 journal: bmc infect dis doi: 10.1186/s12879-014-0698-6 sha: doc_id: 1512 cord_uid: u3u2k8hj background: a total of 453 laboratory-confirmed cases infected with avian influenza a (h7n9) virus (including 175 deaths) have been reported till october 2,2014, of which 30.68% (139/453) of the cases were identified from zhejiang province. we describe the largest reported cluster of virologically confirmed h7n9 cases, comprised by a fatal index case and two mild secondary cases. methods: a retrospective investigation was conducted in january of 2014. three confirmed cases, their close contacts, and relevant environments samples were tested by real-time reverse transcriptase-polymerase chain reaction (rt-pcr), viral culture, and sequencing. serum samples were tested by haemagglutination inhibition (hi) assay. results: the index case, a 49-year-old farmer with type ii diabetes, who lived with his daughter (case 2, aged 24) and wife (case 3, aged 43) and his son-in-law (h7n9 negative). the index case and case 3 worked daily in a live bird market. onset of illness in index case occurred in january 13, 2014 and subsequently, he died of multi-organ failure on january 20. case 2 presented with mild symptoms on january 20 following frequent unprotected bed-side care of the index case between january 14 to 19, and exposed to live bird market on january 17. case 3 became unwell on january 23 after providing bedside care to the index case on january 17 to 18, and following the contact with case 2 during january 21 to 22 at the funeral of the index case. the two secondary cases were discharged on february 2 and 5 separately after early treatment with antiviral medication. four virus strains were isolated and genome analyses showed 99.6 ~100% genetic homology, with two amino mutations (v192i in ns and v280a in np). 42% (11/26) of environmental samples collected in january were h7n9 positive. twenty-five close contacts remained well and were negative for h7n9 infection by rt-pcr and hi assay. conclusions: in the present study, the index case was infected from a live bird market while the two secondary cases were infected by the index case during unprotected exposure. this family cluster is, therefore, compatible with non-sustained person-to-person transmission of avian influenza a/h7n9. electronic supplementary material: the online version of this article (doi:10.1186/s12879-014-0698-6) contains supplementary material, which is available to authorized users. human infection with avian influenza a/h7n9 virus was first identified in march 31 of 2013, in china, a total of 453 confirmed cases were found in the world up to date [1] . the seasonal epidemiology is characterized to occur from november through april in china, coinciding well with both seasonal human influenza and h5n1 in birds [2] . almost all cases were hospitalized, and 1/3 of cases died. the fatality is much higher than that for seasonal influenza in the china (0.04%), but it is lower than for cases of h5n1 (60%) [3, 4] . current evidence suggests that human infection appears to be associated with exposure to infected live poultry or contaminated environments, including markets where live poultry are sold [5] [6] [7] . in the light of this opinion, the closure of live bird markets (lbm) has been associated with a reduction in the incidence of human infections [8] . despite the fact that h7n9 remains to be a zoonotic infection of avian origin, there are concerns that the virus show genotypic and phenotypic evidence of partial adaptation to mammals [9] . compared to other subtypes of avian influenza virus, h7n9 virus show increased binding affinity to mammalian-type receptors, and their amount grow up rapidly at the temperatures that are close to the normal body temperature in mammals (although it is lower than that of birds). in addition, they possess pb2 gene mutations that are associated with adaptation to mammals [10] [11] [12] . whilst sequence analyses had shown that the haemagglutinin (ha) and neuraminidase (na) genes of h7n9 virus detected in china show very high homology, whereas the genes for coding internal proteins are diversified [13] . ferret and mouse models confirm that strains isolated from humans could replicate efficiently in both mammalian and human airway cells, with efficient transmissibility by direct contact and modest transmissibility by respiratory droplets [14, 15] . given these signatures of partial adaptation to mammals, it is imperative to closely monitor and investigate all clusters of human h7n9 virus to determine the transmissibility and severity of virus infection, as well as its potential host and pathogen determinants. a few of family clusters of h7n9 infections (in shanghai, jiangsu, shandong, guangdong and beijing) have been described involving two family members. it was concluded that limited person-to-person transmission may occur following close, prolonged, and unprotected contact with the symptomatic index case, while sustained transmission was not found [16] [17] [18] . here we describe an additional cluster, comprised of three laboratory-confirmed cases of human infection with h7n9 virus reported in zhejiang province in january 2014. this is the largest reported cluster of virological confirmed h7n9 cases, and the full genome data of the virus were isolated from all cases and associated with clinical and epidemiological data and their close contacts. all three h7n9 confirmed cases and 25 adult contacts and surveillance cases had provided written consent for the participation in this study and the publication of their individual details. data collection for h7n9 cases was determined by the national for h7n9 cases was determined y f man of china, as a part of the continuing public health outbreak investigation; therefore, it was exempt from assessment by institutional review board. the protocol for collecting epidemiological data and conducting serological test of close contacts were approved by the institutional review board of the china cdc. suspected cases of human infection with h7n9 virus are identified through the chinese surveillance systems for influenza-like illness, severe acute respiratory illness (sari), pneumonia of unexplained origin, and clinical diagnostics of cases of pneumonia. based on the chinese guidance, an individual could be considered as a confirmed case of h7n9 virus infection if the presence of the h7n9 virus is verified by real-time reverse transcriptase polymerase chain reaction assay (rt-pcr), virus isolation, or serologic testing [19] . epidemiological and clinical data were collected through interviews and reviews of medical records between january 13 and 25, 2014. all three cases and their relatives were interviewed by public health staff to record their exposure history during the two weeks before the onset of symptoms, to validate the timeline of events and to identify close contacts. respiratory tract samples were collected from the index case, case 2, and case 3, on january 18, 22, and 23, respectively. environmental samples were collected from the lbm (a1 market) and the secondary wholesale markets (b1, c1, and d1 markets) and from a neighboring household where several chickens were bred. all samples were placed in sterile viral transport medium and shipped within 24 hours to the laboratory of zhejiang cdc at 4°c for h7n9 testing. viral rna was extracted using qiagen rneasy mini kit. real-time rt-pcr was used to detect influenza type a, subtype h7 and n9 using the protocol, specific primer and probe sets provided by china cdc [20] . specimens were also tested by rt-pcr for the presence of seasonal influenza virus (h1, h3, and b) and h5n1 virus. complete genomic fragments of the h7n9 virus were amplified directly from clinical samples, and sequencing was performed using an abi 3730xl automatic dna analyzer. the nucleotide sequences were determined by dideoxy sequencing using an abi prism bigdye terminator cycle sequencing kit as previously described [21] . nucleotide sequences were analyzed with the dnastar package (lasergene, madison, wi, usa). phylogenetic analysis was done by neighbor-joining method with mega (version 5.2). close contacts were placed under daily active surveillance for fever and respiratory symptoms, which was last for seven days after their last exposure to the h7n9infected case. close contacts were defined as individuals who had close contact (<1 meter) with any case without the use of personal protective equipment at any time before illnesses onset to the time of isolation of the case in hospital. antiviral chemoprophylaxis was neither recommended nor provided to contacts. following written informed consent, a structured questionnaire was used to gather demographic information and data on use of personal protective equipment, antiviral chemoprophylaxis, symptoms, and potential risk factors for h7n9 infection during the two weeks starting from their last exposure to h7n9-infected cases. respiratory specimens for h7n9 testing were taken from close contacts with a febrile respiratory illness occurred during the 7-day observation period. contacts were asked to provide a single convalescent serum collected ≥ 3-4 weeks after their last exposure to a case with h7n9. h7n9 serological testing was done by hi assay using a modified horse red-blood-cell assay, recommended by the who. the antigen used for the hi assays was the a/zhejiang/1/2013(h7n9) strain. a hi titer ≥ 1:40 in single serum sample and a four-fold or greater rise in titer in paired sera was defined as seropositive. the index case, a 49-year-old farmer with type ii diabetes, taking antidiabetic drugs for one year, had been unwell since january 13, 2014, with fever (39.6°c) and flank pain. after consulting a health care clinic (a1 clinic) on january 14 and 15, he was treated as an outpatient with ciprofloxacin and intravenous amoxicillin/ clavulanate potassium. on january 16, he made a further consultation at a local hospital (b1 hospital) owing to persistent fever. chest radiography showed a leftlower-lobe pneumonia; meanwhile, treatment with ciprofloxacin was continued. peripheral blood cell count was normal. on january 17, index case's condition was worsened and again medical advice was sought; therefore, index case was admitted to a different hospital (hospital c1). upon admission in hospital c1, he had severe leucopenia, lymphopenia and thrombocytopenia (table 1) . he was diagnosed with community acquired pneumonia with a left pleural effusion. on january 18, he consulted at hospital d1 (a more advanced hospital) where a sputum sample was collected and sent to zhejiang cdc for microbiologic testing. on january 19, h7n9 virus-specific rna was detected by rt-pcr (ct value 29) in the sputum sample. once the h7n9 virus infection was confirmed, the patient was transferred from hospital c1 to d1 immediately. at hospital d1, he was isolated in a single room, where he was intubated, mechanically ventilated and commenced on oseltamivir (75 mg, twice daily by nasogastric tube) and peramivir (600 mg, once daily, intravenously). on january 20, the patient died of acute respiratory distress syndrome (ards) and multi-organ failure (table 1, figure 1 , figure 2 , and additional 1: figure s1 ). case 2 (index case's daughter, figure 1 ), a 24-year-old female with no underlying diseases, developed a throat sore and cough on january 20, the day her father died. she initially consulted the healthcare clinic in hospital a1 due to constant fever on january 22, where she was treated with antibiotics (amoxicillin) and then transferred to hospital d1 for further examination, where sputum and throat swab samples were taken. rt-pcr was conducted on the samples of sputum and throat swabs on the january 23, and the influenza a/h7n9 specific rna (ct value 34) was positive. afterwards, case 2 was admitted directly to an isolation room at the hospital d1 and commenced on oral oseltamivir (75 mg, twice daily), and intravenous peramivir (300 mg, once daily). on admission her peripheral blood count, serum blood biochemistry, and chest ct scan were normal (table 1) . she was given supplemental oxygen via nasal cannula with a flow rate of 1-3 l/min, whereas her oxygen saturation was 99%. her condition remained stable, and symptoms were improved during hospitalization. later, she was completely recovered and was discharged on february 2 after sputum samples tested negative for h7n9 rna by rt-pcr on january 30 and february 1 (table 1, figure 1 ). case 3 (index case's wife and case 2's mother), a 43year-old female farmer, with no underlying diseases, developed an acute cough with expectoration on january 23. she attended the hospital d1 where a throat swab was collected and an rt-pcr assessment was conducted on the throat swab sample, which was positive for h7n9 (ct value of 37). she was admitted to the hospital d1 on january 23. although chest radiography was normal, she was treated empirically with oral oseltamivir (75 mg, twice daily) and intravenous peramivir (300 mg, once daily). results of peripheral blood cell count, serum electrolytes, renal and liver function, and coagulation profiles were normal. arterial blood gas results were normal while the patient was breathing room air. the case remained stable during her admission and then she was discharged on february 5 after sputum tested h7n9 negative by rt-pcr on february 4 (table 1, figure 1 ). the husband of case 2, who had been in close contact with the index case and case 2, had no respiratory symptoms, and throat swabs and paired serum samples were negative ( figure 1 ). rt-pcr-positive throats swabs or sputum samples were obtained on 5 days, 2 days and 1 day of illness for the index case, case 2, and case 3, respectively. from these samples, four complete full genome sequences were amplified. sequence analyses indicated that the four isolates were highly homologous the other h7n9 strains previously identified in shanghai, jiangsu, anhui province, and with candidate vaccine strains (sharing 99.61 00% identity in amino acid sequences of all 8 segments). the four sequences from the three confirmed h7n9 case shared 96.4~99.6% homology with the animal isolates (a/chicken/zhejiang/sd019/2013), and phylogenetic analysis showed that the four isolates were almost genetically identical to other h7n9 virus isolated from the other provinces and chickens. furthermore, amino acid analyses showed that the ha gene of all four strains possessed the mutation 226 l, indicating high affinity to human receptor alpha 2-6 sialic acid receptors. it showed that the four isolates were entirely of human origin, and na protein possessed amino acid sequences associated with susceptibility to neuraminidase inhibitor antiviral drugs (h294 and e120 and h276 in na). the 8 fragments isolated from the index case, and two secondary cases were identical except for three non-synonymous amino substitutions identified in the index case. these were g574a nucleic acid substitution (aa mutation v192i) in the ns gene, g1095a nucleic acid substitution in the pb1 gene (nonsense amino mutation) and c839t nucleic acid substitution (v280a) in the np gene. (figure 3 , additional file 1: figure s2 , table 2 and table 3 ). the index case, his daughter (case 2), his wife (case 3), and his son-in-law (the husband of case 2) lived in separate rooms of one large house with three floors. there were no domestic animals and birds within the home or in the immediate vicinity of the home. however, two neighboring families located 100 meters and 500 meters respectively from the cases' homebred ducks and chickens, and there were a several free-range domestic poultry in the village. the index case and case 3 worked in the lbm (a1 market), selling vegetables and bird eggs between 4 am and 7 pm during the two weeks prior to the illness onset in the index case. furthermore, two weeks before the illness onset, the index case had visited a wholesale lbm (d1 market) to buy vegetables twice per week (each time he stayed there for 3 hours). the last known exposure date of the index case in a1 market was january 12 (15 hours), and the last exposure date of case 2 to a1 market was on january 17 for around three hours. in total. case 3 had been exposed to the live bird market on three occasions for a total of 22 hours from january 17-20 as follows: case 3 visited d1 market for a total of three hours between january 16-18 and she worked in a1 market for 8, 10 and, 1 hours on january 17, 18, and 20, respectively. the index case became ill on january 13 and was admitted to hospital on january 17. between january 13 and 17, cases 2 and 3 lived together with the index case in one house. furthermore, case 3 and index case had very close contact between january 13 and 17, sleeping together in one room. case 2 had three hours faceto-face contact with the index case on january 14. on january 17, case 2 provided bedside care in the hospital for the index case for approximately 12 hours. between january 17 and 19, cases 2 and 3 provided bedside care to the index case in hospital without any personal protective equipment for approximately 30 hours and 7 hours, respectively, including washing, cleaning his body, change his clothes and disposing urine and feces of the index case. during this period, the index case had high fevers (39.9°c), frequent coughing, and extensive sputum production. after the index case had been confirmed h7n9 infection on january 19, he moved to icu for treatment and isolation. cases 2 and 3 visited the index case for four hours on january 19, wearing facemasks. case 3 had frequent close contact with case 2 during the funeral ceremony of the index case on january 21-22. case 3 visited case 2 on the january 23 when case 2 was hospitalized with mild symptoms; case 3 wore a facemask during this visit. a summary of the cases' exposure to each other was shown in table 4 . the results of rt-pcr assay of environment samples were listed as follows: 4 swabs of chicken and duck eggs from the index case working site (a1 market) were h7n9 negative; 3 of 5 environmental samples from secondary live wet market (b1 market, a wholesale for a1 market) were positive for h7n9; 1 of 2 sewage samples from c1 market (located nearby b1 market) were positive for h7n9; 10 of 20 environmental samples taken from d1 market through routine avian influenza surveillance were h7n9 positive; 12 environmental samples from the area where neighbors were breeding poultry were all h7n9 negative; 11 of 26 environmental samples from different live birds markets under routine surveillance in xiaoshan district were h7n9 positive during january 2014 (source: unpublished data from the zhejiang avian surveillance system, additional file 1: figure s3 ). none of the 25 close contacts developed acute respiratory symptoms during the seven days surveillance period. throat swabs collected from all twenty-five close contacts on january 24 were negative for influenza a/ h7n9 virus by rrt-pcr, and all serum samples tested negative for h7n9 antibodies (titer < 1:40) by microneutralization and horse red-blood-cell hi assays (see table 5 ). no close contacts were reported taking oseltamivir chemoprophylaxis. here we describe a family cluster of three confirmed cases of h7n9 virus infection, involving a fatal index case, his wife and daughter (both survived). the index case presented with severe pneumonia and died of ards and multi-organ failure. the presence of chronic diseases has been associated with an increased risk of hospitalization with h7n9 virus infection [22] , and the index case had pre-existing diabetes, which requires oral anti-diabetic medication. another factor that may have played a role in the severity of disease was the late diagnosis of h7n9 virus infection and the late commencement of anti-viral therapy. the efficacy of neuraminidase inhibitors (nais) in reducing the risk of mild influenza infection progressed to severe illness has not been fully assessed in randomized controlled trials; however, observational data suggest that early treatment with nais of hospitalized patients with influenza infection is associated with better outcomes [23] . the other two cases i308v 308 i i i v i i i i i t618k 618 t t t t t t t t t single letters refer to the amino acid (aa) found in the noted protein at a specific site. *the numbering starts with the first condon of methionine for these proteins. were previously reported healthy, and presented with lower viral loads and mild symptoms that did not progress. both patients received early antiviral treatment, but it is not possible to determine whether the lack of clinical progression was result from antiviral treatment or as a consequence of a naturally indolent course [24] [25] [26] . since there were no functionally important differences in the genotype of the virus infecting the three cases, viral virulence is not likely to contribute the differential severity. who evaluates all clusters of human cases of nonseasonal influenza virus to determine whether humanto-human transmission or common exposure to infected animals or contaminated environments may have occurred [19] . the homology of all eight gene segments was between 99.6~100%, suggesting it was either a common source exposure or a person-to-person transmission. whilst all three individuals were exposed to potentially contaminated market environments within a putative maximum incubation period of 7 days, case 2 and case 3 had extensive unprotected exposure to the index cases when he was ill. we believe that most likely explanation for this family cluster is that the index case was infected from the live bird market, and the virus was transmitted directly from the index case to his daughter and his wife. several reasons could explain for this conclusion, as follows: (1) 7 days prior to illness onset in the index case, he had not been in contact with any people with a febrile illness and other confirmed cases, but was frequently exposed to the a1 live bird market for 9 hours daily and to the d1 secondary live bird market. although the a1 market was h7n9negative based on the environments samples collected on january 24, 2014, the samples from wholesale market b1 that supplied a1 market were h7n9 positive. furthermore, 42.30% (11/26) of environments samples from different live bird markets under routine surveillance in xiaoshan district during the same period were h7n9 positive (source: unpublished data from the zhejiang avian surveillance system); (2) case 2 stayed with the index case and provided beside bed medical care frequently on the january 14, 16, and 17-19. she had close unprotected contact with the index case for cleaning and washing his body on january 18 without any personal protection when the index case had severe symptoms such as high fever and cough. although case 2 had visited the a1 live bird market for three hours in three days prior to her illness onset, she reported no direct contact with live birds or poultry products. (3) there were multiple potential sources of infection for case 3, including the index case, the live market a1, and case 2. however, the index case and case 3 shared the same room every day and worked closely together after the illness onset in the index case. most importantly, case 3 provided beside bed care to the index case including washing his body, dealing with his secretions, and changing his clothes for him, without any personal protective equipment. the day numbers between the onset of illness in the index case and the onset of illness in the secondary cases (the serial interval) was 7 and 10 days, [27, 28] . furthermore, sequence analysis showed that four strains isolated from the three cases were genetically similar to each other. all four isolates possessing amino acids q226l and g228s in the ha segment were associated with increased affinity for human receptors (α-2, 6linked sialo-saccharides) [29] . virus from all three cases possessed p42s in ns and e627k and d701n in pb2 (which were associated with increased virulence in mice) and i368v and h99y in pb1 (which was associated with aerosol transmission of avian virus between ferrets) [7, 8] . there were only two amino differences (v192i in ns and v280a in np) between the virus infecting the index case and the secondary cases. those two mutations are not associated with any known functional change. therefore, field investigation and h7n9 full genomics analyses supported the secondary cases acquired infection most likely from the index case. person-toperson transmission of h7n9 has been reported [30, 31] . previous animal experiments (ferrets, mice, and pigs) also indicate that h7n9 virus possess the capability to bind to both avian and human receptors and it might be transmissible by respiratory droplets under certain conditions [12, 15] . our findings indicate that the virus has not gained the ability for efficient sustained transmission from person to person [12] . in this study, four close contacts and 21 frequent contacts were negative for h7n9 infection by hi testing and rt-pcr. although the husband of case 2 had close contact with the index case, case 2, and case 3 without any personal protective equipment, he showed no evidence of infection with the h7n9 virus. there were several limitations in this paper. firstly, h7n9 positive samples in environmental or bird samples were not found from a1 live bird market where the index case and case 3 were working. secondly, the full genetic sequence of h7n9 virus detected in the environment and live birds could not obtained. thus it is not able to compare human, avian, and environmental strains. on the basis of experiences of controlling of h5n1 and h7n9 virus, continued risk assessment, surveillance, and vigilance are required. a high degree of clinical awareness is necessary for people with possibility of h7n9 infection, especially for health workers who are occupationally exposed to poultry and for people with respiratory illness following recent contact with live poultry or live bird markets [32, 33] . here we report a largest size of the family cluster with confirmed h7n9 in china, in which the index case was fatal while the secondary cases were mild. in the term of an infectious source, the index case was infected from a live bird market while the index case infected the two secondary cases during unprotected frequent exposure. this family cluster supported that the transmission of avian influenza a/h7n9 was limited and not sustained. all of h7n9 referred isolates in additional materinals were download from genbank (http://www.ncbi.nlm.nih. gov/nuccore/?term=h7n9++and+china) and the global initiative on sharing all influenza data (gisaid) (http://platform.gisaid.org/epi3/frontend#50dda5). additional file 1: figure s1 . family pedigree showing three h7n9 affected individuals and their close contacts. figure s2 . phylogenetic analysis of six segments (mp, np, ns, pa, pb1, and pb2) from the four h7n9 isolates in three confirmed cases of a family cluster in hangzhou, zhejiang province, china, in january of 2014. figure s3 . all authors have declared: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. authors' contributions hd, yc, zy, pwh, ec, and hy designed the study. fw, jh, and xy conducted the field investigation and analyses. hm, sq and cc collected and tested the samples, performed and sequence analyses. sl and pwh wrote the first draft and all authors contributed to review and revision of the report. ec and hy are guarantors. all authors read and approved the final manuscript. human infections with avian influenza a(h7n9) virus h7n9: preparing for the unexpected in influenza writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza av pandemic characteristics and controlling experiences of influenza h1n1 virus 1 year after the inception in hangzhou amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel a/h7n9 influenza virus in mammalian hosts dynamic reassortments and genetic heterogeneity of the human-infecting influenza a (h7n9) virus human infection with avian influenza a(h7n9) virus re-emerges in china in winter live-animal markets and influenza a (h7n9) virus infection pathogenesis and transmission of avian influenza a (h7n9) virus in ferrets and mice limited airborne transmission of h7n9 influenza a virus between ferrets novel avian-origin human influenza a(h7n9) can be transmitted between ferrets via respiratory droplets the k526r substitution in viral protein pb2 enhances the effects of e627k on influenza virus replication environmental connections of novel avian-origin h7n9 influenza virus infection and virus adaptation to the human epidemiology of human infections with avian influenza a(h7n9) virus in china family outbreak of severe pneumonia induced by h7n9 infection surveillance of the first case of human avian influenza a (h7n9) virus in beijing probable person to person transmission of novel avian influenza a (h7n9) virus in eastern china, 2013: epidemiological investigation one family cluster of avian influenza a(h7n9) virus infection in characterization of h7n9 influenza a viruses isolated from humans infectivity, transmission, and pathology of human-isolated h7n9 influenza virus in ferrets and pigs human infection with a novel avian-origin influenza a (h7n9) virus comparison of patients hospitalized with influenza a subtypes h7n9, h5n1, and 2009 pandemic h1n1 determinants of antiviral effectiveness in influenza virus a subtype h5n1 risk assessment on the epidemics of human infection with a novel avian influenza a (h7n9) virus in jiangsu province global concerns regarding novel influenza a (h7n9) virus infections analysis of the clinical characteristics and treatment of two patients with avian influenza virus (h7n9) comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a population-based study of laboratory-confirmed cases probable longer incubation period for human infection with avian influenza a(h7n9) virus in jiangsu province receptor binding by an h7n9 influenza virus from humans probable person-to-person transmission of avian influenza a (h5n1) three indonesian clusters of h5n1 virus infection in 2005 genomic signature and protein sequence analysis of a novel influenza a (h7n9) virus that causes an outbreak in humans in china human infection with avian influenza a h7n9 virus: an assessment of clinical severity we thank all of staff at zhejiang provincial and hangzhou municipal cdc, xiaoshan district cdc, for their help in field investigation and collection of environmental samples. the views expressed are those of the authors and do not necessarily represent the policy of the china cdc. note: data are median (iqr) or n (%). *including direct contact (touching), preparation, cooking, and consumption of well-appearing poultry. key: cord-002972-ge7qt256 authors: torner, núria; martínez, ana; basile, luca; mosquera, mmar; antón, andrés; rius, cristina; sala, m. rosa; minguell, sofia; plasencia, elsa; carol, mónica; godoy, pere; follia, núria; barrabeig, irene; marcos, m. angeles; pumarola, tomàs; jané, mireia title: descriptive study of severe hospitalized cases of laboratory-confirmed influenza during five epidemic seasons (2010–2015) date: 2018-04-14 journal: bmc res notes doi: 10.1186/s13104-018-3349-y sha: doc_id: 2972 cord_uid: ge7qt256 objective: the plan of information on acute respiratory infections in catalonia (pidirac) included the surveillance of severe hospitalized cases of laboratory-confirmed influenza (shclci) in 2009. the objective of this study was to determine the clinical, epidemiological and virological features of shclci recorded in 12 sentinel hospitals during five influenza seasons. results: from a sample of shclci recorded during the 5 influenza epidemics seasons from 2010–2011 to 2014–2015, cases were confirmed by pcr and/or viral isolation in cell cultures from respiratory samples. a total of 1400 shclci were recorded, 33% required icu admission and 12% died. the median age of cases was 61 years (range 0–101 years); 70.5% were unvaccinated; 80.4% received antiviral treatment (in 79.6 and 24% of cases within 48 h after hospital admission and the onset of symptoms, respectively); influenza virus a [37.9% a (h1n1)pdm09, 29.3% a (h3n2)] was identified in 87.7% of cases. surveillance of shclci provides an estimate of the severity of seasonal influenza epidemics and the identification and characterization of at-risk groups in order to facilitate preventive measures such as vaccination and early antiviral treatment. electronic supplementary material: the online version of this article (10.1186/s13104-018-3349-y) contains supplementary material, which is available to authorized users. influenza is an infectious disease affecting mainly upper respiratory tract worldwide. influenza virus causes between three and five million severe cases and an estimated 250,000-350,000 deaths annually. in the european union, there are between 40,000 and 220,000 annual deaths attributable to influenza. however, mortality is only the tip of the iceberg in terms of the disease burden, since influenza also causes a decrease in functional status and increased dependency in the elderly [1] . estimating the burden of disease caused by influenza is difficult because many cases do not require medical care, or no confirmatory laboratory tests are widely performed to all influenza like illness' cases [2, 3] . in catalonia, influenza surveillance is conducted through the plan of information on acute respiratory infections in catalonia (pidirac) based on the network of sentinel physicians, who provide information on patients with influenza symptoms [4] . given the situation generated by the 2009 pandemic caused by the new influenza a (h1n1) pdm09 virus, the pidirac sentinel network included surveillance of severe hospitalized cases of laboratory-confirmed influenza (shclci) to assess severity. the pidirac sentinel surveillance network has a primary care sentinel network made up by 60 gps and pediatricians who inform on a daily basis of all ili attended and perform sampling of respiratory swabs for confirmation. this information allows to plot weekly ili incidence and 12 sentinel hospital facilities who notify on a weekly basis all influenza confirmed cases that meet the ecdc definition for severe influenza [5, 6] . this surveillance allows the clinical and epidemiological characteristics and risk factors associated with greater severity to be determined, and the emergence of influenza virus strains with clinical characteristics and behaviours outside the normal range to be detected, in order to correctly prioritize and direct preventive and control measures during the influenza season [7] . the aims of shclci surveillance are to provide an estimate of the severity of seasonal influenza epidemics to identify and characterize the risk groups that may present serious complications as a result of infection by circulating influenza viruses or their association with some underlying diseases and to identify the virological characteristics of viruses associated with these severe cases, such as genetic changes and/or antigenic changes that lead to increased virulence. the aim of this study was to describe the clinical, epidemiological and virological characteristics of shclci based on data collected in five influenza seasons in catalonia. epidemiological surveillance of severe cases of influenza in catalonia during five epidemic influenza seasons (2010-2015), beginning on week 40 of the season until week 20 of the following year, with the recording by twelve hospitals (covering 95,3% of the population) from the pidirac sentinel network of shclci reported to the epidemiological surveillance units corresponding to each hospital [5, 7] . shclci cases were cases with previous influenza like illness symptoms (sudden onset of symptoms and/ or fever; malaise; headache; muscle pain; and/or cough; sore throat; shortness of breath) who presented to a hospital facility and complyed with shclci case definition. shlcic was defined as a severe case of laboratory-confirmed influenza due to the influenza virus (a, a (h1n1)pdm09, b, c) that required hospitalization because of pneumonia, septic shock, multiorgan failure or any other severe condition, including icu admission or who developed clinical signs during hospitalization for other reasons. influenza diagnosis was confirmed by polymerase chain reaction (pcr) and/or culture of nasopharyngeal swabs. respiratory tract samples were processed within 24 h of receipt at the laboratory. a 300 μl aliquot was taken for total nucleic acids extraction and eluted in 25 μl of rnase-free elution buffer using the automatic qiasymphony system (qiagen, hilden, germany) according to the manufacturer's instructions. subsequently, two specific one-step multiplex real-time pcr was carried out using the stratagene mx3000p qpcr systems (agilent technologies, santa clara, ca, usa), were used for typing a/b influenza virus and subtyping influenza a virus [8, 9] . for each reported case, an epidemiological survey was made to collect anonymized demographic variables (age and sex); risk factors; icu admission; day of onset of symptoms, of hospital admission and discharge; vaccination history; influenza virus type and subtype; and outcome at hospital discharge. epidemiological survey was conducted by preventive medicine physician from data in medical history registry and public health epidemiologist in charge. we studied all data on shclci from five influenza seasons in pidirac sentinel network hospitals and made a comparative analysis of viral types and subtypes. the strain identified in > 50% of cases in each season was considered the predominant strain. duration of hospital stay was divided into two categories < 10 days and 10 days or more. the statistical analysis was made using the chi square test and student's t test with 95% confidence intervals (ci) for continuous variables and the anova test for categorical variables. during the 2010-2015 seasons 1400 cases of shclci were recorded, 462 (33%) required icu admission and 167 (12%) died: 778 (55.6%) were male. the median age was 61 years (range 0-101 years-mean 55.2 (sd 26.7 years). the most-affected age group was the ≥ 65 years age group with 633 cases (45.2%) ( table 1 ). the median age of the ≥ 65 years age group was 79 years (range 65-101) and the mean age was 78.7 years (sd 7.8 years): 296 (47%) were aged ≥ 80 years. of deaths, 111 (66.5%) occurred in patients aged ≥ 65 years and 55 (33.3%) in patients aged > 80 years ( table 1) . the distribution by type of influenza virus was: 87.7% (1228) influenza virus a, 531 (37.9%) of which corresponded to the a (h1n1)pdm09 subtype and 410 (29.3%) to a (h3n2), and 20.5% to influenza a that remained unsubtyped: 172 (12.3%) of cases were influenza b (additional file 1). there were significant differences in the mean age of cases according to the virus type, with a higher prevalence of virus a (h3n2) in older patients and virus a (h1n1)pdm09 in younger patients with mean age of cases 66.9 and 46.8 years (p < 0.001) and those with death as outcome 78.8 and 60.2 years, respectively (p < 0.001) ( table 1) . in 1384 (98.9%) of shclci there was a known risk factor. the most prevalent risk factors were cardiovascular disease, chronic obstructive pulmonary disease and diabetes (25.5, 23.4 and 20.5%, respectively). the most prevalent complication was pneumonia in 992 (71.7%) cases, of these 304 (30.6%) presented bacterial superinfection. for cases with known immunization for influenza, 682/967 (70.5%) of cases were not vaccinated for the current season included in the study (missing data on vaccination status: 433 (31%). the age group with highest vaccine coverage was the older than 65 age group (57%) and cases with at least one risk factor had low vaccination coverage (20.5%). vaccine proved effective in reducing intensive care unit (icu) admission [or = 0.64 (95% ci 0.47-0.88) p = 0.003] ( table 2) . of the 21 pregnant women hospitalized as shclci, all were unvaccinated, 14 (66.7%) required icu admission, 19 (90.5%) received antiviral treatment and none of them had any underlying disease or risk factor other than pregnancy. the mean hospital stay was 13.8 days (sd 17.9) with a median of 9 days (range 1-374 days). the mean stay by age group was: 0-4 years 7. a total 1125 cases (80.4%) had information on antiviral treatment, 1113 (99%) received oseltamivir and 12 (1%) zanamivir. 863 of these cases (79.6%) received treatment in the first 48 h after admission. antiviral treatment administered before 48 h on admission was associated with a shorter length of stay (los) (or 0.25: ci 0.18-0.34, p < 0,001) nosocomial cases (41) were excluded from the analysis (table 3 ). after the 2009 influenza virus a(h1n1)pdm09 pandemic, among the lessons learned was the need to expand surveillance of seasonal influenza to include severe cases in order to determine the characteristics of shclci caused by seasonal influenza viruses circulating during each season. the results obtained by the pidirac sentinel surveillance system during five post-pandemic seasons underscore the importance of prevention by vaccination in order to avoid serious complications such as ards and icu admission of the most vulnerable persons, while showing the need for increased vaccination coverages in groups such as pregnant women, in whom the proportion of icu admission is 66.7% while vaccination is zero [7, 10] . in our study no significant differences between influenza a and b virus infections among hospitalized cases was observed, except for younger age for a (h1n1) pdm09 cases; similar results also found by other studies in the united states and australia [11] [12] [13] . although the number of hospitalizations associated with influenza a virus infections was greater than the number with influenza b virus infections this fact can be explained by greater prevalence of influenza a viruses circulating in the community during the seasons included in the study. the delay in the administration of antiviral drugs at symptom onset in people with an identified risk of complications, such as the elderly or people with medical conditions that worsen the prognosis of influenza or make a longer hospital stay likely, also demonstrates the need to confirm influenza in primary healthcare and administer treatment within 48-72 h for it to be effective. influenza remains an important global public health problem in spite of scientific evidence which support immunization to protect those at high risk for complications, such as the elderly [1] . predominant influenza type/subtype circulating each season, influenza vaccination policies and coverage, influenza vaccine strain match/mismatch and vaccine effectiveness significantly influence the % of hospitalised influenza cases and cfrs in all age groups, including older age groups. however, the high percentage of hospitalizations (45.2%) and mortality (17.5%) in the ≥ 65 years age group, especially in people aged > 80 years, where mortality is higher (33.3%), reflect the consequences of increased life expectancy. early administration of antiviral treatment has proven to diminish length of stay. healthcare providers should start antiviral treatment as soon as possible, before 48 h from onset of symptoms is the recommendation, [11] unfortunately this is not feasible. yet if treated as soon as patient is admitted to the hospital facility and influenza is confirmed, shorter length of stay and prompt recovery can be attained [12, 13] . this makes it necessary to deepen our knowledge of the effect of aging and its interaction with the most prevalent chronic diseases in the elderly and the immune response in order to implement preventive measures to provide better protection of this population group [11] . it is necessary to improve some surveillance aspects, especially with regard to data collection, in order to avoid a loss of information that makes some variables impossible to assess, such as risk factors such as smoking, which was not recorded in 91% of cases as well as lack of information on the vaccination status, which was more than 30% [14, 15] . a limitation to this study is that only shclci cases were recorded during the study period. this unables global hospitalization burden estimates caused by seasonal influenza nor the estimation of seasonal differences in vaccine effectiveness to prevent severity and death. the system identifies the epidemiological and virological characteristics of severe forms of influenza that show changes in their virulence, but comparison between severe and non-severe cases is not feasible. the proportion of shclci cases admitted to icu and cfrs are potentially higher than other surveillance systems that monitor all hospitalised cases of confirmed influenza. this is particularly evident with regard to pregnant women because of the small number of cases. yet, in all shclci surveillance provides an estimate of the severity of seasonal influenza epidemics, and provides ad hoc information to identify and characterize the groups at risk of complications and take appropriate preventive measures. abbreviations ards: acute respiratory distress; icu: intensive care unit; cfr: case fatality rate; ci: confidence interval; ecdc: european center for disease control; ili: influenza like illness; los: length of stay; or: odds ratio; pcr: polymerase chain reaction; pidirac: plan of information on acute respiratory infections in catalonia; sd: standard deviation; shclci: severe hospitalized cases of laboratoryconfirmed influenza. • fast, convenient online submission ready to submit your research ? choose bmc and benefit from: vaccine effectiveness in older individuals: what has been learned from the influenzavaccine experience influenza illness and hospitalizations averted by influenza vaccination in the united states predicting clinical severity based on substitutions near epitope a of influenza a/h3n2 public health agency of catalonia. department of health. pla d'informació de les infeccions respiratòries agudes a catalunya (pidirac) estratègia de vigilància dels casos greus de grip hospitalitzats vigilancia de casos graves hospitalizados confirmados de virus de la gripe the global influenza hospital surveillance network (gihsn): a new platform to describe the epidemiology of severe influenza global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis virological surveillance of influenza and other respiratory viruses during six consecutive seasons from severe influenza in 33 us hospitals, 2013-2014: complications and risk factors for death in 507 patients early administration of oral oseltamivir increases the benefits of influenza treatment effectiveness of antiviral treatment in preventing death in hospitalized cases of severe influenza over six influenza seasons increased antiviral treatment among hospitalized children and adults with laboratory-confirmed influenza estimating the burden of seasonal influenza in spain from surveillance of mild and severe influenza disease estimated influenza illnesses, medical visits, hospitalizations, and deaths averted by vaccination in the united states seasonal influenza (flu) cdc. centers for disease control and prevention, national center for immunization and respiratory diseases (ncird) the members of the pidirac working group for the surveillance of severe nt conceived and wrote the manuscript, am and mj reviewed the manuscript and cr, ib, nf, pg, ep, sm, ms, mc, mm1, mm2, aa and tp were involved in case management. all authors read and approved the manuscript. the authors declare that they have no competing interests. the raw data supporting this study are publicly available as additional file. not applicable. ethical approval was not necessary as the study uses routinely collected, anonymous surveillance data. the study was partially funded by agaur (agència de gestió d' ajuts universitaris i de recerca) grant 1403 and ciber epidemiologia y salud pública ciberesp and by fondo de investigación sanitaria pi 11/01864 and recercaixa 2010acuo_00437i of the catalan association of public universities (acup). springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-034961-4lpjo9a5 authors: dos santos, bruno pereira; de gouveia, giovanna cristiano; eller, sarah; pego, ana miguel fonseca; sebben, viviane cristina; de oliveira, tiago franco title: is covid-19 the current world-wide pandemic having effects on the profile of psychoactive substance poisonings? date: 2020-11-08 journal: forensic toxicol doi: 10.1007/s11419-020-00558-3 sha: doc_id: 34961 cord_uid: 4lpjo9a5 nan recent times have shown an increasing number of intoxication cases, resulting in damaging public health issues all around the globe, and brazil is not an exception [1] . most of the cases observed denoted the use of psychoactive substances, such as recreational drugs and medication for psychiatric use [2] . currently, with the covid-19 pandemic, both strict lockdown and general social distance rules have been recommended. the application of such public health control measures resulted in personal behavior alterations, considering that, during quarantine, individuals find themselves deprived of any social interactions, which can ultimately modulate the profile of poisonings [3] . the present study seeks to verify possible temporal changes in the poisoning profiles of rio grande do sul state, southern brazil, due to the covid-19 outbreak. for this purpose, data was gathered from cases of intoxication by the five main classes of psychoactive substances: anticonvulsants, antidepressants, antipsychotics, benzodiazepines, and recreational drugs, arriving at the toxicological information center of rio grande do sul, from march to july 2019 and from march to july 2020 ( table 1 ). the data analyzed are comprised of a wide range of incidents, verified by the many variables obtained, such as age, gender, circumstance of exposure, and location of exposure. out of the 8588 cases attended by our team during the quarantine, a reduction of − 6% in comparison with the same period in the last year was observed; 3179 cases were included in the five classes of substances evaluated. as for the four classes of psychiatric drugs tested, their combined number decreased by − 8.4%, while illicit drugs and alcohol intoxications alone rose by + 41.4%. the reported numbers clearly indicated a change in poisoning profile as compared to 2019. within recreational drugs (alcohol, amphetamine, cocaine, ecstasy/mdma, inhalants, lsd, and marijuana), the highest incidence of cases were alcohol, from 94 to 137 cases (+ 45.7%) and cocaine, from 62 to 91 cases (+ 46.8%). a substantial increase was also found for other substances such as marijuana which rose from 17 to 27 cases (+ 58.8%) and ecstasy/mdma, from 5 to 9 cases (+ 80.0%). situations generated by the pandemic, such as stress, isolation, and financial insecurity can result in increased alcohol and drug consumption and misuse [4, 5] , thus explaining an expected rise in intoxication cases. unlike recreational drugs, a medication used for psychiatric treatment showed a decrease. the class of antidepressants presented an expressive reduction, of − 17.6%, followed by anticonvulsants (− 10.1%), benzodiazepines (− 2.1%), and antipsychotics (+ 1.0%), given that the last two maintained a certain stability in comparison with the remaining substances. within the class of antidepressants, the compounds with the highest incidence were fluoxetine, which decreased from 302 to 266 cases (− 11.9%); amitriptyline also showed a reduction from 255 to 204 cases (− 20.0%), and finally sertraline from 157 to 135 cases (− 14.0%) reported. as for anticonvulsants, these have shown a much lower absolute reduction, with carbamazepine, from 158 to 143 cases (− 9.5%), valproic acid, from 143 to 137 cases (− 4.2%), phenobarbital, from 47 to 43 cases (− 8.5%) and topiramate, from 46 to 34 cases (− 26.1%). as it can be seen, benzodiazepines showed much less decrease than antidepressants in absolute numbers during the interval studied of quarantine. clonazepam, the group's main representative, showed a decrease, from 749 to 686 cases (− 8.4%), while diazepam increased from 255 to 302 cases (+ 18.4%). the main variation observed with antipsychotics were chlorpromazine, with an increased from 167 to 171 cases (+ 2.4%) and risperidone, from 155 to 158 cases (+ 1.9%). within this scenario, the reduction in medication for psychiatric treatment was mainly guided by a decrease in suicide attempts reaching a decline of − 9.8%. as shown in table 1 , antidepressants presented a decrease of − 18.9% followed by anticonvulsants with − 14.2% and benzodiazepines with − 7.7% for suicide attempts. the analyzed data indicates a reduction on suicide attempt rates that can be correlated to a possible modulation within the intoxication profile of individuals due to social isolation, where people have been asked to stay confined within their homes with family and friends, which can be a protective factor associated with the occurrence of suicide attempts [6] . however, we observed an increase in the number of cases with recreational drugs as suicidal agents (2019, 101 cases; 2020, 138 cases). in addition, the cases of self-medication increased for all classes of medicines evaluated in this study. while quarantine can be effective in reducing the number of suicide attempts, the number of individual accidents has increased expressively (table 1) , mainly associated with recreational drugs, anticonvulsants, and benzodiazepines, especially accidents with children up to 5 years old. the age variable is one of the most relevant parameters, as it shows a clear tendency of who, in fact, is more vulnerable in these cases of intoxication. for instance, children aged 0-5 years showed a serious increase in cases of intoxication by recreational drugs, of + 275%, followed by more than + 30% in anticonvulsants and benzodiazepines. much likely, due to the implementation of the isolation measures, which involved the closing of schools, resulting in children staying home for a much longer period. according to the results obtained, the use of recreational drugs during quarantine has increased, as well as the number of accidents associated with children and with the same compounds. furthermore, individuals over 19 years old, showed a substantial increase in the consumption of recreational drugs, with + 48.0% and only + 3.0% in antipsychotics, as opposed to other classes which decreased, justifiable by a reduction in suicide attempts. considering the exposure site, cases at home had an expressive increase simply for recreational drugs. despite the possibility of an increase in home poisonings, this number has not changed significantly, as most cases already occurred at home. however, in both the site and circumstance variables, large numbers that stood out were the "unknown" results, increasing in most classes, which causes serious difficulties for the medical staff to deal with in cases of intoxication since these variables are of extreme importance for a rapid and effective medical intervention. another variable that has considerably changed was gender. within the recreational drugs group, for instance, cases involving women increased by + 80.3%, while cases with men rose with + 17.0%. nonetheless, other classes showed a reduction in the number of female attendances and, curiously, male attendances in benzodiazepines and anticonvulsants increased, even considering the general decline of the groups. in conclusion, we observed a change in the poisoning profile during quarantine, possibly due to the modification in habits and behavior of the population caused by social isolation. a significant increase in the incidence of cases by recreational drugs has been noted, in almost all aspects, while the cases involving medication for psychiatric use decreased, mainly because of the decline in suicide attempts, in a way as a possible protective effect. the statistics observed in 2020 contradicted our pre-pandemic period epidemiological data in 2019, indicating a strong influence of the quarantine. therefore, the results show how the environment and social activities can modulate human behavior and that some factors such as gender and age can influence the intoxication profile. these numbers are of paramount importance for toxicovigilance and can be used as a projection for other states and countries because measures to prevent poisoning cases during quarantine can be adapted to reduce the damage caused indirectly by the outbreak of covid-19. a fast and simple approach for the quantification of 40 illicit drugs, medicines, and pesticides in blood and urine samples by uhplc-ms/ms multivariate analysis applied in dataset of poison control center of são paulo control centre members, descatha a (2020) covid-19: home poisoning throughout the containment period [the corrected version first appears in lancet public health alcohol use and misuse during the covid-19 pandemic: a potential public health crisis? lancet public health 5:e259 the covid-19 pandemic and its impact on substance use: implications for prevention and treatment suicide mortality and coronavirus disease 2019-a perfect storm the authors would like to acknowledge the financial support from coordination of improvement of personal higher education -brazil [capes -finance code 001]. conflict of interest the authors declare that they have no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord-201798-doi5w7tb authors: seto, christopher; khademi, aria; graif, corina; honavar, vasant g. title: commuting network spillovers and covid-19 deaths across us counties date: 2020-10-02 journal: nan doi: nan sha: doc_id: 201798 cord_uid: doi5w7tb this study explored how population mobility flows form commuting networks across us counties and influence the spread of covid-19. we utilized 3-level mixed effects negative binomial regression models to estimate the impact of network covid-19 exposure on county confirmed cases and deaths over time. we also conducted weighting-based analyses to estimate the causal effect of network exposure. results showed that commuting networks matter for covid-19 deaths and cases, net of spatial proximity, socioeconomic, and demographic factors. different local racial and ethnic concentrations are also associated with unequal outcomes. these findings suggest that commuting is an important causal mechanism in the spread of covid-19 and highlight the significance of interconnected of communities. the results suggest that local level mitigation and prevention efforts are more effective when complemented by similar efforts in the network of connected places. implications for research on inequality in health and flexible work arrangements are discussed. the coronavirus disease 2019 pandemic has dramatically impacted societies globally, with over 32 million confirmed cases and over 980,000 covid-19 deaths worldwide at the time of this writing 1 (hopkins, 2020) . consequently, a growing body of research seeks to understand the social and demographic predictors of this disease at the community level, identifying local etiological factors such as age structure , population density (sy et al., 2020) , and racial composition of the residents (millett et al., 2020) . in addition to local factors, equally important it is to understand the role of social contacts within and across communities, such as the extent to which the movement of people between communities facilitate the transmission of this infectious disease. one important type of such movement is commuting for work, a routine mobility activity that millions of people in the us engage in, typically on a daily basis (mckenzie, 2015) . many of the local and state level mitigation and prevention policies have involved some form of social distancing recommendations to "flatten the curve", in recognition that close physical proximity among people (in the regular course of their daily activities such as in the workplace, at church, or in school) can contribute significantly contributor to the spread of this disease. research on the transmission of this disease across space, between places such as work areas and residential areas is still in its infancy, yet important evidence is starting to emerge. for instance, (bai et al., 2020) analyzed inter-county commuting flows in the state of new york and found that "community spreader" counties were characterized by high commuting flows to and from other counties. these findings are consistent with prior research focused on the spread of other infectious diseases which finds that commuting is an important mechanism through which 1 as of 09.25.2020 diseases may be transmitted to new populations. for example, (xu et al., 2019) linked road traffic among chinese cities to the incidence of influenza a (h1n1) during the 2009 pandemic. understanding how exposures to coronavirus in an area's commuting network affects local cases and deaths is important in guiding thinking and policy in support of remote working schedule and other flexible work arrangements. because many types of jobs do not permit remote work, certain populations, often underpaid and socioeconomically vulnerable minority groups, are disproportionately affected both at work and at home by increased risk of exposure to this disease. moreover, these same groups are further disadvantaged disproportionately by school closures and the need to find alternative arrangements for the care of school age children and other dependents. we contribute to the extant literature on the social and spatial dynamics of covid-19 by analyzing population across united states (us) counties which we consider to be linked via a network of commuting ties. we assess the extent to which county rates of covid-19 deaths and cases are predicted by covid-19 cases in linked counties, controlling for relevant structural and sociodemographic characteristics and spatial contiguity. we leverage methodological strategies from computational statistics to assess model fit and estimate significance while accounting for spatial and network dependencies within the data. our findings demonstrate that commuting networks are an important determinant of the spread of covid-19, as measured by deaths and confirmed cases. we analyze a population of all us counties. data on total number of covid-19 confirmed cases and total number of covid-19 deaths are drawn from a database maintained by usa facts 2 , which is updated daily and contains counts by county and state. we utilize 3-level mixed effects negative binomial models, analyzing covid-19 cases and deaths of county-time periods (n=31,380), nested within counties (n=3,139), nested within states (n=51, includes dc). these models are implemented using the menbreg command in stata 16 (statacorp, 2019). negative binomial models are well suited to predicting overdispersed count outcomes (osgood, 2000) , making them well suited to this research application. we incorporate state-level random intercepts to account for cross-state variation in covid-19 outcomes which may have been driven by statelevel policy differences (e.g., different masking requirements and enforcement of business lockdowns) and county-level random intercepts to account for unmeasured variation across counties in covid-19 susceptibility and response. county-time periods, our first-level units of analysis, are based on the number of new covid-19 cases and deaths for a given county within a given two weeks. within each county are nested ten of these county-time periods, ranging from april 1st to august 18th, 2020. we use total county population (based on the 2018 american community survey 5-year population estimates) as an exposure term for all models, making the model coefficients interpretable as population rates. all models are estimated using huber-white robust standard errors. as a result of the network and spatial interdependencies which we hypothesize to exist among counties, conventional, analytic tests of statistical significance may fail to produce accurate confidence estimates (lesage, 2015) . instead, we utilize a permutation testing, a flexible, simulation-based approach (breiman, 2001; graif et al., 2019) . for each predictor, we conduct 100 permutations in which the values of the predictor are randomly permuted across all observations, breaking any association with covid-19 mortality rates. each permuted dataset is used to calculate model error, generating a distribution of what model error would look like if the predictor had no effect. the observed error is then compared to this distribution in order to assess the contribution which the predictor makes to model fit. a relatively low proportion of permuted cases which produced a lower error than that which was observed shows a significant contribution to model fit. in these permutation tests, we use mean arctangent absolute percentage error (maape) to capture average model error. maape is computed by averaging the arctangent of the ratio of error to observed value for each observation, as shown in equation 1. maape has the advantage of capturing error as a percent, making it less sensitive to outliers than mae, while also being robust to observations for which the true value of y is 0 (an advantage over mape) (kim & kim, 2016) . (1) we used data on intercounty commuting, used to construct a weighted average of networklagged covid-19 exposure, were drawn from the lehd origin-destination employment statistics (lodes) dataset, which is publicly available from the u.s. census bureau (us census 7 of 20 -lehd) (graif et al., 2017; kelling et al., 2020) . this measure was created according to equation 2, where a given home county (h) is connected to w work counties. ℎ− represents the number of commuters from county h who commute to county w, while ℎ− represents the total number of outgoing commuters from county h. an additional measure of covid-19 exposure was also created based on spatial proximity using average rate of confirmed cases of all (queen) contiguous counties (e.g., equation 3 for a county which borders b counties). we incorporate a temporal lag into the construction of these measures by using cases from the prior two-week time period. we also incorporate measures capturing network and spatial change in covid-19 cases from the prior to current time period which use identical weighting (equations 4 and 5). finally, we control for each county's own covid-19 case rate during the prior 2 weeks. we used the rubin-neyman causal inference potential outcomes framework (rubin, 2005) to estimate the causal effect of each of the county-level characteristics, including economic disadvantage, percentage of population over the age of 65, etc. (see table 1 for details), on the number of deaths by covid-19 in that county. to estimate the causal effects, we applied the wellestablished weighting procedure in causal inference by following a two-step mechanism: first, we weight each data sample so as to adjust for the effect of confounding and generate a weighted population that we can consider "as if randomized." second, we perform a weighted regression where we regress total number of deaths by covid-19 against county-level characteristics. we repeated this two-step procedure for each county-level characteristic separately, each time designating a characteristic as "treatment," and estimated the causal effect of that characteristic on total number of deaths by covid-19. we used the following state-of-the-art weighting methods for causal inference from observational data. each of the three methods that we applied use a different methodology for computing the weights (in the weight model). (i) covariate balancing propensity score weighting (cbpsw): propensity score is defined as the probability of receiving the treatment given the covariates and is used in estimating the causal effect of binary treatments on outcomes. propensity density is its counterpart for coping with continuous treatments. cbpsw was recently proposed and it estimates the weights based on the propensity score (for binary treatments) and propensity density (for continuous treatments) while maximizing covariate balance between the treated and controlled via an additional balancing constraint in the optimization (fong et al., 2018; ratkovic, 2014) (ii) inverse probability of treatment weighting: this method weights each of data samples proportionate to the inverse of the propensity score (robins et al., 2000) . (iii) super learner: this method offers a doubly robust estimate of causal effects computed through an ensemble of propensity score estimators (pirracchio et al., 2015; van der laan et al., 2007) . the methods that we have used have been shown to be reliable, effective, and efficient in estimating causal effects from observational data in various applications , 2020 . the weighted outcome regression model determines the causal effect of each county level characteristic on deaths by covid-19 through statistical hypothesis testing. we tested for the null hypothesis that each such causal effect is zero. a statistically non-significant p-value would determine a non-significant causal effect. a statistically significant p-value shows a significant causal effect and the degree (and sign) of the causal effect is determined by the magnitude (and sign) of the estimated coefficient for the treatment in the outcome regression model. we used 0.05 as the statistical significance level. research has indicated that communities that have lower socioeconomic status can have more preexisting health conditions, lower access to healthcare, lower access to high-speed internet that could enable remote work, and are less able to engage in social distancing during the covid-19 pandemic (chiou & tucker, 2020; weill et al., 2020) . for these reasons, several sociodemographic controls are included in the analyses. these measures were drawn from the 2014-2018 american community survey (acs) 5-year estimates. note that these measures are county-level attributes, i.e., considered to be invariant over time for the two-week time periods defining the level-one units. economic disadvantage was measured as the first principal component produced following an analysis of unemployment rate, median income, percent in poverty, percent female-headed households, percent college graduates, percent owner-occupied housing units, and percent vacant housing units (eigenvalue = 3.2). we also include the percent of residents of 65 years or older, as well as binary indicators of whether the county is above average regarding (1) percent non-hispanic white, (2) percent non-hispanic black, and (3) hispanic. finally, we include a measure of the percent of the county with urban residence, as measured in 2010. table 1 shows descriptive statistics for all measures described above. table 2 displays coefficient and standard error estimates from multilevel negative binomial models predicting total deaths and total confirmed cases using the full analytic sample. results from these models are consistent with prior literature and theoretical expectations. as shown, the commuting network-based measures are robust predictors of both total deaths and total cases. this is true for both the network measure based on confirmed cases at the prior time period and the network measure capturing change in cases from the prior time period. note that, when these network measures are taken into account, spatial contiguity is not a strong predictor of covid-19 spread across counties. estimated coefficients for other measures are also consistent with extant research and our expectations. economic disadvantage, concentration of racial/ethnic minority groups, and urban population are associated with higher rates of covid-19 cases and deaths, while a higher concentration of non-hispanic white population is associated with lower covid-19 cases and deaths (tai et al., 2020) . population percent aged 65 years and older is negatively associated with cases, but positively associated with deaths (le couteur et al., 2020) . as expected, a county's case rate at the prior time point is a strong predictor of cases at the current time point. the permutation test results shown in table 3 provide further support for these findings. the p-values shown in table 3 we conducted several tests to assess how findings changed with alternative model specifications. in order to better separate network effects from possible unmeasured spatial confounders, we re-estimated the models using network measures based on (1) only contiguous counties and (2) only non-contiguous counties. tables 4 and 5 show estimates from these models (respectively). as shown, the network effects persist in both cases, further supporting our finding that commuting networks matter for the spread of covid-19 beyond spatial proximity. notes: exposure = county race-specific population 2014-2018, acs 5-year estimates; nhb = non-hispanic black; ***p < .001; ** p < .01; * p < .05; † p < 0.10 to aid our causal inference, we also conducted several analyses using different weighting strategies on a cross-sectional version of our data in which outcomes are cumulative counts of a county's cases or deaths, and network and spatially lagged measures are based on these cumulative counts. results from these models are shown in table 6 . as shown, these alternative model specifications produced substantively similar results with regard to the commuting network effects, offering further support for our conclusions. results of all of the causal effect estimators consistently show that the percentage of population over the age of 65 and economic table 5 . negative binomial models (with state and county random intercepts) predicting covid-19 outcomes across 10 time periods based on network, spatial, and time lagged cases. network based on only non-contiguous counties. total confirmed cases beta se beta se network lagged confirmed case rate (tn-1) .0020 *** (.001) .0020 *** (.001) δ network lagged confirmed case rate (tn-1 -tn) .0004 (.000) .0029 *** (.000) spatially lagged confirmed case rate (tn-1) .0013 *** (.000) .0019 *** (.000) δ spatially lagged confirmed case rate (tn-1 -tn) .0006 *** (.000) .0022 *** (.000) confirmed case rate (tn-1) .0019 *** (.000 given the growing research suggesting that vulnerable populations are less able to work remotely and engage in physical distancing during this pandemic, our results also indicate the acute need for work level protections, such as providing paid sick days, increasing minimal wages, providing health safety equipment to essential workers, to assisting with childcare for working parents who have to work while the schools are closed or in remote mode. these necessary provisions will not only help save the lives and health of workers who cannot afford to socially distance themselves from their work environments, but they have the great potential to spillover and improve the fates of whole communities that their workers go back home to. as expected, an area's socioeconomic disadvantage contributed to both higher death rates and cases relative to the local population. the area's concentration of whites was associated with a protective effect against both infection cases and covid-19 deaths. the concentration of minorities, both above average share of hispanics and non-hispanic blacks was associated with higher rates of confirmed cases, consistent with a large body of work that has documented the many challenges associated with covid-19 risk that burden minority communities, including the higher likelihood to be in frontline occupations and in other low paid occupations that have little flexibility and cannot be easily be transitioned to remote work format. 3 these data come with several limitations. the analytical focus was on counties in part due to restrictions regarding the covid-19 data availability across the country. to the extent that the data access and granularity expands in future months, analyses at more local levels will be very valuable. still, analyses on other important transmittable diseases like influenza have examined place-to-place transmission patterns for geographic units as large as states and counties (bozick & real, 2015) with important lessons that have inspired further research. the network measures used in this study were limited by the data access constraints to information updated on an annual basis, and thus they do not capture the fast-occurring changes during this ongoing pandemic. while these measures captured the commuting network prior the pandemic, those links have likely been weakened by layoffs or remote work transitions. still, the information on the covid-19 rates within the commuting network was captured as it changed over time. given that the pandemic likely contributed to weakening rather than strengthening preexisting commuting links across places, the fact that nevertheless we still see strong effects suggests to us that adjustments in the future to these data to reflect the rapid changes in employment status will likely reveal even stronger effects of commuting exposures to covid-19. many businesses across the country have restricted their employment during the covid-19 pandemic, some have even closed temporarily or permanently, while others allowed employees to work remotely for the purpose of "social distancing" and in the hope of "flattening the curve" (bartik et al., 2020) . understanding how these mobility changes and restrictions contribute to containing the covid-19 transmission is an important next step for future research. moreover, it is known that some population groups are more likely to be in occupations (e.g., health care providers, grocery workers, bus drivers, meatpacking workers) that have been on the frontlines in the fight against covid-19, unable to comply with social distancing recommendations and policies. understanding how workplace networks and risk transmission differentially affect disadvantaged and minority populations is of great importance in future research. importantly, also understanding the types of workplace connections and other social network-based distancing strategies that can work best to contain the pandemic risk without further isolating the most vulnerable populations and communities is essential. mapping the intercounty transmission risk of covid-19 in the impact of covid-19 on small business outcomes and expectations 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algorithmic bias in recidivism prediction: a causal perspective a causal lens for peeking into black box predictive models: predictive model interpretation via causal attribution a new metric of absolute percentage error for intermittent demand forecasts covid-19 through the lens of gerontology spatial econometrics. in handbook of research methods and applications in economic geography who drives to work?: commuting by automobile in the united states assessing differential impacts of covid-19 on black communities poisson-based regression analysis of aggregate crime rates improving propensity score estimators' robustness to model misspecification using super learner marginal structural models and causal inference in epidemiology causal inference using potential outcomes: design, modeling, decisions stata statistical software: release 16 population density and basic reproductive number of covid-19 across united states counties the disproportionate impact of covid-19 on racial and ethnic minorities in the united states super learner social distancing responses to covid-19 emergency declarations strongly differentiated by income impacts of road traffic network and socioeconomic factors on the diffusion of 2009 pandemic influenza a (h1n1) in mainland china key: cord-206391-1dj285h8 authors: yan, donghui; xu, ying; wang, pei title: estimating the number of infected cases in covid-19 pandemic date: 2020-05-24 journal: nan doi: nan sha: doc_id: 206391 cord_uid: 1dj285h8 the covid-19 pandemic has caused major disturbance to human life. an important reason behind the widespread social anxiety is the huge uncertainty about the pandemic. one major uncertainty is how many or what percentage of people have been infected? there are published and frequently updated data on various statistics of the pandemic, at local, country or global level. however, due to various reasons, many cases were not included in those reported numbers. we propose a structured approach for the estimation of the number of unreported cases, where we distinguish cases that arrive late in the reported numbers and those who had mild or no symptoms and thus were not captured by any medical system at all. we use post-report data for the estimation of the former and population matching to the latter. we estimate that the reported number of infected cases in the us should be corrected by multiplying a factor of 220.54% as of apr 20, 2020. the infection ratio out of the us population is estimated to be 0.53%, implying a case mortality rate at 2.85% which is close to the 3.4% suggested by the who. with the quick spread at the global scale, the covid-19 pandemic has become one of the most tragic disasters in human history, with a worldwide confirmed cases of 2.74 million and death toll at 192k as of april 24, 2020. rising trend of these numbers still remains in multiple countries right now. the most risky aspects about the coronavirus are the long incubation period and the existence of a large number of asymptomatic cases. these cause a substantial proportion of infected cases not tracked by medical systems. for better policy making and disease control, as well as to reduce the widespread speculations among the public about the extent of the disease spread, it is of significant interest to give an estimate on the missing counts. specifically, when the pandemic gradually becomes under control, the world is considering the resume of normal business. this requires a prudent assessment of the potential risk. inevitably, such an assessment would involve the estimation of the number of asymptomatic cases when such cases are still active. however, the task of estimating the number of those undocumented cases is very challenging, exactly because of the long incubation period and those asymptomatic cases. in this work, we will present a structured approach for such an estimation task and give an approximate estimate at the us national and state level. the remainder of this paper is organized as follows. in section 2, we will describe our approach. this is followed by a presentation of results in section 3. finally we conclude in section 4. statistically, the estimation of the number of unreported cases is related to the problem of inference with missing data [5] or censored data [2] . however, certain characteristics of the coronavirus epidemiology allow us to take a different approach. we adopt a structured approach, inspired by the diagnostic analysis of remote sensing studies [8] where the errors in the land use classification were decomposed according to their sources. our approach is illustrated by figure 1 . the missing counts in the reported numbers come from two sources. one is those cases for which, at the report date, the symptoms were not severe enough and the affected individuals would not test for infection; however, they would eventually visit some medical facility and test for potential coronavirus infection. we call such cases the type i cases, and the waiting period before the onset is termed as the incubation (or dormant) period. this is illustrated as the filled blue bars in figure 1 . at the time of report, all such cases are still in dormant status thus are missing in the reported number. the second source of unreported cases are those who were infected but are either not aware of it or with symptoms too light to visit the medical facility, and later on recovered without any particular medical treatments. we call such cases the type ii cases. the type ii cases never show up in any reported numbers, thus leaving too little clue for estimation. but we cannot overlook such cases, be-cause the number of such cases could be potentially large and such individuals form an important infecting source. for the rest of this section, we will describe our method for the estimation of each of the two cases. the estimation of the number of type i cases is facilitated by a crucial observation. though not included in the reported number while in the dormant period, such cases would eventually be reported when the symptoms become so severe that the individuals have to seek medical treatments. by that time, those previously missed cases at the original report date (which was a few days ago) would be counted towards infected cases at some later report dates (though one would not know at which particular report date). such numbers should be included at the original report date but surface only several days later; for this reason we call them delayed counts. if there is a way to estimate such delayed counts or their total, then one can estimate the number of type i cases for the original report date. it will be instructive to consider a simple ideal case where all infected cases have a dormant period of 7 days. in this ideal case, the numbers d 1 , d 2 , ..., d 6 in figure 1 are exactly the number of cases who were at their 6 th , 5 th , ..., 1 st day of infection, respectively, when counted at the original report date. if we assume that the incubation period is 7 days, then these are all the number of type i cases missed at the original report date but reflected perfectly later in the number of newly reported cases during the 6 days time window following the original report date. so the total number of type i cases at the original report date can be calculated by their sum, the reality is, however, complicated. first, the length of the dormant period varies for individual cases. also, during the post-report time window, newly infected cases may arise and be reported. thus the number of newly reported cases at any particular day within this time window might be mixed, in the sense that it would include both cases that are infected both before (but were in dormant period) and after the report date. the former case will not pose a problem as anyway such cases would be counted towardsd type1 though cases infected at the same day may now contribute to different d i 's. the latter case is undesirable but could be corrected, to a certain extent, by the truncating effect when we only sum up the counts in the post-report time window up to a length of t days. that is, those cases with a dormant period extending more than t days post-report will be truncated and not included ind type1 , with the total count of such truncated cases being 'cancelled out' by the newly infected cases within the post-report time window of a properly chosen length t . this leads to an estimate for the number of type i cases aŝ where d i are now the number of cases reported at the i th day after the original report date. we can let t take a value around or slightly larger than the mean of the incubation periods. in the appendix, we give a justification on why our estimate,d type1 , would be a reasonable one. if we can keep track of the delayed estimated type1 through time, then we can get a time series which, upon smoothing, could be used to estimate the current missing type i counts. for such an estimation to be feasible, we have two requirements. one is that the daily reported counts through time would not change too abruptly. thus, our approach would not work well when the infection trend still rises very rapidly. during such a period, the safest strategy might be to strictly enforce social distancing. but as the overall situation is gradually under control, our estimation would apply. the other is knowledge of the duration of the incubation period. according to many studies [3, 1, 4] , the incubation periods has a median of around 4-7 days. while further studies or data analysis is required to confirm this, we take t = 7 in our estimation. additionally, it should be cautious that our estimation is valid assuming that the test of coronavirus is sufficiently carried out for the population of interest; insufficient test would render an underestimate of the number of type i cases. in figure 2 , we plot the ratio of estimated type i cases w.r.t. the reported number of cases for connecticut (ct) and massachusetts (ma) since mar 8, 2020. these two states were chosen as they are similar in many aspects, so we expect their ratio of type i cases out of reported cases would be similar. in figure 2 , there is an initial difference in ratios of type i cases in these two states, which we attribute to the late response and the small number of cases tested in ct. later, these two states exhibit strikingly similar trend, which is quite xpected. we also explore the effect of using different values of t where 6 and 7 are used. again, initially the resulting estimation are fairly different, which indicates the rapid spread of coronavirus and the rapid rise of infected cases. gradually, the difference in the resulting estimations diminish, which implies that the choice of t = 7 leads to a fairly stable estimation at late stages of disease spread. similar observation can be made for the estimation of type i cases in us. this is shown in the right panel of figure 2 . the estimation of the number of type ii cases is extremely challenging, as there is barely anything observable. our main strategy in the estimation is based on the matching of population statistics-using what we see well to infer what is missing. when we group reported infection cases, we notice a significant discrepancy in the count statistics by age groups between reported cases and the us population. we expect that, while people in most age groups in the population have a similar chance of being infected, those type ii cases occur much more often in age groups 20-64 but rarely for people of age 65+. this is because people at age over 65 typically have a relatively weaker immune system along with some pre-existing medical conditions. once they are infected with coronavirus, a slight symptom would prompt them to seek medical treatments. as a result, such cases are very likely to be discovered. thus reported counts about such age groups would be more accurate and can serve as a reference to correct counts for other age groups. on the contrary, cases for the 20-64 age group are easy to be overlooked or not noticed, unless their status is deteriorated. the reported counts for these age groups thus require a correction (termed as age correction). the age group of 85+ is more vulnerable to infections, as they typically live in the senior centers or extended-care nursing facilities which, as a matter of fact, have a very high risk of infection. the case statistics for this age group would be very thorough, but many in this age group get infected simply because they share a very confined living space with many other equally vulnerable seniors, and the infection of any one (including staff) in a senior center will quickly spread to the rest (to certain extent, one may think of this as a party of many people during covid-19). so statistics in this age group would not be a reliable reference for population match, since people in other age groups have a very different mobility pattern (the infants interact with the world through their parents thus have a chance of infection not so different from the general population). the main assumption we use for population match is that all the people, with an age in the range 0-84, have similar chance of being infected. as a result, the counts at different age groups would be proportional to their respective percentage in the population; we call such an approach population matching. let r pop and r case be the proportions of the reference group in the population and in the reported cases, and x pop and x corrected be the the respective proportions for the target group, respectively. then r case : r pop = x corrected : x pop , and the corrected percentage in the infected cases for the target group can be calculated accordingly. as we argue before, the case statistics for age groups 65-74 and 75-84 are reliable, but those for ages 0-64 are incomplete and consist of substantial missing data, and we will use the reliable portion of the data to infer or correct statistics about the incomplete part of the data. a simple calculation reveals that age groups, 65-74 and 75-84, according to table 1, have a similar ratio of cases percentage: population percentage, i.e., 9.00 : 4.70 ≈ 17.00 : 9.32. thus, we can pool counts from these two groups and obtain r case : r pop = (9.00 + 17.00)/(4.70 + 9.32) = 1.8544. this yields the corrected ratio as the bottom row of table 1 . adding up numbers in the bottom row gives a total of 187.94%, implying that we should expand the reported counts by 87.94% in order for the reported case counts to match the population statistics across age groups. this gives the ratio of type ii cases over the reported cases. an interesting question is, will estimated counts of type ii overlap with that for type i cases? we claim that this will not, at least not significantly, so the addition of estimated counts for type i and ii cases is valid. the reason is that, type i cases still contribute to the reported numbers, at a delayed time though. these delayed cases can be thought of as a sample from the reported cases (assuming that the reported cases have a stable proportion when breakdown by age groups). the inclusion of type i cases will not change the age-breakdown proportions. thus, after the inclusion of type i cases, we still have the same age-breakdown proportions and thus require an age correction. we apply our approach to each of the 50 states and the us. the data are available from wikipedia [7] . due to the large variation of the population at different states, we calculate the ratio of missing cases out of the number of reported cases for individual states. the ratio for type i cases is shown in figure 3 . due to the lack of reported case data for individual states by age groups, we use the overall estimate, which is 87.94% according to discussions in section 2.2, for the ratio of type ii cases for all the states. the overall ratio for type i cases for the us is estimated to be 32.60%. combining with the 87.94% ratio for type ii cases, this gives an estimated ratio of missing cases versus the reported number at 120.54%. in other words, the reported number should multiply by a factor of 220.54% to reflect the true number of infected cases. with the unreported numbers estimated, we can estimate the infection ratio, defined as the ratio of the number of infected cases out of the population. the overall infection ratio of the us is estimated to be 0.53%, or 1.75 million, as of apr 20, 2020. if we use the associated death toll at about 50k, then the case mortality rate is calculated as 2.85%, which is close to the who suggested estimation of 3.4% [6] . the infection ratio for individual states are visualized as heatmap in figure 4 . heavily hit states are ny, nj, ct, ri, ma, and la with infection ratio estimated at 2.61%, 2.11%, 1.22%, 1.15%, 1.31% and 1.04%, respectively, as of apr 20, 2020. the trend of infection ratio and cases by time for these states is shown in figure 5 . it can be seen that, except la, the infection ratio for all other five states are still rapidly increasing. nj shows a similar growing pattern as ny, while the three new england states, ct, ri and ma, are similar. we have proposed a structured approach for the estimation the number of infected cases not included in the reported number at a given date. we distinguish two types of 'missing' cases, those cases which were infected but are still during the dormant period and those asymptomatic cases which later self-recover without medical treatments. the number of these two types of cases are estimated by accumulating reported counts within a properly chosen post-report time window and by population matching. the reported number, as of apr 20, 2020, of infected cases in us should be corrected by multiplying a factor of 220.54%. the overall infection ratio of the us is estimated to be 0.53%, with a case mortality rate of 2.85% which is close to the recommended 3.4% by who. our estimation can potentially be used for risk assessment. the infant age group may worth further consideration as people in this group are much less risky than other age groups as they interact with the rest of the world through their parents, so the number of cases for this group may need to adjust accordingly to reflect the true risk. denote the number of cases that were infected one day, two days and so on before the report date (for which we use n 0 ). here we limit to type i cases as we can conveniently assume that type ii cases have an infinite incubation period. then the expected number of cases that are discovered during the time window of t days following the original report date is calculated as for simplicity, we would assume that all the en −i 's take a constant value n . we feel that this should not be too unrealistic as we would expect that the number of newly infected cases per day do not vary too much when the pandemic reaches a stable stage (those at the very far distant past would be small, but they carry a very small fraction of the total number so could be ignored). also, we abuse the notation a bit by using d . 's to also indicate the expected value of the associated random variable; the exact meaning will be determined by the context. then equation (1) can be rewritten as under the same assumption, the number of new cases generated during the post-report time window of length t days is (t − i) · p (i − 1 ≤ x < i) + n · p (x < t ) thus the total number of reported cases during the t days post-report time window is calculated as d a + d new = (t − 1) · n + n · p (x ≤ t ) = t n − n · p (x > t ). assuming that random variable x has a finite mean, then we have p (x > t ) ≤ ex/t µ/t, implying that the estimated number of type i cases satisfies the actual number of cases that have accumulated but not being discovered before the report date consists of missing cases during the previous t days and those even earlier cases, which has an expected value (2) indicates that the mean number of type i cases equals the product of the mean daily infected cases of type i and the mean length of the incubation period, which is consistent with the ideal case discussed in section 2.1. let t = (1+ǫ)µ, then we have the following error bound for the estimated number of cases of type i |d type1 − d type1 | ≤ µn · max(ǫ, |ǫ − 1/t |). it follows that the relative error of the estimate satisfies |d type1 − d type1 | d type1 ≤ max(ǫ, |ǫ − 1/t |) = max(ǫ, |ǫ − 1/((1 + ǫ)µ)|). for a given µ, one can pick ǫ to optimize the above bound. for example, when µ = 7, one can take ǫ = 0.07 to achieve a relative error bound of about 7%. incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china nonparametric estimation from incomplete observations the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data statistical analysis with missing data world health organization. coronavirus (covid-19) mortality rate covid-19 pandemic in the united states a structured approach to the analysis of remote sensing images in this appendix, we will give a justification on our estimation algorithm. we show that the error between our estimate,d type1 , of the number of type i cases and its actual value d type1 is small in expectation under reasonable assumptions about the distribution of the incubation periods.denote by random variable x the length of the incubation period, and for simplicity we further assume that x ≥ 0 takes integer values. let n −1 , n −2 , ... key: cord-006882-t9w1cdr4 authors: nan title: royal academy of medicine in ireland date: 2012-07-22 journal: ir j med sci doi: 10.1007/s11845-012-0833-6 sha: doc_id: 6882 cord_uid: t9w1cdr4 nan the tuberculin-skin-test is the most commonly used test to screen for tuberculosis worldwide. in most cases it is administered by the most junior member of the medical team. there is some anecdotal evidence to suggest that junior doctors have limited knowledge of how to administer and interpret this test correctly. the aim of this audit was to assess the proficiency of interns and senior-house-officers in st. vincent's university hospital at performing the tuberculin-skin-test and improve standards. a multiple choice questionaire was used to assess doctors' knowledge of tuberculin-skin-test administration, interpretation, alternatives and the availability and awareness of information regarding the tuberculin-skin-test within the hospital. 45 interns and senior-house-officers were assessed. of those questioned 75.6 % correctly identified intradermal as the method of administration. 66.7 % knew to correctly assess the induration at 48-72 h, but only 29 % knew that the induration should be measured across the forearm. only 11.6 % were aware of the information leaflet within the hospital. 92.9 % of senior-house-officers correctly identified intradermal injection as the method of administration. it is apparent that the tuberculin-skin-test is often administered and/or interpreeted incorrectly. we recommend formal teaching for junior doctors in this area, coupled with improved availability of the information leaflet. mucinous tubular and spindle cell carcinoma (mtscc) is an extremely rare type of kidney tumour that has only recently been described, with less than eighty cases in the literature. this was only recognized as a specific entity in the world health organization 2004 classification of renal cell carcinoma (rcc). mtsccs are polymorphic renal neoplasms characterized by small, elongated tubules lined by cuboidal cells with cords of spindled cells separated by pale mucinous stroma. we report the case of a 57-year old lady who had an incidental finding of a mass in her right kidney. the radiological features were consistent with a rcc and following a multidisciplinary team discussion she underwent a laparoscopic radical nephrectomy. macroscopic examination revealed a well circumscribed 6.5 9 6 9 6.5 right lower pole mass. histologically it was composed of elongated tubules, small tubules and papillary structures with a necrotic centre. the cells demonstrated cuboidal and spindle cell morphology. histological grade was fuhrman grade 2. subsequent ct thorax abdomen and pelvis staged the tumour as pt1b. the majority of mtsccs are indolent, and there is only one report of a distant metastasis which responded favourably to adjuvant sunitinib. to date there is no international consensus on long term surveillance of these patients. due of the favourable prognosis with this type of tumour, mtscc must be differentiated from papillary renal cell carcinoma to avoid administration of excessive adjuvant treatment to patients. this is the first recorded case of this recently classified, rare tumour in ireland. this incidental finding of solid pseudopapillary neoplasm (spn) was discovered when a 59-year-old female underwent a chest x-ray to investigate a wheeze. a subsequent ct abdomen revealed a 10 cm well circumscribed mass adjacent to the tail of the pancreas. this neoplasm had reached a significant size of 10 cm appreciable on radiological imaging and yet was asymptomatic and not palpable on physical examination. laparatomy revealed a highly haemorrhagic and calcified mass emanating from the pancreas. this was adherent to the omentum, distal pancreas and splenic vessels. distal pancreatectomy was performed with en bloc resection of the mass. repeated ct scans at 3, 6 and 12 months failed to demonstrate recurrence. solid pseudopapillary neoplasms are rare entities accounting for between 0.13 and 2.7 percent of pancreatic tumours. this neoplasm has a predilection for females under the age of 35. these tumours are indolent and usually reach a large size before detection. diagnosis is confirmed on histology and complete surgical excision of localised tumours is curative. we aimed to assess the prevalence of smoking among patients with vascular disease and the role of the health care profession in encouraging smoking cessation. 100 patients who attended the vascular outpatient department were surveyed over a 2 month period in 2011. patients gave verbal consent to partake in the audit and the surveyor entered the responses into a standardised questionnaire response sheet. 29 % of patients were current smokers, 39 % ex-smokers and 32 % had no history of smoking. 38 % smoke over 30 cigarettes per day and 66 % had a smoking history spanning over 30 years. just 58 % of smokers who are under the care of the vascular service have been advised to give up smoking in the past by a healthcare professional. smoking has long been established as a major modifiable risk factor for the development of atherosclerosis however 29 % of patients attending the vascular service continue to smoke. just half of patients who were offered smoking cessation advice found it was effective. therefore a system needs to be put in place where all vascular patients are advised of the benefits of smoking cessation and the manner in which information is dispensed needs to be revised. to investigate the optimum location for the teaching of procedural skills to medical students english n, o'flynn s introduction: procedural skill training is a vital component of medical education. traditionally it has been teaching hospital based however general practice rotations may provide greater opportunities than previously thought. aims: this study aimed to ascertain whether a general practice setting or a teaching hospital setting provided a better environment for acquiring procedural skills in terms of opportunity to practice and the variety of skills performed. the correlation between end of year osce results and the amount of procedural skill exposure was also looked at. methods: a cross-sectional quantitative study which included all 107 3rd year medical students at ucc was conducted. a log book listing 28 procedural skills was made available to all students before beginning both general practice and teaching hospital rotations. students were instructed to indicate on the log when they performed any of these skills and in which location. logs were returned to medical school. data was obtained and analysis performed using spss17. results: a response rate of 80 % was achieved. 92.9 % of students performed more skills at the gp setting. 40.5 % (n = 34) did not perform any skills while in a teaching hospital 0.17 skills were performed significantly more frequently in a gp setting while 5 were performed more frequently in a teaching hospital. students who performed a high number of skills in one location were no more likely to perform a high number in the other. conclusions: students were able to take greater advantage of procedural skills opportunities in a gp setting. as this was the students first clinical year it is likely that the one-to-one teaching scenario provided them with a more suitable location to practice skills for the first time. this study also highlighted the diverse nature of procedural skills which a general practice setting can provide. accuracy of sentinel node biopsy in determining the requirement for second axillary surgeries in t1-t2 breast cancer with retrospective application of z0011 criteria background: lymph node status is the most important prognostic marker in breast cancer management. in tandem with breast conser-vative surgery, surgical approaches to the axilla have also become less invasive thus decreasing the morbidity associated with axillary clearance. the acosog z0011 trial reported no difference in survival in patients undergoing sentinel lymph node biopsy (slnb) alone versus axillary lymph node dissection (alnd) in t1-t2 tumours. our aims were to establish whether sentinel lymph node biopsy was a true representative of axillary burden. we also analysed whether retrospective application of criteria from z0011 trial would have prevented patients undergoing second axillary surgery. methods: all patients with t1-t2 tumours undergoing sentinel node biopsy were included in our study (n = 1019). analysis of our prospectively updated breast cancer database was performed. minitab version 16.0 was used to carry out statistical analysis of the data results: 1019 slnb procedures for t1 & t2 tumours were performed over a 7 year period. 730 patients were reported as histologically negative and 289 were positive. of the lymph node positive group, 223 patients progressed to axillary clearance. staging of 149 patients remained unchanged with only 74 patients having [2 axillary lymph nodes reported as positive. 72 patients from the slnb negative group also had an axillary clearance. 5 of these patients had further axillary disease with 1 patient being upstaged having [2 axillary lymph nodes positive. with retrospective application of z0011 criteria 66 % of patients would have avoided second axillary surgery. conclusions: sentinel node biopsy is a strong indicator of axillary tumour burden. this study highlights the accuracy of sentinel lymph node biopsy in staging disease and representing overall tumour burden. flaherty ra, kelly bd, coyle d, quinlan mr, d'arcy ft, rogers e, jaffry sq we report the first case of a spontaneous right nephrocutaneous fistula (ncf) with an accompanying fistula limb communicating with the right ureter. a 65-year-old man presented with a groin mass, which was initially diagnosed as a hernia. he was scheduled for an inguinal hernia repair. upon incision there was extravasation of urine from the wound and the procedure was abandoned. a ct urogram identified a ncf running from the right lower pole calyx, anterior to the psoas muscle and emerging on the right groin skin with an accompanying fistula limb communicating with the right ureter. during the course of investigation it was discovered that the patient was suffering from chronic indolent calculus pyelonephritis which led to the formation of both aberrant pathways from the kidney and the ureter and that both had calculi located at their origins. the patient was first treated with a nephrostomy and ureteric stenting to relieve urinary obstruction and after this failed to resolve the fistula, was successfully treated with percutaneous nephrolithotomy for removal of the calculi and fibrin glue injection into the fistula. this case is one of only a few reported cases of spontaneous nephrocutaneous fistula and the anatomy of the fistulous tract in this case is very unusual and posed a particular challenge for surgical management. this case report further advocates the use of fibrin glue in the management of complicated ncf. this is a retrospective case study. there were six cases of ocular tuberculosis over the 6 year period, one annually, four of whom are women, with ages ranging from 17 to 46 years old. two were foreign-born. all patients presented with reduced visual acuity. four developed posterior uveitis, one anterior uveitis and one panuveitis. this was also complicated by vitritis, retinal detachment and retinal vasculitis in four. the median duration of symptoms until commencement of treatment was 3 months. all cases had a positive mantoux and one case had evidence of pulmonary tuberculosis on chest x-ray. tuberculosis was isolated in two cases. the intended duration of anti-tuberculous therapy for all patients was 9 months. vision improved in all cases. ocular tuberculosis is rare in developed countries, with prevalence ranging from \1 to 7 %. however, it is important to be considered in all cases of uveitis. despite the use of pcr, most cases are presumptive. this leads to delayed commencement of therapy causing further complications. a high index of suspicion is required. we describe the case of a 45-year-old gentleman who presented to our emergency department (ed) with a very unusual complication of central venous catheterisation. this resulted in spontaneous extrusion of a retained intravenous guide wire from the base of the occiput. this has been described only once previously in the literature, but not at such a delayed time interval from insertion [1] . this 45-year-old gentleman presented to the ed reporting that he felt the point of a sharp object irritate his finger in his midline occipital area. he had successfully retrieved approximately 3 cm of a thin metal wire. he had a history of rheumatic fever and had undergone an elective aortic valve replacement 5 years previously, necessitating central venous cannulation. he had remained asymptomatic up to this time. plain radiography of his neck revealed a short segment of wire in the posterior spinal musculature. this segment of wire (approximately 25 cm) was removed manually with minimal force and minor manipulation. the procedure was uncomplicated and the patient was discharged shortly afterwards. retained foreign bodies may migrate slowly over many years eventually extruding from the body, without any serious complications. events such as retained or lost guide-wires are rare. this phenomenon may become more frequent with increasing complexity of medical care and with increasing use of cv catheters in the treatment of sepsis and other emergent critical conditions. physicians should be aware of the possibility of retained foreign bodies and should be somewhat re-assured by reports of simple uncomplicated removal. we present the case of a 79-year-old gentleman who was recently admitted with symptomatic right heart failure and new onset atrial fibrillation. our patient had been treated in the community for symptoms suggestive of ccf but had not previously been investigated. of note, he has no history of a chronic inflammatory condition and no symptoms suggestive of an underlying neoplastic process. on presentation he was also noted to have evidence of an arthropathy affecting his knees and ankles and bipedal oedema. renal function was abnormal with a urea of 17.9 and a creatinine of 116. urinalysis was positive for protein and 24 h urine collection for protein is ongoing. liver enzymes were also elevated and revealed a cholestatic picture. echocardiogram showed a reduced ejection fraction of 30 % and findings consistent with amyloidosis. biopsy of abdominal fat pad at time of writing is pending. amyloidosis refers to an uncommon group of disorders characterised by extracellular tissue deposition of a variety of proteins in an abnormal fibrillar pattern which are resistant to degradation. it can occur alone (primary) or can complicate many chronic inflammatory conditions (secondary). the major sites for clinically reported amyloid deposition are the kidneys, heart and liver. clinically patients present more often with right heart failure; pulmonary oedema is rare. amyloid infiltration results in increased echogenicity on echocardiogram and gives a ''sparkling'' appearance to the myocardium. biopsy is diagnostic. this was achieved using a retrospective review of all children receiving gh therapy (n = 53) over a 5-year period (october 2006-october 2011). 33 of 53 patients on gh therapy had ghd. of these, 18 had ighd (15 male) and 15 had cphd (8 male). all had appropriate work-up and follow-up. age at presentation to endocrinology was older in the ighd group (mean 8.2 years) than in those with cphd (mean 4.4 years). 17/18 children with ighd presented with short stature, compared to only 4 with cphd; the remainder presenting with clinical features of other pituitary hormone deficiencies. the mean height centile at diagnosis was lower in the ighd group (0.4th) versus the cphd group (9th). mri brain/pituitary was abnormal in the majority of patients (14/15) with cphd, compared with 1/18 with ighd. both groups responded well to treatment and height increased by one centile on average at 12 months. all patients diagnosed with ghd at temple st had appropriate work-up and follow-up. children with ighd presented later than those with cphd, and had shorter height centiles at diagnosis. there was a strong male predominance in children presenting with ighd, which may reflect psychosocial factors. structural pituitary abnormalities were more common in those with cphd, and their clinical presentation was more varied. response to therapy was similar in both groups. background: out of hospital cardiac arrests have poor survival rates approx 1-9 %. improving outcomes in ireland have been seen in the past decade. better outcomes are seen if arrest is witnessed and when bystander basic life support was initiated. worse prognosis is seen in a rural setting due to delay in paramedic response times and in administration of advanced cardiac life support. case report: a 60-year-old donegal male experienced chest pain in his rural home and subsequently cardiac arrested. his spouse, whom 3 months prior had trained in basic life support as part of a fas course contacted the 'out of hours' gp and ambulance service and commenced cpr. the gp failed to reach the house and the first ambulance broke down. on arrival of second ambulance, one person cpr had been administered for [40 min. paramedics delivered 10 dc shocks and intubated the patient. in the regional hospital pc was admitted to the intensive care unit for 11 days being managed with acute respiratory distress syndrome (ards). transoesophageal echocardiogram on day of admission showed ef 45 %. ct brain carried out showed no acute pathology. once stable, angiography was carried out showing multivessel disease. discussion at st james's hospital (sjh) cardio-thoracic conference resulted in plan for transfer and pci. in sjh pressure wire study of left anterior descending (lad) coronary artery was positive and stenting (drug-eluting) commenced. lad 96 stents, left circumflex 92 stents and right coronary artery (rca) 92 stents. patient is currently well with no overt signs of hypoxic brain injury and is enrolled in cardiac rehabilitation programme. discussion: this is an incredible case of an out of hospital cardiac arrest. elapsed time in the chain of survival events would predict a negative outcome. however, adequate cpr was administered preventing long term brain injury and certain death. this highlights the need for a greater community-based cpr skill base. recently citalopram and escitalopram have been reported to cause dose dependent qtc prolongation. prescribing guidelines have since changed including contraindication of co-prescription with other qtc prolonging agents. domperidone is a dopamine antagonist widely used as an anti emetic. qtc prolongation and ventricular arrhythmias have also recently been highlighted with domperidone and, since november 2011, caution advised when prescribing domperidone, particularly in patients [60 years of age, or at doses [30 mg/day. in this audit, we aimed to study whether information on qtc prolongation affects prescribing practice by looking at the prescription of a commonly used medical drug, with recently highlighted qtc effects, and its co-prescription with psychotropics. a list of drugs with substantial evidence for qtc prolonging effects was obtained. a kardex review was completed from acute medical and surgical; long stay and rehabilitation wards. kardexes with domperidone were reviewed for dose, age, gender and co-prescription of other qtc prolonging agents. of 820 surveyed kardexes, 10 % (n = 81) were prescribed domperidone. 63 % were[65 years. 38 % were on[60 mg/day. coprescription with another qtc prolonging agent seen in 37 % of cases; of these 77 % were psychotropics, most commonly citalopram (n = 8). four patients were co-prescribed [1 qtc prolonging agent. qtc prolonging agents were commonly co-prescribed with domperidone, which continues to be used even in at-risk groups. psychotropics were the most likely class to be concurrently prescribed. further work in this area is necessary to inform clinical psychiatric practice and encourage responsiveness to new evidence regarding cardiac risk. the development of a mathematical model to predict the time to osteoporosis (tto) using dexa scanning background: dual-energy x-ray absorptiometry (dexa) is the gold standard used for measuring bone mineral density and such readings are currently used to predict osteoporosis and osteoporotic fractures. however, no similar prediction model has been developed to identify the time it will take to become osteoporotic based on dexa scanning. objective: the aim of this study was to develop a mathematical model to determine the tto based on two or more dexa scans with tto defined as the age at which the patient will enter the osteoporotic t-score range. methods: fifty patients who had previously undertaken five dexa scans were identified from the dexa database. t-scores were graphed against patient age using graphpad prism software. straight line curves for the most recent scans and cumulative scans were generated with the age at which the curve intersects t = -2.5 being classed as tto. results: the mathematical model developed successfully predicted the time to osteoporosis for each patient, as well as creating a cumulative osteoporotic trend based on total dexa scans performed. additionally, if the patient was classified as osteoporotic following dexa scanning, the model also successfully predicted the time out of osteoporosis. implication: the tto provides a simple and informative parameter of dexa scanning that a patient can immediately comprehend and understand, while also providing a more simple measure to monitor response to therapy. based on the results presented tto can be incorporated into future dexa scans result summaries. further research will involve validation of this tool. an audit of clinical outcomes in transcervical resection of the endometrium compared to outpatient balloon thermablation anglim bc, von bunau g department of gynaecology, adelaide and meath children's hospital, tallaght, dublin thermablation was introduced to the coombe in november 2009 and thus far it has provided a quick and effective means of treating women with menorrhagia refractive to medical treatment. a retrospective audit was carried out over a 2 year period in tallaght hospital from november 2009 to october 2011. the aim of the study was to compare the efficacy of balloon thermablation compared to transcervical resection of the endometrium (tcre) with or without mirena coil insertion, in the treatment of menorrhagia. 48 patients in total were studied, 24 of which underwent a tcre, and 24 of which underwent balloon thermablation. out of those who underwent a tcre 16 had successful treatment of the menorrhagia and 6 and 12 weekly follow up, 5 had continued menorrhagia which may require a future hysterectomy, however one of which was due to a large fibroid, and one patient described a reduction in menorrhagia however an increase in dysmenorrheoa. out of those who underwent thermablation 15 were treated successfully, 6 had continued menorrhagia to be considered for hysterectomy, 2 had reduced bleeding but increased dysmenorrhoea and one patients symptoms had resolved however she then developed idiopathic thrombocytopenia purpura which led to a recommencement of symptoms. one can therefore conclude that there are both pros and cons to both procedures, tcre being less expensive, however it requires general anaesthesia and may require mirena insertion. thermablation is more expensive however it is a quick outpatient procedure (2 min, 8 s) and is done under local anaesthetic. akinmoluwa s, tormey s department of breast surgery, mid-western regional hospital, limerick breast pain is a common problem especially among women of reproductive age. it accounts for a great percentage of gp visits by young women. it represents a huge proportion of gp referrals to the breast clinic. the palpable effects of this include, among others, an increase in waiting time, increase in healthcare cost, stress on the limited resources and ultimately a decrease in quality of care. in this era of unfavorable economic climate, it is prudent to sanitise our healthcare systems by way of identifying and eliminating practices that have not been proven to alter the course of care. in this study, i reviewed the number of breast pain cases referred to ms tormey's breast clinic in the month of march. the objective of this study is to determine whether or not all breast pain complaints should be referred for specialist review. to achieve this objective, i reviewed all the cases of breast pain referred to the breast clinic in march. the table represents my findings. it is evident from the study that hormonal mastalgia accounts for majority of breast pain complaints in women of reproductive age while a few other cases are attributed to musculoskeletal and other benign disorders. these women, with no risk factors, only need reassurance and pain relief. they do not require specialist intervention. alrashed d introduction: anaemia is a common finding in the elderly population. it may be a sign of chronic disease, underlying malignancy, nutritional status, or blood loss. depending on the classification of anaemia, further investigations such as haematinics and endoscopy may be warranted, as replacing the haemoglobin deficit is never a definitive treatment. objective: to determine the prevalence of anaemia in a population of elderly in-patients and whether further screening was performed. methods: this was a cross-sectional review of all patients 65 years and older under a gastroenterology, a rheumatology, and three geriatrics services at a large teaching hospital. patients' full blood counts were reviewed during their current admission. anaemic patients were then categorised based on anaemia subtype and whether haematinics were investigated. results: out of 116 patients under the five teams, 83 were 65 years and older. 37 out of 83 of these elderly patients were anaemic. none of these subjects had microcytic anaemia during their current admission. 27 out of 37 of these patients had normocytic anaemia. 10 out of 37 of anaemic patients had macrocytic anaemia. haematinics were investigated in 17 out of 37, including 13 out of 17 patients with normocytic anaemia and 4 out of 10 patients with macrocytic anaemia. one patient had abnormal haematinics after being investigated for macrocytic anaemia. conclusion: anaemia was very prevalent in the patients selected for this audit, with the normocytic subtype being the commonest. haematinics were investigated in half those patients. anglim b, murphy c aims: to determine the nature of surgical management of ovarian cysts in the adolescent and paediatric population over a 5 year period. methods: a retrospective audit was carried out over a 5 year period in tallaght hospital from january 2007 to december 2011. this audit reviewed cases of ovarian cystectomy, oopherectomy and salpingooopherectomy using both a hospital online database and records of theatre procedures to identify these patients. results: a total of 103 cases were identified. the commonest presentation was due to pelvic pain. there was a total of 43 ovarian cystectomies, 7 fimbrial cystectomies, 8 oopherectomies, 1 bilateral oopherectomy, and 4 salpingo-oopherectomies. a total of 13 appendicectomies were performed in conjunction with these. histology varied from functional and non functional cysts to dermoids and cystadenomas. there were a total of 30 functional cysts, 13 of which were hemorrhagic. there were 7 follicular cysts, 5 fimbrial cysts, 4 paratubal cysts, 8 dermoid cysts, 2 endometrial cysts, 9 cystadenomas, 6 ovarian torsions and 2 fimbrial torsions. of the total amount of procedures performed 24 were done by a paediatric surgeon, and 52 by a gynaecologist. notably there were fewer cases of benign histology in those procedure performed by gynaecologists. conclusions: adnexal surgery is commonly performed in adolescents and children. pathology is frequently benign. there may be a role for more conservative management. we suggest that imaging of the pelvis and tumour markers should be used more frequently in the pre-operative period. protocols may be developed for future implementation. anglim bc, crowley p day surgery is an efficient way of using hospital beds, provided patients are discharged as planned on the day of surgery. unplanned overnight stay following day surgery places an extra burden on a hospital with the busiest accident and emergency department in ireland. a retrospective audit was carried out of one years day case admissions to determine the incidence and causes of unintended or unplanned overnight stay. 692 women were admitted as day cases over the period of 1st july 2009 to june 30th 2010. a total of 129 diagnostic laparoscopies, 67 operative laparoscopies, 23 diagnostic hysteroscopies, ninety-three operative hysteroscopies, 4 tension free vaginal tapes (tvt) and 26 miscellaneous minor procedure were carried out during this time period. 20 women (2.89 %) were retained overnight. the main reason for overnight stay was excessive post-operative pain. additional reasons included voiding difficulties, reactions to spinal anaesthetic, asymptomatic tachycardia and the need for intravenous antibiotics. there was no evidence of inappropriate selection amongst the laparoscopies and hysteroscopies, however 50 % of the patients undergoing tvt required admission. one can conclude from this study that most patients were appropriately selected for day case admission. patients undergoing tvt surgery should be scheduled for a 24 h hospital stay. a vulval clinic is an ideal and efficient way of detecting patients with vulval cancer. once potential patients have been flagged by general practice clinicians or other specialities within the hospital, immediate steps can be taken to rule out malignancy. a retrospective audit was carried over a 10 month period on a new vulval clinic which commenced in tallaght hospital on 26/01/2011. the aim of the study was to determine the need for a specialised vulval clinic for detection of vulval cancer. a total of 29 patients were referred to the four clinics which took place over this time frame. the majority of referrals were from general practice, other referrals were from dermatology, gynaecology and colposcopy clinics. the main reason for referral was vulval pruritis and pain. nine patients were referred with suspicious lesions on clinical examination. a total of 18 biopsies were taken, two of which showed vulval intraepithelial neoplasia (vin). amongst the other biopsies were 4 cases of lichen sclerosis and the remaining 12 biopsies showed non specific dermatitis. one can conclude from this study that a combined dermatological-gynaecological clinic would be of benefit. in addition a 6.9 % detection rate of vin was achieved and therefore highlights the necessity of this clinic. the prevalence of renal disease in patients aged above 65 with normal serum creatinine balasubramanian i, peters c, lyons d and o'connor m department of ageing and therapeutics, mid-western regional hospital, limerick background: the prevalence of chronic kidney disease (ckd) increases with age. older patients have lower lean muscle mass and therefore using serum creatinine alone as marker of renal function can lead to underdiagnosis of ckd. objective: the aim of this study was to review the prevalence of ckd amongst a cohort of elderly patients with normal serum creatinine. methods: doctot application on the smartphone was used to calculate egfr in a cohort of patients over 65 years with a normal serum creatinine on admission. 40 patients were included. this application is based on the mdrd formula (includes age, sex, ethnicity and serum creatinine). patients were then classed into the various stages of ckd. results: of the 40 patients reviewed, 35 had renal disease. interestingly, only 5 had a diagnosis of renal impairment recorded in the medical notes. 20 of the 35 patients had stage 1 ckd and the other 15 had stage 2 ckd. 18 of the 35 patients with renal impairment especially stage 2, were found to be frail females over 75 years. this group also had a number of co-morbidities including diabetes and hypertension. conclusion: egfr is better than serum creatinine alone for assessment of renal function in the elderly. it is important not only for diagnosis but also for appropriate medical investigation and drug prescribing. as the mdrd formula excludes bmi, further research is warranted to compare measurement of egfr using mdrd formula with the cockcroft and gault equation in this older population. chronic obstructive pulmonary disease (copd) is increasingly prevalent worldwide and the main responsibility for it's prevention and management lies with general practitioners. the aim of this audit was to analyse current standards of care of copd patients in a suburbanrural general practice by examining icgp criteria and comparing results with best practice guidelines. the existing coded population of active patients with copd were telephoned and consent was obtained to ask a set of questions designed to examine certain criteria chosen from the icgp copd quick reference guide [1] . . of the patients included in the audit (n = 39), 64 % of patients were male, the mean age was 71 years (sd = 11.6) and 82 % were general medical service (gms) patients. there was poor recording of smoking status, high uptake of influenza vaccines compared to international figures, a lower uptake of pneumococcal vaccinations and an increased need for osteoporosis prophylaxis. vaccination reminders, smoking cessation advice and information leaflets have been posted to these patients. development of protocols for coding and management have been implemented. in conclusion, general practitioners must focus on ensuring optimum managment of copd in the community. clinical audit is a useful tool to initiate change. we assessed the accuracy of continuous non-invasive haemoglobin measurement using the sphb pulse co-oximeter ã� when compared to traditional laboratory haemoglobin assessment in an outpatient antenatal population. a total of 125 women were recruited. traditional laboratory haemoglobin samples were taken and quantified in the hospital laboratory. the sphb pulse co-oximeter ã� was calibrated and the mean of three non-invasive measurements of haemoglobin were recorded prior to venipuncture. bland-altman plots were used to determine acceptability of the new non-invasive test as a replacement for invasive testing in a clinical setting. the mean gestation at haemoglobin estimation was 20.8 (8.6) weeks. laboratory haemoglobin values ranged from 8.8 to 15.1 g/dl with a mean of 12.1 (1.0) g/dl. the range for the sphb pulse co-oximeter ã� assessment was 9.1 to 15.8 g/dl with a mean of 12.6 (1.3) g/dl. non-invasive haemoglobin measurement provides a clinically acceptable accuracy compared to traditional haemoglobin testing. pressure wound therapy (npwt). in this review we examine the role of npwt in wound healing, compare the products available to clinicians in irish hospitals and explore cost implications today. we achieved this through review of online data, peer reviewed articles regarding efficacy, collection and assessment of data from suppliers of npwt and examining the use and cost of npwt in the mater misericordiae university hospital. we summarise the mechanism of action of npwt, patient selection and indications for its use. the products available on the irish market are compared. through examination of these elements we clarify a role of npwt in management of complex wounds and identify flaws in the management of this service that are both wasteful of money and hospital services and create barriers to discharge. potential strategies to correct the issues identified are detailed, for example, funding of the product by the treating hospital rather than by local authorities in the community or selection of less costly devices in negotiation with suppliers by local health authorities. the solutions we outline will potentially have a financial benefit to the hospital, will lead to the more efficacious running of the hospital system and as such will benefit the patient. we conclude that this is a fundamental service and that there are alternative approaches to implementing use of the product in a more efficacious manner. poster 10 q fever: questions to be answered? brandon l, bannon c, fleming c department of infectious diseases, university hospital galway q fever, an aptly named condition, describes infection with gramnegative bacteria coxiella burnetti. q denotes a question, and there are many to be answered in this rare, but not unknown, condition. take mr. m.c, a 45-year-old farm worker, who had an aortic valve replacement in 1994, for congenital aortic valvular disease. he next presented to medical services in 1999, with fevers, sweats, fatigue and weight loss. investigations at the time diagnosed autoimmune hepatitis, following liver biopsy. he commenced prednisolone and azathioprine. in 2002, again symptomatic, he had another aortic valve replacement. post-operatively, he required 6 weeks of antibiotics for a culture negative valvular infection. in 2003, still on immunosuppression, he developed culture negative meningitis, requiring 2 weeks of antibiotics. azathioprine was discontinued. a renal biopsy revealed proliferative glomerular nephritis in 2004, carried out for macroscopic haematuria. he commenced high dose prednisolone and cyclophosphamide. throughout this time, he regularly presented to medical personnel with high fevers, up to 40 c, present since 1999. they responded to steroids but relapsed on doses below 40 mg. in 2004, the fevers were investigated with a toe, and vegetations seen on the aortic graft. he was diagnosed with culture negative bacterial endocarditis, and subsequently tested positive for q fever. this case highlights the q behind q fever, and raises important issues for medical personnel. when should we remember it? when should we test for it? and what can we do to ensure high risk populations dont slip through the cracks, as this gentleman did? previous point prevalence studies of antimicrobial use in sch have consistently produced the same conclusions and recommendations pertaining to prescribing habits, highlighting doctors' failure to meet ideal standards of antimicrobial prescription. the aim of this study was to assess antimicrobial prescribing habits from the doctors' point of view, to compare this to available prescription data and to raise awareness of the principles of prudent antimicrobial prescribing. a multiple choice questionnaire was used to examine antimicrobial prescribing habits with regard to documentation of indication, documentation of a stop/review date, awareness of local empiric guidelines and other principles of prudent antimicrobial prescribing. 40 trainee and consultant doctors were surveyed. of those questioned, 38 % claimed they always ensure that an indication for commencing antimicrobial treatment is documented in the patient's healthcare record. only 10 % always document a stop/ review date when prescribing antimicrobials, while 69 % indicated that they had failed to do this at least once in the preceding month. 20 % of those surveyed sometimes or never consult local guidelines. when switching patients from intravenous to oral therapy, 90 % believed oral bioavailability to be an important factor, with only 45 % citing cost as being relevant. identifying doctors self-reporting of their deficits allows us to target appropriate interventions to these deficits. our survey identifies areas where awareness of diverted resources and safety issues could be used as a fulcrum for changing prescribing practices. we recommend formal teaching for doctors in this area, with particular emphasis on prudent prescribing and the correct use of empiric guidelines. venous thromboembolism (vte) is a cause of inpatient morbidity and mortality which may be reduced by appropriate thromboprophylaxis. it is well established that vte risk assessment and thromboprophylaxis prescribing may often be inadequate. recently it has been estimated that as many as 14,000 deaths per year due to hospital-acquired vte in england may have been prevented with appropriate prophylaxis [1] . in the current study, a cross section of inpatients was examined to establish concordance with current evidence-based guidelines for vte prophylaxis. data was collected from inpatient charts and drug kardexes relating to patients on three medical wards. laboratory data was also obtained from the hospital it system. data relating to patient mobility was obtained from medical charts, nursing staff, observation, and the patient themselves. sixty-three medical patients and 8 surgical patients (including one patient under obstetrics and gynaecology) were included in the study. 13 (61.9 %) out of 21 at-risk medical patients who were suitable candidates for thromboprophylaxis had sub-cutaneous heparin prescribed, whereas 2 out of 2 of the suitable at-risk surgical patients were prescribed thromboprophylaxis. 4 medical patients (6.35 %) and 4 surgical patients (50 %) were prescribed anti-embolism compression stockings. prescribing of thromboprophylaxis is relatively thorough in this patient population although it remains less than optimal. there exists some evidence of disagreement amongst clinicians regarding the optimum vte prophylaxis strategy [1] . implementation of hospitalspecific guidelines regarding thromboprophylaxis is recommended in keeping with recognised guidelines [2] . ali sheikh a, chandra r, gardezi a, o'hare j mid-western regional hospital, limerick background: good documentation represents good medical practice. objectives: to assess our current standard of documentation of allergy in admission notes, synchronicity with risk alert bands, information given by the patient, documentation on the front allergy alert section of medical notes and drug kardex. methodology: we assessed five parameters i.e. drug kardex, alert band, medical and admission notes and gathered information from each patient staying in medical and surgical services. there were 371 patients in hospital. results: 19 % of the patients admitted under medical and surgical teams had allergy or allergies to different drugs. 21 % had single allergy, whereas 81 % had multiple allergies. penicillin allergy was the commonest 7.2 % followed by opioids 3.7 %. furthermore, it is found that recording of allergies was under par as 24 % was on front page and 50 % appeared in medical notes. more than half of allergy information was found on drug kardex 77 %, patient knowledge 77 % and allergy bands 67 %. it is well documented that use of elastic compression stockings (ecs) prevents post thrombotic syndrome in patients with prior deep venous thrombosis (dvt). a 50 % reduction in these complications has been noted, with 2 year duration of therapy suggested [1] . the advice given to patients, their understanding of the benefits of this therapy and adherence issues has not been documented at sligo general hospital (sgh), this research aimed to address this. a short patient questionnaire was undertaken. this consisted of demographic information, and questions regarding the advice and use of ecs. the population consisted of patients with prior dvt attending the warfarin clinic at sgh. this data collection took place from october 2011-february 2012. the questionnaires were collated and results identified using microsoft excel with simple statistical analysis. eleven patients were included in the study, 36 % were not advised to wear ecs, and only 27 % wore the ecs daily. reasons for nonadherence include; difficulty fitting, discomfort and no benefit noted. improvement in adherence could be achieved if advice was given promoting use, the benefits explained, optimal frequency/duration of use advised and correct measurement. as research strongly supports use of ecs, it is essential adherence is encouraged to reduce the risk of post-thrombotic syndrome and future dvt. opiate injecting drug use is a well-established phenomenon in inner city dublin. the complications arising from this practice affect a predominantly young cohort of patients, who under different circumstances would be expected to enjoy good health. acute infections, acute vascular issues such as pseudoaneurysm, and chronic medical conditions such as hepatitis c and hiv are well recognised and frequently encountered by medical physicians who care for these patients. we present the case of a lady in her thirties with a long history of opiate injecting drug use. approximately 6 months prior to presentation, she underwent left sided pseudoaneurysm repair. she presented to the emergency department in a drowsy opiate induced state. on waking, she stated that she had lost some needles while injecting into her groins. plain radiology of pelvis revealed the two ''lost'' needles ( fig. 1) . on closer questioning she admitted to significant manipulation of the needle injecting path and angle in the weeks prior to presentation. she had attributed this to her previous surgery and the duration of her injecting drug use. follow-up duplex sonography revealed bilateral intact femoral arteries. further surgical management was non-operative with the focus on addiction counselling and further attempts at facilitating cessation of heroin use. bilateral ''lost'' needles is an unusual complication of injecting drug use and certainly would not rank as one of the protean manifestations of such practices. the aim of the study was to investigate patients' recall of their surgery and influencing factors. a questionnaire was given to patients at outpatient follow up and surgical details were recorded. 165 patients completed the questionnaire. the median age was 59 years (range 28-85) and the median follow up was 26 months (range 1-168). the extent of surgery did influence patients' recall with those having an alnd (n = 51, 31 %) having significantly more accurate recall of their surgery as opposed to those who had slnb (n = 114, 69 %), p = 0.007. the presence of ongoing postoperative symptoms also significantly improved recall, p = 0.004. almost half the patients who had slnb (46.5 %) could not accurately remember the extent of the surgery they had but 55.3 % were more careful of their arm or would not allow cannulation. the patient's consent process influenced patient accuracy. patients who filled the consent at both the outpatient consultation and in the hospital were significantly more accurate than those who had signed the consent at the clinic or hospital alone, p = 0.01. patients who have minimally invasive surgery, such as slnb are not accurate at recalling their surgery. this misinformation results in confusion over the subsequent vigilance of their upper limb. the consent process may have a role in improving patient recall. introduction: waiting times can exceed 100 days for general surgical clinics and can reach up to 18 months in different surgical specialities. many outpatient slots are lost by patients who do not attend (dna) to their scheduled appointment. we sought to ascertain whether a reminder text message (rtm) could decrease the number of patients who dna to surgical outpatients. methods: a single text message was sent to patients 4 days before their scheduled appointment, reminding them of the date and time of their upcoming surgical outpatient visit. this incentive was initiated in january 2011. outpatient appointment scheduling and attendances for a single surgical team were analysed over a 1 year period, encompassing two 6 month periods before and after implementation of the rtm service. data was exported to spss v17 for statistical analysis with p \ 0.05 considered statistically significant. results: over the 12 month period there were 1,287 scheduled outpatient appointments for the surgical service, with 653 attending prior to the implementation of the reminder text message service and the remaining 634 attending in the 6 months following its implementation. the percentage of dna patients did not differ significantly (21. classically, focussed assessment with sonography in trauma (fast) addresses a yes/no binary question as to whether fluid is present in the context of trauma. fast generally concentrates on four areas: perihepatic, peri-splenic, pelvic and a sub-xiphoid view of the pericardium. we report on two patients who were the victims of trauma. both patients had normal haemodynamic parameters. in both patients, the initial fast ultrasound scan was technically negative but it exhibited other signs of intraperitoneal injury. in the first case, a young gentleman sustained a penetrating injury to his right upper quadrant area. morison's pouch (the interface between the liver and the right kidney) did not exhibit any fluid. there was, however, a thin anechoic strip around the gallbladder. ct confirmed the suspicion of peri-cholecystic fluid and this patient required urgent laparotomy and repair of his hepatobiliary injury. in the second case, a gentleman in his thirties sustained a blunt injury to his left upper quadrant. ultrasonography exhibited heterogeneous echogenicity of the spleen. this patient proceeded to have urgent laparotomy and splenectomy for this shatter-type injury. as experience with fast techniques grows, the binary question of whether intra-peritoneal fluid is present becomes more nuanced. the objective of this audit was to review the hospitals compliance with hospital guidelines, to get an overview of how fluids are being prescribed in the hospital and to produce quality improvement plans. thirty drug kardexs were chosen randomly from wards around the hospital, both medical and surgical. if a kardex was found to have no fluid prescription, an alternative kardex was chosen in its place. note was taken on whether the prescription had the patient name and hospital number, the date, name, dosage and strength of the prescription, the route of administration and the frequency and rate of administration. the main areas of non-compliance were found to be: name: only 34 (30.4 %)orders out of 112 had the name on the order medical record number: only 34 (30.4 %)orders out of 112 had the mrn on the order, and the route of administration was not present on any of the 112 orders checked. in conclusion, this audit would suggest that there is a lack of compliance with detailing the patients name and mrn on fluid orders, that the route of administration was not written on any kardex, however the back page of each is exclusively dedicated to iv fluid prescription and also that non-approved abbreviations are being used when prescribing fluid orders. spontaneous hip fractures, or fractures without a fall have been described in up to 6 % (1, 2) of cases of hip fracture. an upsurge in such cases was recently observed in our emergency department. we present these in the form of a retrospective case series. patient 1 is a 43-year-old ex intravenous drug user who presented with non-traumatic right-sided hip pain over a period of weeks. initial plain films did not reveal fracture. over 1 week her symptoms deteriorated to the extent that she became unable to weight-bear. patient 2 is a 66-year-old gentleman with increasing left sided hip pain following a seemingly innocuous fall 3 months prior to index presentation. again initial radiographs did not reveal an abnormality. patient 3 is an 83-year-old bed-bound nursing home resident with end-stage alzheimer's disease. she was noted by nursing staff to have bilateral hip symptoms post seizure. the patient was unable to mobilise independently and had not fallen out of bed at any stage. patient 4 is a 29-year-old lady who presented with unilateral sacroiliac pain following a recent intensive exercise program including kickboxing 1 week previously. in each of these cases, subsequent review and plain films demonstrated fracture and in one case bilateral fractures secondary to seizure were demonstrated. our cases highlight the need for diagnostic vigilance and a structured approach in dealing with possible radiologically occult hip fractures, even in patients with no proximate antecedent history of trauma. delerium, or acute confusional state, is a common presentation to our emergency departments, and occurs in up to 30 % of hospitalised patients. we describe the case of acute deterioration in mental status, on a background of alzheimer's disease, with an interesting aetiology. mr k's family sought emergency medical review of 5 days deterioration; withdrawal, somnolence and general disorientation. he is a 73-year old with moderate alzheimer's disease. history and initial investigations were unremarkable. he was mildly dehydrated and physical exam showed only mild truncal ataxia. further investigations to elucidate cause included lumbar puncture, mri brain and immunological and vasculitic parameters. serology revealed human immunodeficiency virus (hiv) infection with acute seroconversion pattern. a history obtained with help of his family identified several casual heterosexual partners within past year. this included a contact who may be an intravenous drug user, with involvement in commercial sex work. symptoms abated within a week of admission, following pattern of hiv viral load. he has subsequently commenced antiretroviral therapy. this case highlights several areas of interest. sexual history is often overlooked in the older patient, which can be deleterious to outcomes. trends of hiv infection in ireland include primary infection in the older person, in addition to greater longevity of people infected in earlier adult life. we would advocate opt-out testing within the emergency department, and this is currently under study in our tertiary emergency department. comparison of comorbidities in patients with pre-diabetes to those with diabetes mellitus type 2 the management of type 2 diabetes and its complications are well researched. the prevalence of these complications in pre-diabetes has not been researched to the same extent. there has been no research comparing the prevalence of complications in pre-diabetes and type 2 diabetes in ireland. a cross sectional study performed on 309 pre-diabetes and 309 type 2 diabetes patients, selected from the diabetes interest group database (a database of the diabetic patients in 30 general practices in cork region) using stratified sampling for age and gender. a questionnaire was designed and completed in each practice assessing the presence of diabetes related complications in pre-diabetes and type 2 diabetes patients. data was analyzed on spss. the prevalence of complications was determined and the chi square test performed to see is there a statistically significant difference in the prevalence of these complications between pre-diabetic and type 2 diabetic patients. the prevalence of ischaemic heart disease and autonomic neuropathy is actually higher in pre-diabetes but the prevalence of renal disease and cerebrovascular disease is higher in type 2 diabetes. none of these differences in prevalence are statistically significant. the prevalence of peripheral vascular disease, eye disease and peripheral neuropathy is higher in type 2 diabetes, this difference being statistically significant. the prevalence of many of the complications in pre-diabetes is as high as in type 2 diabetes which may have implications for the screening and management of these conditions and the related comorbidities. the both groups were evenly matched. the median age was 73 and median homocysteine level was 11 (range 5-34.9). results: in group b, immediate clinical improvement was equivalent between the normal homocysteine group and treated hyhc group. median time to binary restenosis in hyhc was 29 months and in normal homocysteine was 50 months. p = 0.4335. secondary endpoints and all cause survival showed no significant difference. pre-treatment multivariate logistic regression for group a; depicts that hyhc is the main culprit of graft occlusion and limb loss p \ 0.0001. multivariate logistic regression for treatment group reports that corrected hyhc is no longer a significant factor of operative outcome. conclusion: patients with treated hyhc have similar outcomes compared to those with normal homocysteine. it is therefore crucial to measure homocysteine in all patients with cli and correct aggressively prior to intervention to improve outcomes. the efficacy of clinical guidelines in promoting co-prescription of bone protection with glucocorticoids among hospital doctors treating inpatients background: therapeutic glucocorticoids (gc) rapidly decrease bone mineral density, inducing a catabolic shift by promoting osteoclast differentiation and activation and by inhibiting osteocytes. current guidelines (1) direct that bisphosphonates (bp's) and calcium carbonate 1,200 mg (ca ++ co 3 -) with vitamin d 3 (vit. d 3 ) should be given at initiation of gc therapy as it is known that bone catabolism occurs early with steroid usage. we circulated these guidelines within our hospital after auditing the existing practice of the hospitals doctors and 1 year later we sought to measure the efficacy of our intervention by completing an audit loop. methods: a cross sectional audit was performed of all adult medical and surgical inpatients in a tertiary referral centre teaching hospital. it was noted if inpatients had been prescribed gc and if concurrent anti osteoporotic medication had been prescribed. subsequent to the initial audit, guidelines promoting the use of bp's, ca ++ co 3 and vit. d 3 when prescribing gc's were advertised on hospital notice boards, in hospital bulletins, hospital prescribing guidelines and on the hospital website. one year after publishing the new guidelines the audit loop was completed by performing a similar cross sectional audit. results: all inpatient medical records (n = 417) were reviewed in jan 2010 of whom 52 % were female and 58 % were older than 65. 66/417 (16 %) inpatients were prescribed gc's. ca ++ co 3 with vit. d 3 was prescribed for 20 % of patients on gc's with 2 % also receiving bp therapy. 3 % of patients were also receiving-post menopausal hormone replacement therapy. in nov 2011 1 year after guideline publication all 452 inpatient medical records (n = 452) were reviewed of whom 63 % were female and 60 % were older than 65. 55/452 (12 %) inpatients were prescribed gc's. ca ++ co 3 with vit. d 3 was prescribed for 55 % of patients on systemic steroids with 20 % also receiving bp therapy. creation and circulation of hospital guidelines resulted in an improvement in the co-prescription of ca ++ co 3 and vit. d 3 and bp's with gc's by the order of 2.35 and 10 respectively. however 45 % of patients on systemic steroids received no bone protection and 80 % received suboptimal bone protection from steroid induced osteoporosis. conclusion: publication and advertisement of current bone protection guidelines when prescribing systemic steroids resulted in a substantial but suboptimal improvement by hospital doctors in our hospital in the co-prescription of bone protecting drugs to prevent steroid induced osteoporosis. in this audit it appears that the majority of prescribers do recognise the necessity to protect bone health when a patient requires steroids. however a substantial number of patients did not receive any bone protection. it is our perception that most physicians are not aware that short courses of steroids reduce bone mineral density and therefore greater efforts must be made to enhance doctor awareness of the necessity for bone protection to be prescribed at initiation of systemic steroids. there is a trend towards longer total survival for jetflow tcvcs. these results suggest a potential advantage from using this line type, however, further study and formal cost analysis needs to be undertaken prior to changing our practice. with increasing resource restrictions, appropriate ordering of blood tests is vital for medical economic viability. this study evaluated the pattern and cost of thyroid function test (tft) requests and aimed to determine if tsh alone identifies thyroid abnormalities. a retrospective review of tfts performed on in-and-out-patients at a 350-bedded regional hospital was undertaken in january 2011, evaluating the number, results and costs of tsh, t4 and t3 levels. 4055 tsh, 3959 t4 and 28 t3 were ordered. 3456/4055 patients (85.2 %) were euthyroid. tsh abnormalities occurred in 526/4055 (13.0 %) ( table 1) only 82/4055 (2.0 %) patients had a normal tsh despite an abnormal t3 or t4 level. 57/82 (69.5 %) of these patients had known thyroid disease, undergoing treatment with thyroxine or thyroidblocking medications. 9/82 (10.9 %) had t4 levels \1 nmol/l outside the normal range and asymptomatic so were considered to be euthyroid. 16/82 (19.5 %) had a variety of diagnoses, for example, pituitary disease. tft reagents alone cost â�¬10,600. ir j med sci (2012) 181 (suppl 3):s83-s107 this study has identified that non-selective requests for t4 and t3 add little diagnostic value, except in certain circumstances like treatment of thyroid disease, in pregnancy or if pituitary disease is suspected. optimising tfts requests could save in the region of â�¬42,000/per annum. tsh alone would appear to be adequate for the majority of patients. case study: neurodegenerative disorders we present a case with an unusual combination of neurodegenerative disorders. a fit and healthy 60-year-old man, with no history of medical or psychiatric illness deteriorated progressively over a 10 year span, presenting initially with speech and language difficulties, followed by development of extra-pyramidal signs non responsive to levodopa. neurological permacol ã� mesh is an acellular porcine-derived dermal collagen surgical implant used in a wide variety of surgical reconstructions and repairs. we describe two cases where permacol ã� mesh was used to anchor the contents of the femoral triangle in patients undergoing radical block nodal dissection as part of the surgical management for metastatic penile squamous cell carcinoma, one of whom had an atrophied sartorius muscle due to previous infection with poliomyelitis. both patients underwent successful inguinal node dissections and femoral triangle repairs, with permacol ã� proving to be an effective means of protecting the femoral vessels in both patients despite complications related to wound healing secondary to a fixed flexion deformity in one patient. a 65-year-old gentleman, with a past history of vestibular schwannoma requiring a ventriculoperitoneal shunt (vps) was admitted with acute diverticulitis. his condition worsened and required a laparotomy for bowel perforation and faecal peritonitis. this case reports the successful perioperative management of the patient with a vps in situ in the setting of an emergency abdominal surgery. vps placement is an effective treatment of hydrocephalus, diverting cerebrospinal fluid (csf) into the peritoneal cavity. unfortunately, the shunt devices have a high incidence of malfunction mainly due to catheter obstruction or infection and are associated with various complications, 25 % of which are abdominal [1] . incidental pathology unrelated to the vp shunt can also occur such as appendicitis [2] , endometriosis [3] and diverticulitis as in this case. no standard current set of guidelines for perioperative management of vps exists for patients undergoing general gastrointestinal or urologic procedures with varying degrees of contamination [4] . this case reports successful and conservative management of a patient with a vp shunt that underwent contaminated abdominal surgery. there is no consensus on the management of vps in patients undergoing elective or emergent abdominal surgery and further studies are required in this area. the use of antithrombotic therapy on management of atrial fibrillation in an irish general practice malomo k 1 , breen n 2 , dunne l 3 , farrell g 3 , bryne p 3 1 ucd (university college dublin), ireland, now intern, mid-western regional hospital, limerick; 2 general practice, dublin, ireland; 3 pottersway medical centre, bunclody, ireland background and objective: atrial fibrillation (af) is a common cardiac arrhythmia associated with increased risk of stroke events [1] . to assess the use of antithrombotic therapy in patients with known af attending an irish general practice (igp) and use of stratification schemes to assess their suitability for oral anticoagulant therapy. methods and subject: permission to carry out the study was sort from university-college-dublin ethics committee. there were 161 patients with af attending the igp identified using the computerized disease coding system who international classification of disease (icd-10). thirty patients were diagnosed between 01/01/2009 and 21/01/2011 and their data from the computerized medical notes was used to calculate chads 2 , cha2ds 2 -vasc, has-bled scores and identify antithrombotic therapy they were using. results: there were 30 af patients. sixty-three percent (n = 19) were males and 37 % (n = 11) were females (ratio 1.7:1). twentythree percent (n = 7) of patients were aged \65 years, 27 % (n = 8) 65-74 years inclusive and 50 % (n = 15) =/[75 years. two patients with chads 2 score zero were on warfarin although one of them had cha 2 ds 2 vasc score of one. sixty-percent (n = 18) were on warfarin alone, 20 % (n = 6) aspirin alone, 14 % (n = 4) warfarin plus aspirin, 3 % (n = 1) aspirin plus clopidogrel and 3 % (n = 1) on warfarin plus clopidogrel. seven patients were not on warfarin for various reasons. the has-bled score revealed 7 patients at low risk, 12 moderate risk and 11 at high risk of bleeding. implications: ninety-three percent of patients were correctly managed and two patients were on warfarin with chads 2 scores of zero. the use of evidence based management guidelines is necessary to manage patients. keywords: atrial fibrillation, chads 2 score, cha 2 ds 2 vasc score, has-bled score meckel's diverticulum is the most common congenital abnormality of the gastrointestinal tract. only 16 % of meckel's diverticulum are symptomatic [1] . it can cause complications such as ulceration, obstruction, intussusception, haemorrhage and perforation and these complications are more common in the paediatric age group. a 16-year-old has a lifetime risk of 3.7 of developing a complication, this falls to zero over time [2] . adults most commonly present with bleeding [1] . we have a case of a 37-year-old male who presented with a 3 day history of abdominal pain, constipation and anorexia. on examination he had rif tenderness, but no signs of peritonism. a provisional diagnosis of appendicitis was made. the patient was taken to theatre the next morning for laparoscopy and appendicectomy. the appendix was normal and surgery proceeded to laparotomy. an inflamed and perforated meckel's diverticulum was found. a terminal ileum resection with side to side anastomosis was performed. the patient made an uneventful recovery and was discharged to opd follow up. this case illustrates the importance of further evaluation following normal laparoscopy in the case of the ill patient. references: neonatal graves disease is a rare condition, caused by transplacental transfer of thyroid stimulating antibodies from mother to fetus. 0.2 % of pregnant women have graves disease and 1.5 % of their offspring will have overt hyperthyroidism. a further 3 % will have biochemical thyrotoxicosis without symptoms. this is the case of a baby girl with neonatal graves disease. her antenatal course was uncomplicated until 39 weeks gestation. at this point, her mother became clinically thyrotoxic. maternal blood tests showed an elevated free thyroxine level (50 pmol/l) and positive thyroid receptor antibodies. a diagnosis of graves disease was made. she was commenced on treatment but remained thyrotoxic at the time of delivery. the baby was healthy at birth. however, thyroid function tests on day 2 of life showed an elevated free thyroxine (40 pmol/l) and thyroid receptor antibodies were positive. clinically, she remained asymptomatic and examination was normal. treatment with carbimazole was commenced and the dose titrated to maintain her euthyroid. most neonates affected by neonatal graves disease will have biochemical thyrotoxicosis but are clinically asymptomatic. the minority will be severely affected with goitre, eye signs, weight loss, tachycardia, arrhythmias and heart failure. it is a transient disorder, limited by clearance of maternal thyroid receptor antibodies and is usually self-limiting over 3-12 weeks. mortality rates of up to 20 % are reported in untreated cases, usually from arrhythmias and heart failure. this case emphasises the importance of close monitoring of pregnant women with a history of thyroid disorders, before and during their pregnancy, as well as monitoring their babies in the neonatal period. fibreoptic bronchoscopy is considered a safe diagnostic tool [1] . it is suggested however that post-bronchoscopy complication rate increases with age [2] . we decided to study the complication rate and the outcomes of bronchoscopy in patients over the age of 80 years in our institution. a retrospective review of the case notes of patients aged greater than 80 years who underwent bronchoscopy between september 2009 and november 2011 was carried out. data on complications experienced during and after bronchoscopy and the influence of the results on subsequent management of patients were collated and analysed. ninety-six patients were included. the mean age was 82.8 years (sd 2.98). thirty subjects (31.25 %) had a documented lung disease. fifty-nine patients (61.45 %) were current or ex-smokers. indications for bronchoscopy were; to evaluate for malignancy (93.8 %) and to evaluate for tb (6.2 %). post bronchoscopy complications were noted in eight (8.2 %) cases including hypoxia (3.1 %), infection (2.1 %), tachycardia (1 %) haemoptysis (1 %) and pneumothorax (1 %). six patients required treatment including nebulised bronchodilators (2.1 %), antibiotics (2.1 %), and oxygen therapy (2.1 %). malignancy was diagnosed in twenty cases (20.8 %). clinically significant pathogens were detected in six cases (6.2 %). as a result of bronchoscopy fourteen patients (14.6 %) had alterations to their drug therapy, three (3.1 %) received lung cancer treatment with curative intent, eighteen (18.8 %) had palliative care input, seventeen (17.7 %) were referred for further investigation and thirty-seven (38.6 %) had no change to their management. in conclusion, bronchoscopy is relatively safe and has good diagnostic utility in patients aged more than 80 years. patient records were identified from a database of patients who underwent a spinal mri to investigate spinal metastatic disease between november 2006 and april 2009. an analysis of the management of those diagnosed with mscc, specifically radiotherapy and/or surgical intervention was performed. three hundred and sixtyone patient records were identified with one hundred and seventy-one patients having metastatic spinal column disease. of these, thirty-four had mri evidence of metastatic spinal cord compression. radiotherapy alone was the most common therapy employed for patients with mscc. a multidisciplinary team approach was not taken in the majority of cases. a surgical opinion was sought in the minority of cases. this is not congruous with nice guidelines as a management protocol. the complexity of management decisions for metastatic spinal cord compression demands a multi-disciplinary approach. current practise in this major supra-regional cancer centre does not routinely employ this approach. a surgical opinion is sought in the minority of cases. this reflects the national trend with some centres having no spinal surgeons as staff. we recommend the establishment of a care pathway in order to comply with best evidence based practise as outlined by the 2008 nice guidelines. pet ct as a staging modality in primary cervical cancer; to establish the correlation between histological subtype and fdg-18 avidity of the primary lesion purpose: pet ct has become one of the mainstays of diagnostic imaging both in staging and prognosis of cervical cancer. we wanted to establish the link between fdg-18 uptake in the primary lesion and correlation with specific histological subtypes of cervical cancer including squamous cell carcinoma, adenocarcinoma and other rarer subtypes such as clear cell and adeno-squamous carcinoma. methods and materials: the main audit involved working out the fdg uptake in the primary lesions from the cervical cancer database of patients. the patient list was derived from a database of patients collated by the gynaecological services at sjh of all patients who received workup and treatment for cervical cancer from 2006-2011. the computer system at sjh was employed for analysing pet-ct reports and histology reports. microsoft excel was used to store this information parameters and complete statistics on the data. results: the results of this study are to follow. conclusion: there is a correlation between fdg avidity and histological subtype of cervical cancer and this provides valuable information on the reliability of pet-ct findings in a specific cohort of patients with cervical cancer. we present the case of a 44-year-old male with a primary piriform fossa squamous cell carcinoma (scc) who attended for staging positron emission tomography/computerised tomography (pet/ ct) scan. distant to the primary lesion, focused f 18 fluorodeoxyglucose (fdg) uptake was noted in the left iliac bone, without underlying abnormality on the accompanying ct scan. low grade uptake was also noted in subcentimetre upper mediastinal nodes, without any underlying lung parenchymal abnormality. these nodes were felt to be inflammatory or reactive in origin. though an unusual pattern for metastatic head and neck scc, the left iliac bone lesion was concerning for malignancy. thus, a percutaneous biopsy of this region was performed under image guidance. histology revealed non caseating epithelioid granulomata consistent with sarcoidosis. the patient was subsequently able to have potentially curative treatment of his head and neck primary. discussion: sarcoidosis is a chronic inflammatory multisystem condition characterised by the presence of non-caseating granulomas in affected organ tissues. it commonly affects young and middle aged adults with a slightly higher prevalence in women. the disease shows a predilection for adults under 40, peaking between 20 and 29, with a second peak in women over 50 [1] . despite its unknown aetiology, it is felt that t lymphocytes play a central role in the development of sarcoidosis, as they likely propagate an excessive cellular immune reaction. it has been shown that abnormalities with the cd4/cd8 ratio and production of t helper 1 and 17 (th 1/th 17) cytokines such as interferon and tumour necrosis factor (tnf) are found in sites of disease activity [2] . the importance of tnf in sarcoidosis is demonstrated by the efficacy of anti-tnf medications such as pentoxifylline and infliximab [3] . it is estimated that bone lesions occur in 1-13 % of sarcoidosis patients [4] . these figures are however based on radiographic data and are likely an underestimate as the majority of bone lesions would be asymptomatic [5] . varying osseous manifestations of sarcoid have been described; punched out lytic lesions, lace-like destruction and subperiosteal resorption mimicking hyperparathyroidism. commonly, the small bones of the hands and feet (predominantly the middle and distal phalanges) are involved often bilaterally and symmetrically. while pulmonary involvement occurs in 90 % of patients with sarcoidosis [6] , bony involvement is rare without other clinical manifestations of the disorder [4] . indeed our patient had low grade subcentimetre mediastinal nodes. the fdg avidity of sarcoid is a well documented phenomenon. indistinguishable from metastatic disease on f 18 fdg pet scan alone it can lead to false-positive appearance of metastatic disease on pet/ ct. furthermore one-third of pet/ct positive sarcoidosis have osseous abnormalities on pet/ct the majority of which will not be evident on low dose ct [7] . this case serves to remind us of the diagnostic limitations of f 18 fdg pet in the differentiation of inflammatory and metastatic processes. in a patient with an unusual pattern of 'metastatic' disease tissue diagnosis is a necessity. distinct islet auto antibodies against antigens insulin, gad65, ia2 and znt8 have been identified. the presence of autoantibodies has been shown to be predictive of reduced beta cell mass. international data suggests that 85-90 % of patients with newly diagnosed t1dm are positive for at least one of the above antibodies. our aim is to study the prevalence of autoantibody positivity in our population of children with newly diagnosed t1dm over a 5 year period (2007) (2008) (2009) (2010) (2011) . details of all children newly diagnosed with t1dm were collected using the endocrinology department database and chart review was undertaken. children diagnosed elsewhere whose care was transferred to our centre and children who had non type 1 diabetes were excluded. one hundred and thirty-six children were diagnosed with t1dm in our centre, of which 37 (27 %) presented in diabetic ketoacidosis. age at diagnosis ranged between 10 months and 15.7 years. the male to female ratio was 1:2. other autoimmune conditions (coeliac disease, hypothyroidism, addison's) were present in 11 %. ninety-six percent (n = 130) were tested for one of the three antibodies. 76 % were positive for at least one antibody, 30 % positive for two, 5 % positive for all three antibodies. the most common antibody found was anti gad (60 %). positive autoantibodies are helpful in confirming the presence of t1dm and their absence in raising the possibility of monogenic diabetes. the absence of pancreatic islet autoantibodies at diagnosis can be predictive for maintained beta cell function during the 2 years after diagnosis. maternal obesity, based on a body mass index (bmi)[29.9 kg/m 2 , is associated with increased pregnancy complications. moderate exercise during pregnancy is associated with decreased complications such as pre-eclampsia [1] and gestational diabetes mellitus [2] and has a beneficial effect on mood with those who exercise experiencing fewer symptoms of depression and anxiety both during and after pregnancy [3] . the purpose of this study was to determine if obese women exercise less during pregnancy. we recruited 110 women at their convenience after a routine scan confirmed an early ongoing pregnancy. maternal height and weight were measured accurately and bmi calculated. women completed the international physical activity questionnaire. of the 110 studied in early pregnancy, 10.9 % took no exercise, 58.2 % walked only, 21.8 % undertook moderate exercise and 9.1 % undertook vigorous exercise. of the obese women (n = 20), only 10 % reported moderate-vigorous exercise in early pregnancy compared with 34.5 % in women from the normal bmi category (n = 55). also 15 % of the obese group reported doing no exercise compared with 10 % of those with a normal bmi. women with a bmi of 30 or more were found to sit for an average of 453 min per day whereas those with a normal bmi sit for 320 min per day on average. although bmi increases with age and parity, these variables were not found to influence exercise levels in early pregnancy. exercise may be physically challenging in obese women, particularly if morbidly obese, but due to its beneficial effects it should be encouraged antenatally in all pregnant women irrespective of their bmi category. references: angiogram showed an absence of coronary artery disease and echocardiogram ruled out structural abnormality. exercise stress test showed short runs of vt in recovery. further tests included ajmaline and adrenaline challenges. cardiac mri showed right ventricular outflow tract scarring consistent with either a primary diagnosis of arvc or secondary with that of myocarditis. sarcoidosis was outruled by further laboratory and radiological means. non-sustained runs of vt on telemetry were noted and a dual chamber implantable cardiac defibrillator was placed. on discharge, medication included atenolol 100 mg daily and patient will undergo genetic screening. follow up for the siblings included phenotyping and mri. discussion: history, presentation and pathology uncovered are consistent with a diagnosis of arvc. suspected paternal inheritance of an autosomal dominant genetic defect predisposed to the ventricular arrhythmias which at first, manifested as self-limiting palpitations however, later caused a near fatal event. long term management may include cardiac transplantation. prevalence of diagnosed atrial fibrillation in adults: national implications of rhythm management and stroke prevention: the anticoagulation and risk factors in atrial fibrillation (atria) study complications of fiberoptic bronchoscopy at a university hospital the relationship between age and process of care and patient tolerance of bronchoscopy central skeletal sarcoidosis mimicking metastatic disease sarcoidosis is a th1/th17 multisystem disorder osseous sarcoidosis treated with tumour necrosis factor-inhibitors: case report and review of literature. spine (phila pa 1976) musculoskeletal manifestations of sarcoidosis multiple atypical bone involvement in sarcoidosis imaging in sarcoidosis. semin respir crit carre med f-18 fdg pet/ct for detecting bane and bone marrow involvement in sarcoidosis patients poster 42 diaphragmatic rupture: delayed diagnosis and its consequences-a case report diaphragmatic rupture: a frequently missed injury in blunt thoracoabdominal trauma patients diaphragmatic rupture due to blunt trauma: sensitivity of plain chest radiographs the introduction: lymphoscintigraphy has been shown to be accurate in identifying sites of potential nodal metastases in melanoma patients. recent guidelines published by the eortc-eanm have defined specific criteria with relation to performing lymphoscintigraphy in melanoma patients. methods: the aim of this study was to audit all patients with malignant melanoma who underwent sentinel lymph node biopsy (slnbx) and lymphoscintigraphy in university college hospital galway between 2005-2010. results were compared with eortc-eanm recommendations. results: 189 melanoma patients underwent slnbx during the study period. 121 patients had preoperative lymphoscintigraphy using intradermal injections of technetium 99 m. sentinel nodes were identified in 102 of 121 patients (84.3 %) on lymphoscintigraphy. 66.94 % of lymphoscintigrams were reported on the same day as the procedure, 23.97 % after 1 day and 9.09 % greater than 1 day postop. obligatory imaging, as defined in the eortc-eanm guidelines, was obtained in 91 % of all patients undergoing lymphoscintigraphy. no nodal uptake was reported in 18 patients, 14 of whom received imaging in accordance with the guidelines. the location of those melanomas with no nodal uptake was 44.4 % on the head and neck, and 38.9 % on the trunk. the overall rate of false-negative lymphatic mapping and sentinel node biopsy was 5.2 %. in patients receiving lymphoscintigraphy the false negative rate was 3.7 versus 7.9 % in patients who did not have lymphoscintigraphy. conclusion: preoperative lymphoscintigraphy is an essential adjunct in identifying the sentinel lymph node in clinically node negative melanoma patients and should adhere to eortc-eanm guidelines. conflict of interest: none. on examination, she was alert, hr 160, bp 105/60. she was tachypnoeic, but reported this to be her baseline. there was a palpable, non-reducible mass in the left upper quadrant. a chest x-ray showed loops of bowel above the diaphragm. ultrasound showed an abscess in the rectus sheath, which drained mucopurulent fluid.mb opted not to have the diaphragm repaired, despite medical advice. she was readmitted 2 weeks later with a recurrence of the abscess. her clinical condition deteriorated, with severe abdominal pain, and oxygen saturations of 70 %. an emergency laparotomy was performed, which showed an obstructing lesion in the descending colon, with large and small bowel above the diaphragm. she had an extended right hemicolectomy, with restoration of bowel to the abdominal cavity and mesh repair of the diaphragm. histology showed an descending colon adenocarcinoma, t3n0m0.traumatic diaphragmatic rupture is a rare problem, occurring in 1-8 % of blunt and penetrating traumas. (1) plain films and ct scans are not always diagnostic in the acute phase, due to concomitant injuries. (2) repair is essential once diagnosis has been reached to avoid herniation of abdominal viscera. patients with ongoing dyspnoea after blunt trauma may benefit from a repeat chest x-ray. a 75-year-old retired veterinary surgeon was referred to tertiary referral with a 2 months history of a painless enlarging neck mass. clinical examination showed a right side neck mass approximately 7 cm 9 5 cm in size which extended through both anterior and posterior triangles. cervical lymphadenopathy was not appreciated and the patient was clinically euthyroid. patient was admitted under the care of the maxillofacial service, where he underwent a needle core biopsy of the neck mass. this was returned showing poorly differentiated spindle cell tumour with large pleomorphic nuclei and abundant abnormal mitoses. the immunoprofile was consistent with metastatic poorly differentiated sarcomatoid carcinoma and the differential diagnosis included origin fro the kidney, lung or thyroid.the case was discussed at the head and neck mdm and a consensus was reached that the patient as developed a sarcoma of the neck, with a level 5 neck dissection the most appropriated intervention.intraoperatively, following the removal of the neck mass it was noted that the right lobe of the thyroid was enlarged. an intra-op fna was performed on the mass in the right lobe of the thyroid. the fna was returned showing bizarre giant cells, suggestive of malignancy. ultimately the patient underwent a total thyroidectomy but, despite surgery the patient died 19 weeks post-operatively. using feedback from the pilot study and analysis of the preexamination consultant and registrar-led teaching schedule for students a further 'intern-led' tutorial timetable was structured. it allowed for a weekly maximum of 9 h of teaching dependent on demand and intern availability. programme duration was 10 weeks, january to march 2012. group sizes were a maximum of 8 students. tutorials were all at the patient bedside. feedback forms were distributed at the end of the programme.sixty-four tutorials were given in total. seventy feedback forms were returned. mean number of tutorials attended per student was 5.8. students rated statements 1-5 (1-strongly disagree, 2-disagree, 3-neutral, 4-agree, 5-strongly agree). median scores were used. scoring showed improvements were made from last year in terms of level of intern preparation for tutorials and importantly, the students own subjective view of their level of preparation for forthcoming examinations. most importantly, students agreed that tutorials improved their history taking skills and strongly agreed that their examination skills improved. matching feedback from the pilot study, students strongly agreed that intern-led teaching is an appropriate adjunct to the final year programme.of the 47 intern working in st james's hospital, 22 participated. seventeen of these had received tutorials on the pilot programme. of the 25 that did not participate, many had never received formal intern teaching.the feedback obtained from the pilot study was invaluable in organising and delivering this teaching programme. ongoing improvements will be made for next year based on this audit. this also highlights that the intern-teaching tool is extremely beneficial, yet largely underused. key: cord-015947-kgyl052w authors: oommen, seema title: emerging respiratory pandemics date: 2016-02-22 journal: clinical pathways in emergency medicine doi: 10.1007/978-81-322-2710-6_45 sha: doc_id: 15947 cord_uid: kgyl052w since the identity of the respiratory pathogen is not known at the time of admission, emergency department personnel and intensive care staff are at the highest risk of exposure while handling such patients. swine fl u is the popular name of the relatively new strain of infl uenza a/2009/ h1n1 that caused a pandemic which began in mexico and spread rapidly from there across the world in 2009-2010. more than 600,000 laboratory-confi rmed cases were reported from more than 200 countries worldwide as of march 2010 with a total of 18,449 deaths as reported by the world health organization (who) in august 2010 [ 2 ] . this is considered an under-representation of the actual numbers as many deaths were never tested or recognized as infl uenza related [ 2 ] . meanwhile new cases of h1n1 are being diagnosed worldwide including india in 2014. the who estimates a total of 676 laboratory-confi rmed human cases of avian fl u (h5n1) infection and 398 related deaths in 16 countries from 2003 to 2014 [ 3 ] . avian infl uenza viruses are divided into the high pathogenicity h5n1 virus with 100 % mortality in the poultry and low pathogenicity h7n9 viruses not associated with severe disease in poultry. cases of h7n9 are reported mainly from the people's republic of china. the most recent respiratory illness was fi rst reported in saudi arabia in 2012 and is of a new strain of coronavirus called the middle east respiratory syndrome coronavirus (mers-cov) that shares a genetic relatedness to a similar virus found in camels. by june 2014 there were around 699 laboratory-confi rmed infections with 209 deaths [ 4 ] . the majority of these cases were from the middle east countries with few cases in the usa, europe, malaysia and the philippines in asia. thus the crude case fatality rate of h5n1 is highest at 60 %, followed by mers-cov (30 %), sars-cov (10 %) and the least h1n1 (0.5 %), the latter being most likely under-represented [ 1 -4 ] . adaptation of the viruses by mutation or reassortment leading to an ability to cross the species barrier into humans, the capability to become established in humans and a sustained ability to pass from one human to the other are the three features needed to cause an infectious disease of epidemic proportions. combine this with the increased mobility of individuals across the world; transfer of these infections from one part of the globe to the other can take place in a relatively short period of time. direct lysis of the host cells is one of the mechanisms of host tissue damage. more important are the indirect consequences of the host immune response which get disrupted, tipping the balance from being favourable to an exaggerated and destructive host immune response leading to an outpouring of pro-infl ammatory chemokines and cytokines termed as 'the cytokine storm' [ 5 ] . cytokines like tumour necrosis factor alpha (tnf α), interleukin 6 (il-6), il-1β and il-8 play a major role in tissue damage [ 5 ] . of this il-1β has been found to be the main cytokine in the broncho-alveolar lavage (bal) fl uids of patient with lung injury [ 5 ] . the net result is local diffuse damage to the alveoli (acute lung injury -ali) due to increased arrival of leucocytes, dilatation of blood vessels and tissue oedema and can swiftly progress to the more severe acute respiratory distress syndrome (ards). spillover of these cytokines into the systemic circulation leads to multisystem organ failure and fi nally death. there is a considerable overlap in the clinical presentation by the common respiratory viruses and other atypical causes of community-acquired pneumonia making arousal of suspicion in the treating physician of an emerging epidemic virus unlikely. hence suitable samples may not be collected at the appropriate time leading to misdiagnosis and a delay initiation of therapy. • the common clinical presentation [ 1 , 6 , 7 ] of most respiratory pandemic viruses is that of an 'infl uenza-like illness (ili)': an acute respiratory infection with sudden onset of fever (temperature of >38 °c or >100.4 °f), chills, myalgia and a non-productive cough. sore throat and rhinorrhoea may also be present. many cases are associated with gastrointestinal symptoms like abdominal pain and diarrhoea. • a history of contact, in the preceding 10 days of symptom onset with poultry or with a known case in the countries detected to have human avian infl uenza cases, has to be elicited. likewise, history of travel to the middle east countries should arouse suspicion of a mers-cov infection. • cases may range from a mild ili to a fulminant viral pneumonia. rapid clinical deterioration may occur with diffuse viral pneumonitis with hypoxaemia, acute respiratory distress syndrome (ards), septic shock, multisystem organ failure and death occurring within a week of onset of illness [ 7 ] . • secondary bacterial pneumonia especially due to staphylococcus aureus, s. pyogenes and s. pneumoniae is a common complication with infl uenza [ 7 ] . • extremes of age; pregnancy; obesity; presence of pulmonary, cardiac, hepatic, renal or metabolic co-morbidities; and underlying neurological conditions are the common risk factors for severe disease [ 1 , 6 ] . • case fatality of h5n1 is much higher than seasonal infl uenza viruses with rapid clinical deterioration mainly due to early involvement of the lower respiratory tract. considering the rapid spread of virus in the past within hospitals and community, methods to rapidly identify infected cases are of utmost importance. • the real-time-based polymerase chain reaction (rt-pcr)-based assay is one such means which has proven its worth both during the sars-cov and the h1n1 outbreak. the only caveat is that appropriate clinical samples need to be collected at the appropriate time during the disease and should be transported to the laboratory in cold chain in a viral transport medium so as to maintain the viability of the nucleic acid. • multiplex pcr can detect simultaneously other viruses causing a similar clinical picture like the seasonal infl uenza a and b, respiratory syncytial virus (rsv) and human metapneumovirus. • the most preferred specimen is a nasopharyngeal aspirate or a swab preferably within 1-2 days of onset of disease [ 6 ] . cotton swabs are not recommended due to presence of inhibitors; rayon-or nylon-fl ocked swabs are used instead. broncho-alveolar lavage, tracheal aspirates and sputum which contains the highest viral loads are the ideal specimens especially later in the course of illness [ 6 ] . • rt-pcr tests may be carried out on serum specimens. • in case of suspicion of mers-cov or sars-cov, stool specimens may also be sent to the laboratory. • many a times these newer molecular assays may not be available even in established diagnostic molecular laboratories and the specimen may have to be shipped under strict biohazard protocols to the national or a regional reference centre for testing. • rapid diagnostic tests available for the diagnosis of infl uenza have high specifi city but low sensitivity and hence a negative result should be interpreted with care [ 7 ] . • though viral cultures don't play a signifi cant role in rapidly diagnosing cases, it is important in confi rmation of emerging and re-emerging cases of viral infection, epidemiological typing of isolates and research into vaccines and newer drugs. • clinical signs on examination are minimal when compared to the radiological fi ndings of the chest. chest x-rays typically show diffuse interstitial infi ltrates, unilateral or more commonly bilateral ground-glass opacities to focal consolidation that is seen early in the disease [ 1 , 6 , 7 ] . these opacities are usually seen in the lower lungs fi rst and may become widespread affecting larger areas as the disease progresses. high-resolution computed tomography may be required in ambiguous cases. • tests done to rule out other infectious aetiology include blood cultures, gram's stain and culture of the sputum and urinary antigen detection for legionella and pneumococci. acute and convalescent serum samples may be collected for antibody detection of various pathogens. healthcare personnel should be on high alert in the present global climate for any clustering of similar cases. picking up a probable epidemic early in its course may limit the spread of the infection within the hospital and the community. treatment is largely supportive for uncomplicated cases, also bed rest and maintenance of hydration, in addition to analgesics and antipyretics. severe cases require supportive measures including ventilation and antibiotics for secondary bacterial infections. • oseltamivir and zanamivir [ 6 , 8 ] are neuraminidase inhibitors that decrease the release of infl uenza viruses from infected cells, thus limiting its spread. it has been used extensively in the 2009 h1n1 pandemic. resistance to oseltamivir has been documented [ 8 ] . best results were obtained when treatment was started within 48 h of symptom onset even before the availability of laboratory results. • the dosage of oseltamivir for persons above 13 years of age and >40 kg weight is 75 mg twice daily for duration of 5 days. • for children <15 kg, the dose of oseltamivir is 30 mg twice a day, 15-23 kg is 45 mg twice a day and >23-40 kg is 60 mg twice daily. • zanamivir is advised for persons above 5 years of age at a dose of 10 mg (two inhalations) twice a day. oseltamivir is the drug of choice to treat human cases of avian infl uenza. • unlike infl uenza there is no specifi c antiviral or vaccine available for the coronaviruses. a combination of ribavarin and interferon 1α shows synergistic action in vivo and has been used to treat mers-cov and sars-cov infections but limited data is available on their effectiveness to combat the disease and clinical trials are needed to demonstrate their effectiveness [ 1 ] . • steroids were used during the sars outbreak to limit the cytokine-mediated lung injury in conjunction with ribavirin but the actual role of steroids has to be elucidated with further studies. steroids are contraindicated in cases of infl uenza pneumonia as it may further predispose to secondary bacterial infection. • the incubation period for most infl uenza viruses including h1n1 is 1-4 days [ 7 ] , whereas the incubation period for h5n1 is slightly longer ranging from 2 to 8 days. • patients with infl uenza are most infectious in the fi rst 2 days of the onset of illness averaging from a day before the onset of symptoms to 5-7 days after the onset of illness. • the incubation period of coronaviruses like sars-cov and mers-cov is around 2-14 days. in contrast to infl uenza cases, they transmit the virus usually after the fi fth day of the onset of disease when viral load maximizes in the nasopharyngeal secretions [ 1 ] . 1. immunoprophylaxis [ 9 ] : exists currently only for infl uenza. it is advised by the advisory committee on immunization practices (acip) that all persons over 6 months of age be vaccinated annually against the predicted infl uenza strains which are most likely to cause infections in the next infl uenza season based on surveillance data. it is available as an annual infl uenza vaccine incorporating three or four live attenuated or inactivated infl uenza strains. it is available for administration as nasal sprays (live attenuated vaccine) and the intramuscular or intradermal route (killed vaccine). 2. chemoprophylaxis [ 7 ] : • oseltamivir and zanamivir (neuraminidase inhibitors) are active against both infl uenza a and b viruses. it is indicated in exposed unvaccinated immunocompromised persons or people with co-morbid conditions who are at a high risk of developing infl uenza. • oseltamivir should be given within 1 day after exposure at a dose of 75 mg once daily for persons 13 years and above of age for a minimum of 10 days after exposure to a recent contact with a known case of infl uenza. • zanamivir is prescribed at two inhalations once daily for people above 5 years of age. if the exposed person develops respiratory symptoms, he should be given treatment doses of the drug. • in case of h5n1, close contacts of strongly suspected cases of human avian infl uenza and personnel handling infected poultry are advised oseltamivir as chemoprophylaxis [ 10 ] . 3. standard contact , droplet and airborne precautions [ 11 ] : oftentimes, emergence of an infection of pandemic potential is not routinely expected by physicians and staff in their regular days' work. but going by the past experience especially in case of sars-cov, healthcare personnel were the ones at high risk of infection given the close proximity to the patient. hence it is important that all staff follow the standard contact and droplet precautions for any case suspected to have a respiratory infection. • contact and droplet precautions include wearing of personnel protective equipment (ppe) such as gloves, gowns, eye and face shields. • there is special emphasis on hand hygiene which must be diligently performed before and after contact with the patient, the potentially infectious material generated by him, before wearing and after removing ppe. • airborne precautions include placement of patients in an airborne infection isolation room (aiir) and wearing of n95 or greater respirators and masks. airborne transmission is especially possible while suctioning a ventilated patient, bronchoscopy, sputum induction, intubation and extubation and cardiopulmonary resuscitation. • pending placement of patient in the aiir a face mask must be placed on the patient and the patient isolated in a single room to prevent spread of infection. • environment infection control must be followed per hospital infection control policy using a suitable disinfectant for disinfection and collection, transport and treatment of all infectious waste generated. patient presenting with 'influenza like illness' and chest radiograph showing signs of pneumonia necessitating hospitalization inform district heath authorities. always maintain personnel protection: contact, droplet and airborne precautions to be practised. elicit travel history to middle eastern countries for mers-cov and or history of contact with poultry for h5n1. physicians and staff to be alert on clustering of similar cases in the recent past collect appropriate specimens to confirm diagnosis and to rule out alternate diagnosis. severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection infl uenza at the human-animal interface. summary and assessment as of 4 middle east respiratory syndrome coronavirus (mers-cov) summary and literature updateas of 11 into the eye of the cytokine storm two years after pandemic infl uenza a/2009/ h1n1: what have we learned? writing committee of the who consultation on clinical aspects of pandemic (h1n1) clinical aspects of pandemic 2009 infl uenza a (h1n1) virus infection antiviral agents for the treatment and chemoprophylaxis of infl uenza. recommendations from the advisory committee on immunization practices (acip) prevention and control of seasonal infl uenza with vaccines who rapid advice guidelines on pharmacological management of humans infected with avian infl uenza a(h5n1) virus. available at guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings key: cord-236070-yao5v598 authors: carneiro, carlos b.; ferreira, i'uri h.; medeiros, marcelo c.; pires, henrique f.; zilberman, eduardo title: lockdown effects in us states: an artificial counterfactual approach date: 2020-09-28 journal: nan doi: nan sha: doc_id: 236070 cord_uid: yao5v598 we adopt an artificial counterfactual approach to assess the impact of lockdowns on the short-run evolution of the number of cases and deaths in some us states. to do so, we explore the different timing in which us states adopted lockdown policies, and divide them among treated and control groups. for each treated state, we construct an artificial counterfactual. on average, and in the very short-run, the counterfactual accumulated number of cases would be two times larger if lockdown policies were not implemented. the evolution of the covid-19 has been posing several challenges to policymakers. decisions have to be made in a timely fashion, without much undisputed evidence to support them. being a new disease, and despite the enormous research effort to understand it, estimates of the transmission, recovery and death rates remain uncertain. nevertheless, these are key pieces of information to assess potential pressures on the health system capacity, as well as the need of a lockdown policy and its intensity if implemented. not surprisingly, similar regions have implemented different strategies regarding lockdowns. the leading example in the media is the looser social distancing policy in sweden versus strict policies in its scandinavian peers. by informally comparing the evolution of the pandemics in sweden and denmark (or norway), many commentators argue that several covid-19 cases and deaths in sweden would be avoided in the short-run were a strict lockdown in place. 1 aiming to provide a quantitative assessment on the short-run effects of lockdowns, this paper takes this exercise seriously in the context of us states. given that the timing us states adopted lockdown policies differs among them, we adopt techniques based on synthetic control (sc) approach of abadie and gardeazabal [2003] and abadie et al. [2010] to assess the impact of lockdowns on the short-run evolution of the number of cases and deaths in the treated us states. 2 more specifically, we consider an extension of the original sc method called artificial counterfactual (arco) which was put forward by carvalho et al. [2018] . due to the nonstationary nature of the data, the correction of masini and medeiros [2019] is necessary. our results point to a substantial short-run taming of the cumulative number cases due to the adoption of lockdown policies. on average, for treated states, the counterfactual accumulated number of cases, according to the method adopted here, would be two times larger were lockdown policies not implemented. a key feature of our approach is that it is purely data-driven. in the beginning of the crisis, the majority of papers written by economists to evaluate the effectiveness of lockdowns relied on epidemiological models for analysis, including the most recent ones that incorporate behavioral responses. 3 these models are hard to discipline quantitatively. many calibrated parameters remain uncertain, 4 and models that incorporate behavioral responses need time to mature and agree on a reliable set of ingredients and moments to be matched. model-free approaches like ours or medeiros et al. [2020] should complement policy discussions or forecasting exercises based on those models, especially from a quantitative point of view. there are related papers using state or county level us data. 5 at least one of them, , uses a synthetic control approach but it is restricted solely to california. other papers, such as brzezinski et al. [2020] , and sears et al. [2020] , use variations in the timing of statewide adoption of containment policies, and difference-in-differences models to document substantial reductions in mobility and improvements of health outcomes. the key identification assumption in these papers is that variations in the timing are random after controlling for covariates. brzezinski et al. [2020] also consider an instrumental-variable approach. fowler et al. [2020] and grassi and j. sauvagnat [2020] follow similar empirical strategies but at county level, and also find substantial reductions in cases and fatalities in counties that adopted stay-at-home orders and state-mandated business closures, respectively. our analysis, that rests on alternative identification assumption and method, should be seen as complementary. as the pandemic evolves, and more data become available, we expect more related empirical evidence to be consolidated. the paper is organized as follows. section 2 describes the data, while section 3 presents the empirical strategy. the results are discussed in section 4. finally, section 5 concludes the paper. additional results are included in the appendix. data on covid-19 (confirmed) cases are obtained from the repository at the johns hopkins university center for systems science and engineering (jhu csse). we consider the cumulative cases for a subset of the 50 us states and the district of columbia. instead of using the chronological time across the states, we consider the epidemiological time, which means that the day one in a given state is the day that the first covid-19 case was confirmed there. the econometric approach adopted here relies on the fact that some states adopted a lockdown strategy (the treatment), whereas others did not adopt social distancing measures (control group) and are used to construct the counterfactual. 6 lockdown strategies include a mix of state-wide non-pharmaceutical measures aiming to limit social interactions, such as restrictions on nonessential activities and requirements that residents stay at home. containment policies. 4 see, for example, atkeson [2020a] on the uncertainty regarding estimates of the fatality rate. 5 there are also related papers for other countries. for example, fang et al. [2020] for china. 6 the timing of those policies at each state were obtained, and double checked, in several press articles, e.g., https://www.businessinsider.com/us-map-stay-at-home-orders-lockdowns-2020-3 and https://www.nytimes.com/interactive/2020/us/coronavirus-stay-at-home-order.html. in this section, we describe how we assign states to control and treatment groups, and then, describe the method used to construct the counterfactuals. aiming to balance control and treatment states, and at the same time obtain enough observations to estimate properly the model before the lockdown policy was implemented, we divide us states into three groups. for a state to be included in the analysis, a state-wide lockdown policy must be established at least twenty days after the first case. we assume that whenever an individual becomes infected, it takes an average of ten days to show up as a confirmed case in the statistics. 7 hence, the in-sample period used to estimate the synthetic control ("before" the lockdown policy) for each treated state (to be defined below) is the number of days between the tenth day after the first confirmed case and the tenth day after the lockdown strategy was implemented. we choose to start the in-sample from the tenth day as a way to smooth the initial volatility of the data. we adopt a criteria that a state must have at least twenty observations in the in-sample period to be included in the analysis. this criteria excludes states that adopted a state-wide lockdown strategy too early, such as connecticut, new jersey, ohio, among others. these are the unmarked states in table 1 , which reports the dates of the first case and lockdown policy, as well the difference in days between them, and also helps visualize the three groups of states. the remaining states must be divided into treated and control groups. the idea is to find a synthetic control for each of the treated states. the group of potential controls should consist of states that adopted a lockdown policy too late (or never adopted), such that counterfactuals are not contaminated by lockdown policies implemented in those states. at the same time, and for a similar reasoning, the lockdown strategies adopted in treated states must be in place during the period of analysis. 8 7 this assumption is motivated by the incubation period of the virus. according to the world health organization, the "[...] the incubation period for covid-19, which is the time between exposure to the virus (becoming infected) and symptom onset, is on average 5-6 days, however can be up to 14 days." see https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200402-sitrep-73-covid-19.pdf. 8 in the appendix a.1, table a .1 shows the reopen dates for the treated states. fortunately, there are horizons that can balance both goals: enough states to build the synthetic controls and a relative extensive period to construct the counterfactuals. in particular, we restrict the analysis up to the 58th epidemiological day. this figure accommodates at least ten control states to build the synthetic controls, 9 at the same time it maximizes the out-of-sample days to run the counterfactuals. in this sense, our analysis concerns the very short-run impact of lockdowns, up to nearly three weeks. the treated states are marked in blue in table 1 , and include twenty states: alabama, colorado, florida, georgia, kansas, kentucky, maine, maryland, mississippi, missouri, nevada, new hampshire, new york, north carolina, oregon, pennsylvania, rhode island, south carolina, tennessee, and texas. the potential control states are marked in red, and include ten states: arizona, arkansas, california, illinois, iowa, massachusetts, nebraska, north dakota, south dakota, and washington. nonetheless, due to the lack of variation within the in-sample period, we exclude four states from this control pool as we explain below. importantly, oklahoma, utah and wyoming only implemented partial lockdowns (not reported in the table). therefore, they are hard to classify as either treated or control states. we opt to exclude them from the analysis. figure 1 illustrates the empirical strategy, which is formalized in the next subsection. it plots the evolution of (log) cumulative cases along the epidemiological time. the first vertical dashed line represents the tenth day after the first confirmed case. the in-sample period is represented in between the first and second vertical dashed lines, which mark the tenth day and the following twenty days, respectively. similarly, the out-of-sample period is in between the second and third vertical dashed lines, which mark the 31th and 58th epidemiological day, respectively. blue lines represent the treated states, whereas the red ones the potential control states. the turning points from blue full-to dashed-lines represent the days lockdowns were implemented (plus ten days) in treated states. note that new york is clearly an outlier among the treated states, exhibiting a huge amount of cases (more on that below). we use the red lines to build synthetic controls for each full blue-line up to the turning point, and then construct counterfactuals by simulating the synthetic controls forward up to the 58th day. the idea is to compare them with the blue dashed-lines that capture actual cases. as figure 1 highlights, some states display lack of variation within the in-sample period. just to give an example, washington had had only one confirmed case for the first 36 days since its first confirmed covid-19 infection. hence, we exclude it from the control group. for similar reasons, we also exclude arizona, illinois, and massachusetts from the control pool. the analysis ended up relying on six control states. we propose a two-step approach using the artificial counterfactual (arco) method introduced by carvalho et al. [2018] with the correction of masini and medeiros [2019] to estimate the number of cases for each us state. let = 10, 11, . . . , 58 represents the number of days after the first confirmed case of covid-19 in a given state. define as the natural logarithm of the number of confirmed cases days after the first case of the disease in this specific treated state, and as a vector containing the logarithm of the number of reported cases for control states also days after the first case has been reported as well as a logarithmic trend: log( ). the inclusion of the trend is important to capture the shape of the curve. the model is estimated as follows. we use the weighted least absolute and shrinkage operator (wlasso) as described in masini and medeiros [2019] to select the control states that will be used to estimate our counterfactual. the goal of the lasso is to balance the trade-off between bias and variance and is an useful tool to select the relevant peers in an environment with very few data points:̂︀ = arg min where = | , |, = 1, . . . , − 1, and = 1. is, for each state, the number of days from the first reported case until the lockdown plus ten extra days, and > 0 is the penalty parameter which is selected by the bayesian information criterion (bic), in accordance with medeiros and mendes [2016] . the weight correction in the lasso is necessary in order to control for the nonstationarity of the data; see masini and medeiros [2019] for a detailed discussion. the counterfactual for = + 1, . . . , is computed aŝ︀ we also report 95% confidence intervals based on the resampling procedure proposed in masini and medeiros [2019] . we are interested in examining the effects of lockdown policies not only on the number of cases, but also on the number of deaths. however, we cannot implement the strategy described above because there is not enough variation in deaths for the in-sample period. some states, for instance, implemented a state-wide lockdown policies before the first confirmed death. thus, we propose an alternative method. we consider a counterfactual state for the number of deaths based on the counterfactual estimated for the number of cases. this is not straightforward as in the traditional synthetic control method because the arco methodology described above includes an intercept in the estimation, which is measured in the log of the number of cases, and not only a convex combination of other states. intuitively, the methodology described above chooses a combination of states that is at a fixed distance from the treated unit at the in-sample period and not a convex combination of states that matches exactly the actual number of cases. the intercept controls for all time-invariant characteristics that define the counterfactual. then, we proceed as follows. let be the number of accumulated deaths in state at the day . also, let be the vector of estimated coefficients for the state as in (1) and used to construct the counterfactual for cases. in addition, let be a vector of the number of deaths for all states in the control pool at time . we define the counterfactual number of deaths in that state as: = −¯+¯ (3) where¯is the day that state implemented the lockdown policies. that is, we maintain the weights estimated above and adjust the intercept so that the counterfactual series for deaths matches the number of actual observed deaths in the beginning of the quarantine. for the sake of exposition, we relegate the results on cumulative deaths to appendix a.4. 10 to illustrate how the method works, figure 2 presents the arco counterfactuals for the states of alabama, colorado, and maine. the timing of the policy intervention ( 0 + 10) corresponds to the lockdown date plus ten days. the counterfactual analysis makes it clear the importance of lockdown policies in mitigating the acceleration of the number of covid-19 confirmed cases in the treated states. as shown in figure 2a , for example, our results point to a substantial increase in the number of cases in alabama if it had not adopted an early lockdown. similarly, figures 2b and 2c reveal the same behavior for the cumulative curves in the other selected states. counterfactuals are constructed with the estimated weights and cumulative cases of the six states that compose the control group. these weights are reported in table a .2 in appendix a.2. in appendix a.3, we present similar counterfactual plots for the remaining treated states. in order to assure that the proposed methodology is producing proper counterfactual analysis, we generate placebo results by producing a "synthetic control" for each control state using the remaining control states as control pool. results are displayed in figure 3 , which shows the ratio of the estimated counterfactual cumulative cases to the actual ones for treated states except new york (black lines), and non-treated states (red lines). we assume that the epidemiological day of the placebo intervention is 0 = 36, marked by the vertical dashed line, which is the median (and the mean) timing of the policy interventions in the treated states. it is reassuring that for half of the placebo counterfactuals the ratios fluctuate around one, whereas for the majority of treated states ratios grew above one at some point (likely around the actual timing of policy intervention). the latter result means that lockdown policies were effective to tame the spread of the virus, whereas the former suggests that results are not driven by chance. regarding south dakota, the only placebo counterfactual that reached a ratio well above one, by using google mobility data (described in appendix a.5), we show that mobility in residential areas increased whereas mobility in outdoor areas decreased substantially once compared to the period before the pandemic (see figures a.41 and a.42 in appendix a.5) . this is suggestive that south dakota's population endogenously decided to stay more at home, and avoided environments prone to the risk of contamination. at the time, a proper lockdown policy was not necessary, and south dakota's non-conformity to the placebo test does not seem to invalidate our approach. in contrast, for nebraska and california, the counterfactuals are pointing to a smaller number of cases than the actual ones, which goes against finding that lockdowns were effective to reduce cases of covid-19. the case of california is quite emblematic, as the number of cases during the estimation window remained very small and with very low variation. however, the number of cases started to grow at a fast rate much after the cut-off date. the state of nebraska displays a similar pattern. to gauge the quantitative impact of lockdown policies, for each state, whether treated or control used as placebo, we compute the ratio of the counterfactual estimated comulative cases ("without" a lockdown strategy in place) to actual ones on the 58th epidemiological day, which is the last day used to compute the counterfactual. table 2 reports the mean and median of the ratios across states, whereas table a .3 in appendix a.2 reports these ratios for each state. the first row corresponds the case in which controls are used as placebos, whereas the second considers the treated states only. as we discuss below, new york is clearly an outlier, whose ratio reached an implausible value of 16.5 as reported in table a .3. hence, our preferred specification is displayed in the third row which excludes new york from the pool of treated states. we also compute other two versions of these ratios using the lower bound (lb) and upper bound (up) of the 95% confidence interval in the numerator. the ratios are clearly above one for the treated units, whether new york is excluded or not. according to our preferred specification, counterfactual estimates suggest that the number of cases would be nearly two times larger were lockdown policies absent. again, it is reassuring that among the controls used as placebo, these average ratios remain around one. regarding the effects of lockdowns on cumulative deaths, we present the results for all treated states in appendix a.4. for some states, the counterfactual cumulative deaths exhibit similar patterns to those regarding cumulative cases. but, for many other states, they are not statistically significant at least for the first days after the policy implementation. one possible explanation is that there is a delay between cases and deaths, as the latter is a consequence of the former. hence, deaths only show up in the official statistics days after cases. perhaps, if we could estimate counterfactuals for longer periods, the synthetic accumulated deaths would further decouple from the actual ones. in addition, since weights on the controls are estimated considering the (log) cumulative number of cases, the counterfactuals for cumulative deaths are arguably noisier. 11 as discussed above and presented in table a .3 in appendix a.2, we obtain an implausible ratio (of counterfactuals to actual cumulative cases) of 16.5 to new york. this section puts a lens on this state. in particular, figure 4 displays the estimated cumulative number of cases for new york "without" lockdown, as well as extrapolations of the cumulative number of cases based on the mean and median growth rate of the last ten days of the in-sample period. as reported in table 1 , among the treated states, new york was the fastest one to react to the pandemic, and established a state-wide lockdown policy only 20 days after the first case. figure 4 extrapolates the last in-sample observations by using both the observed mean and median growth rates for the last ten days, which yields a similar pattern to the result obtained by applying the synthetic control approach. due to the progression of the virus, particularly in new york city, the in-sample observed rates are quite high once compared to other states as illustrated in figure 1 , which can be explained not only by the dynamics of the city but also by its high population density. hence, new york is clearly an outlier and might not be amenable to our synthetic control approach, which justifies reporting results excluding new york above. in this paper, as opposed to most of the early and incipient literature on the lockdown effects during the covid-19 crisis, we conisder a purely data-driven approach to assess the impact of lockdowns on the short-run evolution of the number of cases and deaths in some us states. also, as opposed to some recent papers that use a difference-in-difference approach, we adopt a variant of the synthetic control approach, arco, due to carvalho et al. [2018] and masini and medeiros [2019] . on average, according to the synthetic controls, the counterfactual accumulated number of cases would be two times larger were lockdown policies not implemented in treated states. in the first two columns of table a .1 we show the date of the first confirmed case in every treated state we analyze and its reopen date (plus ten days), whenever available at the time we started to circulate this paper. 12 in the third column, we show the difference (in days) from the first confirmed case and the reopen date plus ten days. these figures illustrate why we had to limit our sample size to only 58 epidemiological days. for example, if we had used 60 days in our analysis, we would have to exclude alabama and maine from our treated states, given that they would not be in a state-wide lockdown in the last days of the out-of-sample period. we report in the first seven rows of table a .2 the coefficients estimated by the lasso model for each treated state for the 0 +10 in-sample period. the last two rows display the mean and the median (across the out-of-sample period) of the ratio between the actual cumulative cases and the counterfactual cases for every state. with only two exceptions (missouri and nevada), every state has an out-of-sample mean and median of the observed-to-predicted ratio below one. this means that, on average, the realized cumulative cases were smaller than the counterfactual, which highlight that lockdowns had a meaningful impact on slowing down the covid-19 spread in these states. table a .3 reports the ratio of the counterfactual cumulative cases to the actual ones on the 58th day after the first confirmed case in each state. it also reports the lower and upper limits of the 95% confidence interval. among the 20 treated states, the ratio is larger than one in 18 of them. for missouri and nevada, there is no evidence on the effectiveness of lockdown policies. for mississipi and south carolina, the impacts of lockdowns are only modest. note that new york is clearly an outlier, with such ratio around 16.5. we discuss this case in the main text. in contrast, among the non-treated states, we obtain ratios close to one for three out of six cases. we assume that the cut-off of the placebo intervention is 0 = 36, which is the median (and the mean) timing of the policy interventions in the treated states. as discussed in the main text, south dakota, which displays a ratio well above one, experienced a large reduction in outside mobility even without official lockdown measures. california and nebraska, which display ratios below one, had very few covid-19 confirmed cases during the period before the cut-off. in this section, in figures a.21 -a.40, we report the counterfactual estimates for cumulative deaths based on the methodology described in section 3.3. as we discuss in the main text, although for some states, the counterfactuals exhibit similar shapes to those regarding cumulative cases, for many other states, they are not statistically significant at least for the first days after the policy implementation. we know that lockdowns affect the covid-19 dynamics by imposing social distancing and mobility restrictions. to help understand the results described in this paper, we analyze the mobility data available at google mobility reports (https://www.google.com/covid19/mobility/). google mobility data show how visits and length of stay at different places change compared to a baseline, before the outbreak of the pandemic. in particular, the baseline is the median value, for the corresponding day of the week, during the five weeks between january 3rd and february 6th 2020. in order to understand how the population in each group (treated and control states) is behaving during the covid-19 crisis, we compute the median of mobility changes across our sample period, i.e. the 48 days following the tenth day after the first confirmed case in each state. also, the data concern mobility changes for six categories, being five of them related to outdoor activities. namely, grocery & pharmacy, transit stations, parks, retail & recreation, and workplaces. the remaining one concerns indoor activities, namely, residential. hence, to capture an idea of outdoor mobility changes, we aggregate the aforementioned five categories into a single one defined as the median of the original five categories. in contrast, mobility changes in residential areas capture indoor mobility changes. the two boxplots in figures a.41 and a.42 present the median of mobility changes in all analyzed states both in residential and in outdoor areas, respectively. we report results for treated and control states separately. regarding mobility changes in residential areas, on average, residents from every state analyzed spent more time in these areas after the pandemic outbreak. however, those from treated states spent even more time indoor. nevertheless, there are outliers. for instance, residents from south dakota spent a lot more time in residential areas than before the pandemic, which helps understand the results found for this state in the placebo test. we found similar results for mobility changes in outdoor areas. clearly, residents from treated states remained in outside areas less often than residents from controls (always compared to the period before the pandemic). in new york, for example, there was a 50% decrease of outdoor mobility. once more, south dakota is an outlier for the control group, reinforcing the thesis that its population voluntarily decided to stay more at home. indeed, residents from south dakota spent almost 20% less time in outside areas, while those from the median state for the control group spent nearly 8% less. the economic costs of conflict: a case study of the basque country synthetic control methods for comparative case studies: estimating the effect of california's tobacco control program pandemic, shutdown and consumer spending: lessons from scandinavian policy responses to covid. working paper, university of copenhagen a simple planning problem for covid-19 lockdown how deadly is covid-19? understanding the difficulties with estimation of its fatality rate what will be the economic impact of covid-19 in the us? rough estimates of disease scenarios an seir infectious disease model with testing and conditional quarantine covid-19 infection externalities: trading off lives vs. livelihoods. working paper 27009 the covid-19 pandemic: government vs. community action across the united states. working paper arco: an artificial counterfactual approach for high-dimensional panel time-series data when do shelter-in-place orders fight covid-19 best? policy heterogeneity across states and adoption time the macroeconomics of epidemics human mobility restrictions and the spread of the novel coronavirus (2019-ncov) in china the effect of stay-at-home orders on covid-19 cases and fatalities in the united states. working paper did california's shelter-in-place order work? early coronavirus-related public health effects costs and benefits of closing businesses in a pandemic optimal mitigation policies in a pandemic: social distancing and working from home the effect of social distancing measures on intensive care occupancy: evidence on covid-19 in scandinavia. working paper counterfactual analysis with artificial controls: inference, high dimensions and nonstationarity ℓ 1 -regularization of high-dimensional time-series models with nongaussian and heteroskedastic innovations short-term covid-19 forecast for latecomers. working paper villas-boas. are we #stayinghome to flatten the curve? working paper key: cord-030870-ao5p3ra3 authors: paul, suman; bhattacharya, subhasis; mandal, buddhadev; haldar, subrata; mandal, somnath; kundu, sanjit; biswas, anupam title: dynamics and risk assessment of sars-cov-2 in urban areas: a geographical assessment on kolkata municipal corporation, india date: 2020-08-25 journal: spat doi: 10.1007/s41324-020-00354-6 sha: doc_id: 30870 cord_uid: ao5p3ra3 sars-cov-2 has been transmitted and outbreak took place in india during the last week before nationwide 1st lockdown took place. urban areas found more vulnerable and reported nearly 65% of cases during every phase of lockdown. mumbai, among four metropolitan cities found huge number of containment zones with nearly 30% of sars-cov-2 cases indicating clustering of cases. most of the containment zones of sars-cov-2 cases in kolkata municipal corporation found a significant relation with slum areas. the study primarily tries considering the nature of sars-cov-2 cases in different urban centres with the help of cartographic techniques. ahp method has been used to determine the factors responsible for such concentration of sars-cov-2 cases with vulnerability assessment (exposure, sensitivity and resilience) and risks. before nationwide lockdown starts, the share of urban centres found 25% which has been transformed into nearly 60% at the end of 3(rd) phase of lockdown. growth rate of sars-cov-2 cases found very high for chennai and thane with less number of doubling time to nation. slum concentration and containment density shows a higher degree of correlation in kolkata municipal corporation. risk map also shows the concentration of cases in central and north kolkata with higher degree of diseases exposure and sensitivity. control measures must be taken by the central and state government to minimise the transmission rate of sars-cov-2 mainly urban areas. as urban area contributing a higher share of sars-cov-2 cases, a proper management plan must be enforce. electronic supplementary material: the online version of this article (10.1007/s41324-020-00354-6) contains supplementary material, which is available to authorized users. in the present day context urbanisation becomes a major driver of demographic change of an area. according to united nations report, world's population living in the urban areas has grown from 43 to 55% during 1990 to 2015 [1, 2] . by 2050, it has been estimated that the world's 70% population will be reside in urban areas. this kind of urban growth and population concentration leads to sprawling and shanty development outside and within the city respectively. high population density, low per capita spacing, concentration of urban poor make significant impact on the epidemiology of the infectious diseases electronic supplementary material the online version of this article (https://doi.org/10.1007/s41324-020-00354-6) contains supplementary material, which is available to authorized users. [3, 4] . association between urban poor and risk of pathogen transmission is very high in this scenario. high human to human propagation can easily be spike with such vulnerable condition [5] . presently, more than 900 million populations in the world are living in slums whereas the figure of india is nearly 104 million [6] . in 1990, worldwide figure of slum populations were nearly 689 million which has been booming to 792 million in next 10 years i.e. in 2000 [7] . in the current worldwide pandemic situation of sars-cov-2, growth pattern, transmission nature and driving factors are the key aspects need to study. december 31, 2019 an outbreak of covid-19 (as known earlier) has been reported from wuhan city of hubei province in mainland china and rapidly spread into the other provinces of china along with 24 countries within end of january, 2020 [8] . wuhan city has been under full lockdown (travel ban and closure of everything except essential services) from 23 january 2020 [9] . but the decision has been taken by the government was too late as the by novel coronavirus (ncov-19) infections has already transmitted in different parts of mainland china and also in the different countries of the world. this episode is highly correlated with the chinese great migration during the january-february when near about 415 million people are moving towards mainland china (within the country and from outside the country) to celebrate their lunar new year. from mid-february countries of european continent, u.s.a, australia facing a terrible spike of sars-cov-2 cases which has been not affected india at a large till the end of march, 2020. nearly 4.9 million populations has been affected by sars-cov-2 and the fatality has reached into 0.32 million (as on 19-05-2020). several countries like united states, russia, brazil, italy, france, united kingdom, germany, turkey, iran has faced a major setback due the this pandemic [10, 11] . india has also reached the mark of 100,000 confirmed sars-cov-2 cases after 64 days of first case found. though the time taken by india to reach one lack infected cases much higher than the other countries, but the exponential triggering has been noticed during last week of 3 rd phase of nationwide lockdown (from 4 may, 2020 to 17 may, 2020). nearly 85% cases are reported from major cities of india and most interestingly, mumbai, delhi, ahmedabad, chennai, thane, pune, kolkata become the most contributing urban centres to sars-cov-2 cases (as on 19 may, 2020). high population density and higher concentration of slum population make an interruption for maintaining the social distancing and lockdown effectively [12] [13] [14] . considering such backdrop the nature of spreading of sars-cov-2 cases in the indian cities need to be analysed. further an attempt has also been made to quantify and assess the hotspot zones along with risks of the concentrated areas of kolkata (one of the metro city) for proper understanding of transmission of diseases in the congested and unhealthy places as a case study [9, 15, 16] . india has only 3 cases of sars-cov-2 up to 3 march 2020, but all cases has transmitted and grow in a slow but steady in different areas of india especially in urban centres. except kasaragode, a rural base area in kerala, urban areas of maharashtra, delhi, gujarat and rajasthan have shown a large number of sars-cov-2 positive cases. in this regard, the mumbai, ahmedabad, chennai, pune, thane, indore, delhi, jaipur, kolkata, surat urban centres have been considered for initial analysis as these urban centres contribute more than 60% of sars-cov-2 cases during nationwide lockdown periods. from the analysis of urban centres contribution of sars-cov-2 cases, four megacities of india (i.e. mumbai, ncr delhi, kolkata and chennai) has been further chosen ( fig. 1 ) to find out the nature of relationship between containment zones and sars-cov-2 cases. as urban centres with high population density and high concentration of slum population faced a risk of rapid transmission of sars-cov-2, a risk analysis have also been assessed on kolkata municipal corporation for the better understanding of driving factors of transmission of sars-cov-2. the sars-cov-2 data set has been obtained from indian council for medical research (icmr) and health website which provide the real time data set on the outbreak of this pandemic. another good source of data has been found from 'how india lives'. this website provides different health infrastructure dataset at district level and uploading the real time covid-19 cases for each day at district and city level. containment zones cities which is a very important source of information to identify the sars-cov-2 hotspot has been taken from health bulletin, govt. of west bengal. 10 major cities of india are taken for primary level study with duration of before lockdown situation to present day (12-05-2020) scenario. the slum data set has been taken from unpublished baseline survey report of 2016 by the department of bustee services, kolkata municipal corporation. as most of the dataset of slum related indicators found from census of india, 2011 dataset, we have to take baseline survey report of kmc. this data base has helped to identify the nature of exposure, sensitivity and resilience of world wise slam households which can assess the nature of risk among the slums. to depict the nature of sars-cov-2 spatial association, local moran's i statistic has been used to identify the cluster and spatial outlier in the neighborhood for 12-05-2020 dataset [16] [17] [18] [19] . moran's i highlights the location based cluster form high to low infection and calculated as follows: where zi the number of sars-cov-2 containment zones at a spatial unit i (ward as an areal unit), z is the overall containment zones in the study area (kmc as a whole) and n is the number of spatial unit which are 144 (no. of wards in kmc) and v is the rate of the variance of sars-cov-2 containment zones in different wards which is computed as below: this method represents high (positive) and low (negative) values. high-high cluster values show the results cluster up of similar values with q-value range from 0.01 to 0.1 (0.1 for 90%, 0.05 for 95% and 0.01 for 99% of confidence level) and low-low cluster shows the clustering of dissimilar values with same q-values. higher the q-values with lower the z-score value shows the perfect significance of the method applied for study the nature of clustering pattern. the p value is a value of probability. for the pattern study, it is the probability which create spatial pattern using some random process. the small p-value value suggests that the observed spatial pattern is an outcome of random process; hence null hypothesis can be rejected. analytical hierarchical process has been developed by satty [20] to facilitate priority setting and decision making. ahp now broadly applied in social science research and specifically in hazard and risk analysis. in this method a pairwise matrix has been developed among the set of scale of choices (table 1 ) on the given alternatives [21] . ahp methods also deliver to judge the nature of consistency of preferences given by the report using consistency ratio when the value has 0.10(cr c 0.10). the consistency ratio is defined as: where cr is consistency ratio, ri represent random number (table) and ci represent consistency ratio is expressed by for the present work, consistency ratio has been found as 0.07, consistency index as 0.04 and the value of random number for n = 3 has been determined 0.58. based on socio-economic data of slum of kolkata municipal corporation and containment zone data and containment zone data from different web sources we have selected the following indicators for quantity exposure, sensitivity and resilience for assessing the risk [22] infector disease like sars-cov-2 (see table 1 ). the study considered the following as exposure indicators, number of sars-cov-2 containment zones/ 0 0000 population (e 1 ), percentage share of slum population to total world population (e 2 ), number of containment zones sq.km (e 3 ), number of slum located in the ward (e 4 ), and percentage slum area to total area of the ward (e 5 ). the study considered the following as sensitivity indicators, hhs size (s 1 ), percentage of hhs size with 5 persons and above (s 2 ), no. of persons in the slum, used community toilet, no. of persons/tube well used (s 3 ), population density (s 4 ) and household density (s 5 ). the study considered the following as resilience indicators, percentage of hhs access to drinking water facility within premise (r 1 ), percentage of hhs access drinking water from treated source (r 2 ), percentage of literate population (r 3 ), average per capita income of the slum located in the ward (r 4 ) and work participation rate (r 5 ). number of containment zones/ 0 0000 population in one of the most important indicators affecting the exposure of sars-cov-2 cases. according to icmr, the sars-cov-2 cases are taken the epicenter of this containment zone [23] . on the basis of data, we have divided the whole data set into 5 categories having high risk factor to low risk factor (above 2.0/ 0 0000 population, 1.51-2.0/ 0 0000 population 1.0-1.5/ 0 0000 population, 0.5-1.0/ 0 0000 population and below 0.5/ 0 0000 population). share of slum population is another prime indicator to explain the exposure of such infections dieses h2h pattern. so the shanty and confected household are very much exposed to such dieses. here, we also divide the whole set of data into 5 categories on the basis of risk factor. density of containment zones in another good indicator for assessing the nature of exposure. as the density is increasing, the exposure of sars-cov-2 to other persons source: computed by the authors becomes very high. slum area to total area is another crucial factor as areal coverage increase, the congestion pattern of living, unhygienic situation are very much exposed. thus higher the percentage signifies higher risk factor. each of the indicators has been categories under five classes of risk factor and weightage of these indicators have been assigned using ahp method. the exposure index of sar-cov-12 has been determined using following formula. exposure index ei ð þ : where e 1c denote the ward data lies or which class [class [1] [2] [3] [4] [5] as mentioned in table 2 and e 1w is weightage assessed using ahp model. here, in this study purpose identifies the factors which can trigger the intensity and probability of spiking up of sars-cov-12 cases. household size undoubtedly increases the extent of severity of such h2h infectious disease. population belonging in a household also led the situation more badly. when social distancing is addressed nationwide, the poor people of the slums cannot maintain such as due to shortage of space in the households. community toilet and tube well use is also promoting the chances of mass gathering in the slams. many people are living under such shanty places and also depend upon these facilities which can aggravate and spread such infectious disease in the community level. sensitivity index can be expressed in the following manner: where s 1c denote the ward data lies or which class [class [1] [2] [3] [4] [5] and s 1w is weightage assessed using ahp model. resilience can be defined as reduction and prevention approach to risk any vulnerability for making an area more socio-economically stronghold [24] . drinking water facility with premises and from treated source, level of literate population are the important indicators for resilience study. on the other hand per capita income and work participation rate is the potential indicator to increase the resilience of the any households. resilience index can be determined as: where r 1c denote the ward data lies or which class [class [1] [2] [3] [4] [5] and r 1w is weightage assessed using ahp model. on the basis of assessed results from exposure index (ei), sensitivity index (si) and resilience index (ri), risk of the selected wards have been estimated using following methodology as prescribed by ipcc [22] framework: where ei w is the exposure index, w e weightage of exposure, si w is the sensitivity index, w s weightage of sensitivity, ri w is the resilience index, w r weightage of resilience. 3 results and discussion outbreak of novel corona virus (earlier it is termed as 2019-ncov and later renamed as sars-cov-2 during the preparation of this manuscript) leading to lockdown (which means entire closure of all services except frontline services and essential services, i.e., banking, fire service etc.) of entire country which took place on and from 24th april, 2020 midnight. till date of the preparation of this manuscript, india already gone through three phases of lockdown (which will end on 17th may, 2020) and during these phases, urban centres have been found the most threating situation contributing nearly 60% of total affected cases of sars-cov-2 from 10 most popular cities of india. nearly it was may 6, 2020 when india experienced 52,469 confirmed sars-cov-2 cases with 1771 death and entered into the list of top fifteen countries of the world. it is only 1.41% sars-cov-2 cases to rest of the world but it was only 0.13% (536 cases) when nationwide lockdown started. the surge of sars-cov-2 cases as seen by usa, italy, france, germany, brazil, russia was not same in india till 19 may, 2020. cases are found to be doubled in every 11 days at that time. but after 20 may, 2020 a large spike has been found till 2 june, 2020 which put india in the same bracket as brazil and russia in terms of upward trend in the infected cases and fatalities. the fact that despite of four lockdown imposed by central and state governments combine, the stringent index has been found to fall from 100 (on 24-03-2020) to 79.2 (26-05-2020) which make a straightway relation of surging the sars-cov-2 cases in the different parts of india (fig. 2) . another issue is the return back of stranded labour from mainly southern and western states to the eastern portion make this spread to rural areas also. based on the dataset and fig. 3 of sars-cov-2 of 10 major urban centres, mumbai has been found most cases with one-fifth (19.35%) to total country's cases up to 12th may, 2020 when the lockdown starts on 24th march, 2020 it was only 6.65% of whole country with a rapid growth of 1.183, 1.085 and 1.049 during 1st phase, 2nd phase and 3rd phase of lockdown respectively. doubling rate of cases is 1.2 times (mumbai doubled the cases in 11.5 days as on 12.05.2020) to country doubling time (9.7 days). ncr delhi also contributes a larger extent of sars-cov-2 cases with 9.78% which was only 5.25% just before lockdown. a single event at nizamuddin marcus (a religions congregation) makes the situation worse during first phase of lockdown [26] . a sharp rise or cases (nearly 13.59% to country's total cases) has been experienced during this time which has been slow down gradually. stringent actions have been taken by the respective state governments which reflect in the higher rate of doubling time (11.8 days) to country's data. after ncr delhi, ahmedabad from state of gujarat originate as a big area of concern for the country which contributing 8.22% of total country's cases at the end lapse of 3rd phase of lockdown but it was only 2.45% at the starting of 1st phase of lockdown. during 2nd phase of lockdown a tremendous spike in the cases of sars-cov-2 has been experienced by this urban centre. during these phase, most of the cases (nearly 70%) are related to travel within the country and related to delhi's religious congregation took place is end of march, 2020 [27]. through having higher value of doubling rate (11.4 days to double) from country's perspective and having decreasing growth rate. number of containment zones gives a clear picture of clustering of cases which signifies the nature of community spreading which is a matter of uneasiness. chennai shows an alarming situation with 5.43% of cases during the end period of 3rd phase of lockdown which was just 1.05% on 24.03.2020 (just before the lockdown). though the growth rate is decreasing (from 1.17% at 1st phase of the lockdown and 1.108% at nearly end of 3rd phase of lockdown) but the doubling time of cases is 6.25 days which signifies a great risk. among all the 10 urban centres under study (which are combine contribute 56:28% of cases), chennai has the lowest doubling time which is a matter of concern. on 14th may 2020, nearly 2600 sars-cov-2 have been outlined to a wholesale vegetable market named koyambedu and authorities have acknowledged it as a coronavirus hotspot [28] . after cases being reported from the popular market of on the other hand kolkata shows a steady but consistency in growth and spreading of sars-cov-2 cases. almost in all the lockdown phases, kolkata shows much below transmission of sars-cov-2 and contributing least to country's tota. doubling rate gives a clear picture of this scenario with almost 12.2 days which is a good indication with a population of nearly 4.5 million and having nearly 26.17% of slum population lived in this region (census, 2011). pune (3.34%), thane (3.19%), indore (2.63%), jaipur (1.70%) and surat (1.26%) contribute nearly 12.06% of the cases to country's total. growth rates of sars-cov-2 in these urban centres are constantly decreasing which is a good pictogram of action taken by the government of those states as well as social awareness with following social distancing. among all the urban centres, thane shows an alarming situation in doubling rate which is 6.9 days and thus thane can be an emerging hot spot like mumbai and chennai if proper action not taken at the earliest. two days before the second phase of lockdown started, india has charted identification of red, orange and green zones which is a strategic approach for defining the area of operation, applying perimeter control, delineating containment and buffer zones. meanwhile ministry of health and family welfare, govt. of india declared 170 districts as 'hotspots' and 207 districts as 'non-hotspots'. ministry also categorises hotspots in two way-(a) clusters-increase in the incidence of sars-cov-2 with less than 15 cases and there must be epidemiologically linked and (b) large outbreak-when more than 15 cases have been found from a defined geographical area and these cases may not be epidemiologically linked. to combat with this pandemic, state governments have begun to experiment the idea of containment zones to deal with sars-cov-2. mechanism of the containment zones is very straight forward, clusters or large outbreak which shown rise of cases and shown rapid transmission either in family or in community must be seated. movement in these zones are very limited which is only for foot line workers and residential movement is completely ban. when large number of cases are found in a smaller number of containment area it may a reflection of large outbreak (ratio is very high) but when large amount of cases with larger number of containment area (the ratio is low) suggest the clustering of disease took place. in case of comparison of confirmed new cases on daily data initiated from the period of first lockdown for four cities is smoothen using five year weighted moving average method. the study considers the weights as defined by using the rule the weighted value of moving average can be calculated from p 5 i¼1 x i w i for the corresponding five values of the series. the study deals with the three types of lock down considering them as phase-i (consist of 21 days starting from 25.03.2020), phase-ii (consist of 19 days starting from 15.04.2020), and the phase-iii (consist of 9 days starting from 04.05.2020). the smoothing data set of new cases of ncovid-19 syndrome is depicted in fig. 4 . the nature of the new cases shows that situation of kolkata is relatively best in compare to other three cities [30] . whatever be the ways the explosion of new cases are found with some controlled behaviour and even with rare fluctuations. the situations of chennai showing some controlled behaviour up to 27.04.2020 i.e. in the middle of phase-iii lockdown but as the relaxation in the lockdown started the outbreak increases with huge rate. similar path also observable for delhi ncr like chennai, but it includes massive fluctuations. as time precedes the frequencies of fluctuations in case of delhi also increases. the situation of mumbai is worse among the all four cities. the periodic up and downs of the new cases puzzle the governance to control it. the linear trend line for cities shows that the line is steepest for mumbai, and it is more than double of delhi. four metro cities shows different pattern of transmission during end ward of 1st phase of lockdown to present time. delhi shows a higher ratio between number of sars-cov-2 cases and containment zones suggesting the larger outbreak for the region. on the other hand mumbai, chennai and kolkata show a clustering scenario of these cases during this time setting. higher number of cases with lower ratio also suggests the expansion of the sars-cov-2 cases in the new areas in faster rate. in 12 may 2020, mumbai and chennai show nearly same value (6.46 and 6.54 respectively) but with the sars-cov-2 cases nearly 3.5 times to chennai. mumbai city shows a huge spreading along different places in this period of time. during this period, delhi shows a much higher rate fluctuating from 35.63 to 42.47 (having a highest ratio of 72.05) and pinpointing the large out break as stated earlier. during this period, kolkata shows a steady pattern having a ratio of 1.9-2.64. the containment zones caused trouble to the citizens, by restricting the mobility almost entirely and have to depend on government officials and selected venders for maintaining the essential services. this methodology of curtailment of rights is temporary for the containing and stops the spreading of disease. but slums in the urban area cannot fully follow the thumb rules of such containment zone. as these people lives in a shanty, unhygienic environment, and using community toilet with large dependency on tube well for water accessibility are very much susceptible for these h2h transmission. mumbai and delhi shows the perfect example of such transmission. dharavi, the world famous slum has been hardly heated by this pandemic. kolkata face a challenge to combat with the spreading of sars-cov-2 cases in the densely slums concentrated areas [7] . most of the wards in kmc having more than 10 containment zones where a large percentage of population living in the slums. increasing number containment zones along with number of cases has proven the spatial dispersal of sars-cov-2 cases in kmc which is needed to be studied further. in kolkata municipal corporation figure 5 shows the time series pattern of the confirmed cases of sars-cov-2 from 1st case detected on 17th march, 2020 to 12th may, 2020 when cumulative number were 1068 and 2173 for kmc and state of west bengal respectively. as kolkata has experienced 1st case of sars-cov-2, here we have taken ward wise containment zone to find out the nature of hot spots located in the municipal area. as per icmr, containment zone confirms the epicentre of cases and due to unavailability of sars-cov-2 ward wise data; we used number of containment zones as proxy indicator [23] . figure 5 also confirms that, up to 1st week of april 2020 kolkata municipal corporation (cmc) contributes a huge percentage share to state's total which has been decreasing afterwards. this scenario again found from starting of fourth week of april when kolkata share nearly 70% of state's total. a huge population of nearly 45 lakh (census of india, 2011) with a high percentage share of slum population (31.35%) is very much vulnerable for transmitting this h2h virus where population density, slum density, using per capita community latrine and tube well are high [31] . figure 6 shows the hot spots for covid-19 using satscan on the basis of two different dates dataset on containment zones result which identifies two primary clusters and three secondary clusters with high confidence value (p-values are found less than 0.01) in kmc. the primary clusters are located covering (a) kareya, tiljala, topsia, tansra, survey park region and (b) jorabagan, burtola, girish park area. secondary clusters have been identified to designate more cases likely to aggregate from kmc which are extent from northern portion to south-western portion. these secondary clusters have also the significance values less than 0.05 (p-value). the highly decentralized nature of incidence of this disease clearly showed limited hotspots within the city of kolkata. ward no. 66 (tiljala/topsia area) has been found a large number of containment zones in a smaller area and it has increasing very sharp. this area has fallen the primary cluster of central kolkata when ward no. 18 and 26 (banstala, girish park region) are fallen under another primary cluster. the sars-cov-2 views caused tremendous pressure situation in the 97 wards in kolkata municipal corporation out of 144 wards (fig. 7a) . 227 containment zones have been found on those 97 wards on 27-04-2020 which has been increased into 338 numbers located in 124 wards. the distribution pattern of containment zones in two different dates has shown a clear increase in spatiality among the wards of kmc. the highest number of zones has been found in the wards located in central kolkata, east suburban and port area and some portion of north kolkata. huge numbers of slums (registered and unregistered) are found in those areas which may play a role of catalyst to spread and transmitted this pandemic [32]. highest concentrations of containment zones have been found in tiljala, kareya, beliaghata, phoolbgan, razabazar, tala, burtola, jorabagan, girish park, bowbazar, entally, muchipara, and survey park area. total number of containment zones in this area have been covered nearly 50% of the total containment zones (fig. 7b) . wards with high containment zones are falling in those wards which have a higher degree of slum population as well as the number. most of the areas under slums are very old and developed before independence [31] . old shanty dwelling slots with higher family size, higher dependency on community toilet/latrine with water availability source as tube well make the area more risky and vulnerable to this sars-cov-2 [33] . as it is already known to everyone that this infection can transmit h2h pattern and ro of 2.5, it is really need to assess the scenario of those slums for better approach to stop the transmission and break the chain of this infection. here need the assessment of the risk of those hotspot areas to make a proper evaluation of vulnerability and steps to be taken in coming days. municipal corporation (kmc) here where ew is weightage of exposure, ei w is the ward-wise exposure index, sw is weightage of sensitivity, si w is the ward-wise sensitivity index, rw is weightage of resilience and ri w is the ward-wise resilience index. on the resulted dataset, risk zones have been categorised into 5 classes from very high risk zone to very low risk zone. of the total selected wards under study (68), 6 are very highly exposed to sars-cov-2 followed by highly exposed wards (16), moderately exposed (22), low exposed (11) and very low exposed (9) categories. the exposure index is composed of sars-cov-2 containment zones density with population, share of slum population and density of slums with areal coverage to ward area made the wards located in north and central kolkata more exposed as these areas are very old and the slums in these wards have an age of more than 75 years. old drainage system, located behind the rail lines and nullas (old sewerage lines) make the situation more worsen. overall the area under gardenreach, metiaburuz (50% of very high exposed wards from this area) with a high percentage of muslim population face the higher degree of exposed of such infections (fig. 8a) . on the initial stage reluctant situation from government end and non-maintaining the social distancing norm during nation-wide lockdown period make this areas face a terrible trouble and have experienced a number of sars-cov-2 cases with a large number of containment zones. sensitivity index analysis showing that of the total wards very high sensitivity has been found among 17 wards followed by 16 wards in high sensitive index, moderately sensitivity found among 13 wards when 15 and 7 wards are found in low to very low sensitivity values respectively. high sensitive wards are mostly lies in the north kolkata and partly in central kolkata (fig. 8b) where population density, household density, latrine and tube well dependency among the slum dwellers are very high which clearly gives the results of very high sensitive zones. these zones settled are much before of independence which has a migration legacy (7, 34) . resilience capability among the selected wards of kmc found to be high as more than 64% of the total wards are characterised by very poor to moderate level of resilience. the wards are very much lagging behind in facilities which can protect them from such infections and the nature of infrastructural development are also found low in nature. figure 8c shows that the most vulnerable wards (high to very high) located in central and eastern portion of the study area. drinking water facilities, per-capita income and work participation rate are very low in these wards. eastern portion wards of the study area are joined with kmc much later during 2000 onwards which shows the less unavailability of facilities in respect to others (33, 19) . risk analysis revealed that very high vulnerability has been observed in 11 wards followed by high risk areas with 21 wards (nearly 30% to total wards under study). as a whole high to very high wards coved nearly 47% of the studied area (fig. 8d) this indicates a disquieting situation for such infectious disease. most of the wards located in central and some portion of north kolkata. four wards also most of the containment zones, high slum population share and density with excessive dependency on community toilet and tube well are the driving forces behind this high risk factor in these areas [34] [35] [36] . on the other hand low working population, low per-capita income, high household density in topsia, tiljala, gardenreach, rajabazar, beliaghata, burabazar, jorabagan area make more hazard prone. earlier evidences of dengue fever also found the same type of hotspots in the past years. unhygienic and close spacing of settlements, not maintaining social distancing and very low per-capita space availability (nearly 5-7 persons in an 80-100 sq. feet room) make the region hotspots and rightly most of the sars-cov-2 cases found from these places. the spatiality of sars-cov-2 has wide-ranging expressively from april to may 2020, and it has exhibited consistency in northern and central part of kmc. a grouping of irregular and epidemic patterns of human-to-human exposure has been observed during this period [12] . by contrast, the distribution curve for cities in india, mumbai has been experienced largest outbreak in india where kolkata has constrains its spike. purely temporal cluster analyses of sars-cov-2 infection illustrated significant clusters in april and may of 2020. this finding is consistent with previous studies of eifan [34] , which showed significant peaks in mers-cov incidence between march and may in saudi arabia in 2014. in this study, sars-cov-2 was observed during mass gatherings in different part of india, which are inconsistent with previous studies [35] . this indicates another knowledge gap regarding the mode of transmission that needs further investigation. though the transmission and outbreak has not a sudden one, major urban centres have been found more vulnerable to transmit this h2h virus. high population density, concentration of high amount of slum population with high household density with low per-capita income shows the main driving factors for such outbreak. the people of those places are compel to break the said social distancing, as the people have a little, very little space for stay in household (in some cases they lived in 80 sq. feet area with 6-7 persons), they used to go and use community toilet, tube well sharing with other hundreds of population make the situation more grave (3, 8, 9) . quick preparation and execution of the containment plan, deployment of adequate human resources (mainly from health workers) at ward level, active surveillance in the well-defined geographical area and higher test (rapid antibody test) can minimise the chance of transmission in community level. as the spacing of households is very congested, such actions must be taken without any interference. revision of world urbanization prospects urbanization and global environmental change: 21st century challenges middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission. the lancet infectious diseases health and health-related indicators in slum, rural, and urban communities: a comparative analysis understanding the spatial diffusion process of severe acute respiratory syndrome in beijing abstract for slums regional disparities of slums, 2013-an overview with special emphasis to kolkata spatial epidemic dynamics of the covid-19 outbreak in china spread of yellow fever virus outbreak in angola and the democratic republic of the congo 2015-16: a modelling study. the lancet infectious diseases modes of transmission of virus causing covid-19: implications for ipc precaution recommendations a mathematical model for simulating the phase-based transmissibility of a novel coronavirus spatiotemporal clustering of middle east respiratory syndrome coronavirus (mers-cov) incidence in saudi arabia the influence of urbanization modes on the spatial circulation of flaviviruses within ouagadougou (burkina faso) spread of yellow fever virus outbreak in angola and the democratic republic of the congo 2015-16: a modelling study. the lancet infectious diseases finding malaria hot-spots in northern angola: the role of individual, household and environmental factors within a meso-endemic area beyond moran's i: testing for spatial dependence based on the spatial autoregressive model spatial clustering of plasmodium falciparum in bihar (india) from 2007 to 2015. spatial information research rapid urban malaria appraisal (ruma): epidemiology of urban malaria in ouagadougou identification of malaria hot spots for focused intervention in tribal state of india: a gis based approach the analytic hierarchy process: planning, priority setting, resources allocation urban flood hazard zoning in tucuman province argentina using gis and multicriteria decision analysis climate change 2014: impacts, adaptation, and vulnerability. part a: global and sectoral aspects. contribution of working group ii to the fifth assessment report of the intergovernmental panel on climate change containment plan for large outbreaks risk assessment of coastal erosion of karasu coast in black sea an impact evaluation study of bsup programme intervention in kolkata metropolitan area (kma), kolkata municipal development authority assessment of water security in socially excluded areas in kolkata, india: an approach focusing on water, sanitation and hygiene a pandemic risk assessment of middle-east respiratory syndrome coronavirus (mers-cov) in saudi arabia a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae slums in kolkata: a socio-economic analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors acknowledge the department of bustee service, kmc, hogg buildings (3rd floor), kolkata for providing data support for the study.authors' contributions sp and sb designed the study; sh, ab and bm contributed to data acquisition; sk and sm carried out the statistical analysis; sp and sb drafted the manuscript. all authors contributed to the interpretation of data and revision of the manuscript. all authors read and approved the final manuscript.funding this work has not been supported by any state or central government funding agencies. key: cord-127109-jdizyzbl authors: bertschinger, nils title: visual explanation of country specific differences in covid-19 dynamics date: 2020-04-15 journal: nan doi: nan sha: doc_id: 127109 cord_uid: jdizyzbl this report provides a visual examination of covid-19 case and death data. in particular, it shows that country specific differences can too a large extend be explained by two easily interpreted parameters. namely, the delay between reported cases and deaths and the fraction of cases observed. furthermore, this allows to lower bound the actual total number of people already infected. the unfolding covid-19 pandemic requires timely and finessed actions. policy makers around the globe are hard pressed to balance mitigation measures such as social distancing and economic interests. while initial studies [3] predicted millions of potential deaths never findings hint at a much more modest outcome [8, 4] . especially the case fatality rate (cfr) and the number of unobserved infections are crucial to judge the state of the pandemic as well as the effectiveness of its mitigation. yet, there estimates are plagued with high uncertainties as exemplified in the quick revisions even from the same institution [3, 4] most studies are based on elaborate epidemic modeling either using stochastic or deterministic transmission dynamics. especially, the susceptible-infectedrecovered (sir) model [10] forms a basic building block and has been extended in several directions in order to understand the dynamics of the ongoing covid-19 pandemic [9, 2, 7, 13] . in this context, it has not only been compared with more phenomenological growth models [12] , e.g. logistic growth, but also been used to quantify the effectiveness of quarantine and social distancing [9, 2] . e.g. social distancing, can be easily included by replacing the infection rate parameter with a function allowing it to change over time. [2] assumes one or several (soft) step functions where the infection rate drops in response to different measures after these had been implemented. such detailed modeling is required in order to capture and forecast temporal dynamics of the epidemic spreading. yet, substantial care is needed as to which parameters can be learned from the data and which cannot. indeed, i show here that sir type models -and others exhibiting similarly flexible growth dynamics -are non-identified with respect to the cfr and the fraction of observed infections. instead, a direct visual exploration of the data leads to valuable insights in this regard. in particular, much of the variability relating reported case and death counts can be explained by two easily interpreted parameters. furthermore, based on three simple assumptions a lower bound on the number of actual infections, including observed and unobserved cases, can be obtained. in turn, confirming recent estimates without the need of complex and maybe questionable modeling choices. covid-19 data are published by several sources, most notably the john hopkins university and the european center for decease prevention and control (ecdc). here, data from ecdc as available from https://opendata.ecdc. europa.eu/covid19/casedistribution/csv are used. figure 1 shows the total cumulative case and death counts of selected countries. these countries are among the eight most effected countries in terms of absolute and relative deaths 1 . in the following, i will focus on relative counts as these are arguably more meaningful when comparing different countries -which could differ widely in terms of population size. assumption 1. death counts are more reliable than case counts. by assumption 1 analysis will start from relative cumulative death counts d t in the following 2 . furthermore, in order to facilitate country comparisons, dates are shifted relative to the first day that relative death counts exceed a threshold θ of 1, 2, 4 or 8 deaths per million inhabitants respectively, i.e. t = 0 is defined such that d t ≥ θ for t ≥ 0 and d t < θ for t < 0. figure 2 shows the resulting time course of relative case and death counts. aligning dates in this fashion shows that several countries exhibit similar time courses, e.g. belgium and spain or china and south korea. as shown in the supplementary figure s1 the remaining country specific differences can be explained by differences in growth rates. re-scaling time according to the estimated doubling time indeed leads to a data collapse as complete as often observed in physical systems exhibiting scaling laws [11] . here, these differences in the precise temporal dynamics of epidemic growth are not required. instead, the relation between relative death and case counts is considered. while relative death counts exhibit similar time courses the corresponding relative case counts c t are more variable when aligned in the same fashion, i.e. relative to the first day that d t exceeds a given threshold. as i will argue now, most of this variability can be explained with two readily interpretable parameters. there is a well defined country specific delay between reported cases and deaths. relative days since one death per mill. estimated cfrτ for varying delays τ ita figure 3 : estimated cfr cfr τ for germany (left) and italy (right) using different delays of τ = 0, . . . , 11 days. note that in each case, there exists a characteristic delay such that estimates are almost constant over time. further note that estimates for all delays will eventually converge to the same final value when enough data are available. figure 2 suggests that relative case counts are not aligned as some countries, e.g. germany, systematically lead the counts reported in other countries, e.g. italy. such a difference could mean that individuals survive longer, e.g. due to differences in medical care, until they eventually. it could also just reflect reporting delays due to bureaucratic reasons. in any case, it is clearly the case that individuals die not immediately, but some days after they had been tested positive previously. this delay also needs to be taken into account when estimating the case fatality rate (cfr). commonly the cfr is defined as cfr = dt ct . not surprisingly this estimate is highly variable and changes systematically over time, especially at the beginning of an epidemic. the observation captured in assumption 2 also explains the surprisingly low cfrs initially announced in austria and germany where reported death counts are simply some days older compared to other countries! thus, taking into account that individuals that had been tested positive will usually not die on the same day but after some delay τ (if at all), i define i.e. comparing current death with previous case counts. figure 3 shows the cfrs estimated for germany and italy in this fashion, i.e. for different delays τ . the estimate using τ = 0 rises over time simply reflecting that due to the reporting delay death counts have not yet caught up with the exponentially growing case counts. interestingly, for each country there exists a characteristic delay at which the estimated cfrs are essentially constant. thus, reflecting the hypothesized delay between reported cases and deaths. this delay can either be estimated by visual inspection or by fitting a linear model on each delay and picking the one with minimal absolute slope 3 . figure 3 shows the delays τ and corresponding cfrs cfr τ , i.e. the median cfr value at this delay, estimated for each country in this fashion. in order to fully relate the observed case with death counts an additional, and stronger, assumption is needed. assumption 3. the true case fatality rate is the same for all countries. while assumption 3 ignores medical, demographic and other differences between countries, i believe it unlikely that the cfr is very different across different countries. in the end, its the same type of virus spreading in all countries. this suggests that differences in estimated cfrs simply reflect differences in the ability of countries to actually observe all infected individuals, i.e. due to more or less effective tracking and testing procedures. to illustrate this effect, a true cfr of 1% is assumed in the following. this is consistent with current knowledge and had also been used in other studies [4] . just from the estimated values any cfr below the minimum of all estimates (about 2% found for austria and south korea) and above 0.1% (which would imply an observed fraction above one for belgium) is compatible with the data. figure 4 shows the country specific estimates of reporting delay, cfr and fraction of observed cases (assuming a true cfr of 1%) obtained in this fashion. in turn, figure 5 shows the implied relative case counts when shifted by the estimated delays and scaled to reflect the unobserved fraction of cases for each country. notably, these implied counts all align nearly as good as the death counts in figure 2 (right panel) even though the initial threshold was based on the deaths counts alone. the supplementary figure s2 shows that this holds also when re-scaling time according to the growth rate of deaths. overall, the collapse of implied case dynamics convincingly illustrates that the relation between case and death counts is fully and reliably captured by two parameters -compatible with three reasonable assumptions. in reality, an additional delay between an infection and its corresponding positive test result can be assumed. therefore, the fraction of observed cases will be even lower than obtained by the analysis above. unfortunately, assuming a sufficiently flexible model for the growth of the actual cases already the cfr and the fraction of observed cases, let alone an additional delay, are not jointly identifiable. the basic sir model [10] , assumes that an infection unfolds when susceptible (s) individuals become infected (i) -which in turn infect further susceptible individuals. finally, infected individuals recover (r) (or die) and are no longer susceptible. in continuous time, the dynamics can be described by the following system of ordinary differential equations (odes): where n ≡ s t + i t + r t is constant over time. model parameters are • the infection rate β • and the recovery rate γ. in this model, the average time of infection is γ −1 giving rise to a basic reproduction number of r 0 = βγ −1 . sir models and extensions are widely used in epidemic modeling. the have also been applied to the understand the dynamics of the ongoing covid-19 pandemic [9, 2, 7, 13] . in particular, models including the possibility of unobserved cases or including a reporting delay have been developed. within the sir framework, both effects can be included in several ways, most easily by assuming that observed cumulative infections are simply a fraction α ∈ [0, 1] of previous total infections i t + r t , i.e. α(i t−τ + r t−τ ). a more elaborate attempt instead considers more detailed dynamics of the form where a fraction α of infected individuals i t is observed (o t ) after an initial delay 1 γ i . in any case, whether observed or not, individuals recover (or die) after an additional delay. in general, the infection rates β i , β o , β u could be different for initial infections and observed vs unobserved cases 4 . in addition, mitigation measures, e.g. social distancing, can be easily included by assuming that β's are functions of time. e.g. αγ i i t . now assume a second model with α = 1 > α which nevertheless exhibits the same dynamics with an additional time shift τ . by using a time varying β (t) such that we obtain exactly the same number of observed cases, i.e. o t−τ = o t . note that as α > α, we have that s t < s t−τ and s t is a sigmoidal function of time due to the sir dynamics. furthermore, when the population is large, i.e. n 1 and s 0 ≈ n the resulting β (t) is mostly driven by the drop in s t+τ as compared to the much smaller change in s t . indeed, figure 6 shows the dynamics of the above model with β = 0.3, γ i = γ r = 2 10 5 , α = 0.1 starting from (n = 10 8 , 1, 0, 0, 0). in turn, assuming α = 1 and τ = 5, the time varying infectivity β (t) is approximated by the best-fitting logistic sigmoid of the form β 1 + (β 2 − β 1 )σ( t−τ t ). note that the number of observed cases is identical, just shifted by τ , whereas the final fraction of susceptible individuals is vastly different. indeed, in the first case the epidemic is stopped by group immunity whereas in the second case effective mitigation measures are imposed. correspondingly, police implications would be vastly different in the two situations even though they are observationally indistinguishable. instead of detailed modeling of epidemic dynamics, which is further complicated due policy actions requiring flexible models with delicately chosen parameters, the present analysis is based on visual inspection of the reported data. overall, relative case and deaths counts (observed for country c) seem to be related as follows: where a c r denotes the actual infections a fraction α c ∈ [0, 1] is observed. a suitable reporting delay τ c can be estimated by visual inspection of the data, but again the fraction of observed cases α c and cfr cfr are not jointly identifiable if there exist sets of parameters such that a t−τ = αa t , as is the case for dynamic sir type models. in the end, any epidemic modeling implicitly or explicitly chooses a parametric form for the latent growth process a t and will not be identified if sufficiently flexible. yet, assumption three of a constant cfr across all countries allows to derive 1. a range of values consistent among all countries, 2. as well as recover the corresponding fraction of observed cases in each country. thereby, assuming a reasonable true cfr value, i.e. from the model implied range 0.1% to 2% which is also consistent with current knowledge, and using the estimated delay, the actual case numbers can be reconstructed. figure 7 shows the resulting actual relative infection counts across several countries. note that despite the simplicity of this analysis, the estimated numbers compare favorable [4] . indeed, i would rather trust these even more as they do not rely on complex modeling assumptions but follow from visual inspection of the data. overall, i have illustrated that much of the variability between observed case and deaths counts between different countries can be explained by two parameters. namely, the reporting delay τ and the fraction of observed cases. especially the reporting delay exhibits crucial differences between countries and needs to be taken into account when comparing data and planning actions. in particular, containment is challenging when long incubation times are involved [1] but a combination of case tracing and isolation policies could be effective [5, 6] . thus, detailed epidemic modeling is certainly needed in order to judge the effectiveness of current mitigation measures across different countries [4, 2] . on the other hand, important parameters need to fixed based on additional knowledge as they cannot be identified within sufficiently flexible models. in the end, data analysis and detailed modeling alone only gets us only that far and more extensive testing is urgently needed to obtain reliable knowledge about the current progression of the covid-19 pandemic. figure s1 : aligned data as in figure 2 , but time is additionally re-scaled to match local growth rate of the epidemics. a data collapse by re-scaling time aligning the data as in figure 2 still shows country-specific differences in the temporal course of epidemic spreading. much of this difference can be attributed to the speed at which the epidemic spreads in different countries. estimating the local growth rate of deaths d log dt dt by the three day running average of observed changes log d t+1 − log d t , relative time, i.e. relative to the threshold of total deaths reached, is re-scaled to match local growth rates. figure s1 shows the resulting data collapse for d t and the corresponding c t dynamics. further, taking the estimated relation between cases and deaths via cfr and country specific delays into account an almost complete data collapse for the cases is obtained. not that as in the main text, data are aligned according to relative death counts only. furthermore, the temporal re-scaling is based on the estimated growth rate from the death counts as well. yet, shifting and scaling case data according to the estimated country specific delay and fraction of observed cases leads to an almost complete data collapse as well. as individual countries can be hard to identify in figures 2 and 5 , the ny times featured panel views where each country is highlighted above a background of all countries. here, i provide similar figures for relative death and case counts using a threshold of two deaths per million inhabitants. note that an sir model already includes a natural delay between infections and recovery (or death). indeed, the total number of cases is given by c t = i t + r t while the cumulative death toll is obtained as cfrr t , i.e. modeling that a fraction of individuals does not recover but dies instead. assuming that only a fraction α of cases is observed, the model is estimated with the following relative days since two death per mill. relative count figure s4 : details of aligned and adjusted case counts for threshold of two deaths per million. sampling distribution thus, observed daily changes are related to the model implement changes via an over-dispersed poisson aka negative binomial distribution. figure s5 shows the resulting estimates assuming β t = β 1 + (β 2 − β 1 )σ( t−τ t ) and cfr = 1% 6 . the sir model assuming a single change point in the infectivity, via the logistic sigmoid sigma(·) in β t reflecting the implementation of social distancing is clearly able to capture the epidemic dynamics. yet, parameter uncertainties, especially about the reporting delay can be large 7 . bayesian estimates have been carried out using stan (full code available from my https://github.com/bertschi/covid repository) and using weakly informative broad normal or student-t prior distributions on all parameters. 6 due to the non-identifiability derived in the main text either α or cfr needs to be fixed. 7 the high uncertainty could also reflect that an sir dynamics is misspecified in that it corresponds to an exponential delay distribution. such additional model assumptions need to be carefully chosen in order to obtain meaningful parameter estimates. figure s5 : model predictions and estimated parameters from sir model fitted to data from italy (top) and germany (bottom). mitigation and herd immunity strategy for covid-19 is likely to fail. medrxiv inferring covid-19 spreading rates and potential change points for case number forecasts impact of nonpharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand estimating the number of infections and the impact of non factors that make an infectious disease outbreak controllable a retrospective bayesian model for measuring covariate effects on observed covid-19 test and case counts substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the sars-cov-2 epidemic. medrxiv effective containment explains subexponential growth in confirmed cases of recent covid-19 outbreak in mainland china scaling, universality, and renormalization: three pillars of modern critical phenomena rational evaluation of various epidemic models based on the covid-19 data of china. medrxiv modeling the epidemic dynamics and control of covid-19 outbreak in china. medrxiv key: cord-020544-kc52thr8 authors: bradt, david a.; drummond, christina m. title: technical annexes date: 2019-12-03 journal: pocket field guide for disaster health professionals doi: 10.1007/978-3-030-04801-3_7 sha: doc_id: 20544 cord_uid: kc52thr8 7.1 humanitarian programs 141; 7.2 security sector 153; 7.3 health sector 158: core disciplines in disaster health 161. primary health care programs 162. disease prevention 162. clinical facilities 164. reproductive health 165. water and sanitation 166. food and nutrition 171. chemical weapons 181. epi methods 184; 7.4 tropical medicine 187: tropical infectious diseases—vector-borne and zoonotic 196. tropical infectious diseases—non-vector-borne 215; 7.5 epidemic preparedness and response 239; 7.6 communicable disease control 242: diarrhea 244. influenza 257. malaria 263. measles 267. meningitis 269. viral hemorrhagic fever 272; 7.7 diagnostic laboratory 275: indications, laboratory tests, and expected availability 276. specimen handling 278; 7.8 acronyms 282; this section provides guidance on technical issues in the health sector. the annexes contain compilations of frequently used reference information. • humanitarian programs-contains conceptual frameworks on global clusters, relief programs, humanitarian financing, and early recovery. • security sector-contains key definitions from the rome statute of the international criminal court • health sector-contains a broad range of core health technical information including environmental classification of water and excreta-related diseases, disease prevention measures, water treatment end points, anthropometric classifications, micronutrient deficiency states, management of chemical weapon exposures, and epi methods. • tropical medicine-contains clinical summaries of tropical infectious diseases with details on disease vector and host, clinical presentation, diagnostic lab tests, clinical epidemiology, and therapy. • epidemic preparedness and response-contains core principles of epidemic preparedness and response. • communicable disease control-contains an overview of selected communicable diseases of epidemic potential including diarrhea, influenza, malaria, measles, meningitis, and viral hemorrhagic fever. • diagnostic laboratory-contains guidance on lab specimen handling and testing. • acronyms-contains acronyms commonly used in disaster management and humanitarian assistance. a. in-kind donations (eg food, seeds, tools, fishing nets, etc) b. types of community projects in food-for-assets programs (1) natural resources development (a) water harvesting (b) soil conservation (2) restoration of agri(aqua)culture potential (a) irrigation systems (b) seed systems (3) infrastructure rehabilitation (a) schools (b) market places (c) community granaries (d) warehouses (e) roads (f) bridges (4) diversification of livelihoods (a) training and experience sharing 2. increase individual purchasing power a. cash distribution b. cash for work (cash for assets) c. vouchers d. micro-credit e. job fairs f . artisanal production g. livelihoods/income generation 3. support market resumption a. market rehabilitation b. infrastructure rehabilitation c. micro-finance institutions goals-protect what's left (1 month), restore the system (6 months), improve the system ( 1. promote transformational development support far-reaching, fundamental changes in relatively stable developing countries, with emphasis on improvements in governance and institutions, human capacity, and economic structure, so that countries can sustain further economic and social progress without depending on foreign aid. focus on those countries with significant need for assistance and with adequate (or better) commitment to ruling justly, promoting economic freedom, and investing in people. reduce fragility and establish the foundation for development progress by supporting stabilization, reform, and capacity development in fragile states when and where u.s. assistance can make a significant difference. 3. support strategic states help achieve major u.s. foreign policy goals in specific countries of high priority from a strategic standpoint. 1. international cooperation to protect lives and health 2. timely and sustained high-level political leadership to the disease 3. transparency in reporting of cases of disease in humans and in animals caused by strains that have pandemic potential to increase understanding, enhance preparedness, and ensure rapid and timely response to potential outbreaks 4. immediate sharing of epidemiological data and clinical samples with the world health organization (who) and the international community to characterize the nature and evolution of any outbreaks as quickly as possible 5. prevention and containment of an incipient epidemic through capacity building and in-country collaboration with international partners 6. rapid response to the first signs of accelerated disease transmission 7. work in a manner supportive of key multilateral organizations (who, fao, oie) 8. timely coordination of bilateral and multilateral resource allocations; dedication of domestic resources (human and financial); improvements in public awareness; and development of economic and trade contingency plans 9. increased coordination and harmonization of preparedness, prevention, response and containment activities among nations 10. actions based on the best available science 1. genocide (article 6)-acts committed with intent to destroy, in whole or in part, a national, ethnic, racial, or religious group a. killing members of the group b. causing serious bodily or mental harm to members of the group c. inflicting on the group conditions of life calculated to bring about its physical destruction in whole or in part d. imposing measures intended to prevent births within the group e. forcibly transferring children of the group to another group 2. crimes against humanity (article 7)-acts committed as part of a widespread or systematic attack against any civilian population, with knowledge of the attack a. murder b. extermination c. enslavement d. deportation e. imprisonment in violation of international law f. torture g. rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence h. persecution on political, racial, national, ethnic, cultural, religious, gender, or other grounds universally recognized as impermissible under international law i. enforced disappearance j. apartheid k. other inhumane acts intentionally causing great suffering or serious injury to body or to mental or physical health 3. war crimes (article 8) a. grave breaches of the geneva conventions of 12 aug 1949 (1) willful killing (2) torture or inhumane treatment including biological experiments (3) willfully causing great suffering (4) extensive destruction and appropriation of property (5) compelling a pow to serve in the armed forces of a hostile power (6) willfully depriving a pow of the right to a fair trial (7) unlawful deportation (8) taking of hostages b. serious violations of laws and customs applicable in international armed conflict (1) intentionally directing attacks against the civilian population or against civilians not taking direct part in hostilities (2) intentionally directing attacks against civilian objects (3) intentionally directing attacks against personnel, installations, material, units, or vehicles involved in humanitarian assistance or peacekeeping mission (4) intentionally launching an attack in the knowledge that it will cause incidental civilian loss of life or severe damage to the natural environment (5) attacking undefended towns, villages, dwellings, or buildings which are not military targets (6) killing or wounding a combatant who has surrendered (7) improper use of a flag of truce, flag or insignia or uniform of the enemy or of the un, or emblems of the geneva conventions resulting in death or serious personal injury (8) transfer by the occupying power of parts of its own civilian population into the territory it occupies, or the deportation or transfer of all or parts of the population of the occupied territory within or outside the territory (9) intentionally directing attacks against buildings dedicated to religion, education, art, science, charitable purposes, historic monuments, hospitals, and places where sick are collected, provided they are not military objectives (10) subjecting persons to physical mutilation or to medical or scientific experiments which are not justified by the medical treatment nor carried out in his/her interest (11) killing or wounding treacherously individuals belonging to the hostile nation or army (12) declaring that no quarter will be given (13) destroying or seizing the enemy's property unless such be imperatively demanded by the necessities of war (14) declaring abolished, suspended, or inadmissible in a court of law the rights and actions of the nationals of the hostile party (15) compelling the nationals of the hostile party to take part in the operations of war directed against their own country (16) pillaging a town or place, even when taken by assault (17) a range of generic prevention measures should be considered for its impact on diseases in a biological "all-hazards" environment. overall, excreta disposal, water quantity, personal hygiene, and food hygiene commonly contribute more to environmental health than do other listed measures. epidemic threats will oblige heightened consideration of disease-specific strategies for prevention and control. c. water treatment (bold text of particular relevance in clinical facilities) 1 ppm = 1 mg/kg (solids) = 1 mg/l (liquids) = 1 ug/ml (liquids) = basic unit of measure for chloroscopes :10,000 ppm = 1% • sam = whz < −3, muac <11.5 cm, or bilateral pitting edema (who). whm not in definition. • sam prevalence worldwide ≈ 20,000,000. • sam mortality ≈ 9x mortality of normally nourished child and its cfr can be 10-50%. • gam = mam + sam • gam = moderate wasting cases, severe wasting cases, or bilateral pitting edema cases (where due to malnutrition) • underweight is not used for screening or surveys in nutritional emergencies. it reflects past (chronic) and present (acute) undernutrition and is unable to distinguish between them. it encompasses children who are wasted and/or stunted. however, weight gain over time can be a sensitive indicator of growth faltering which is easily tracked on road to health charts. • stunting generally occurs before age 2. it is irreversible. • stunting prevalence worldwide ≈ 165,000,000. • stunting is not a good predictor of mortality, but the cfr from ids in cases of severe stunting ≈ 3x the cfr from ids in cases without stunting. reference standards can be absolute muac, centile, % of median reference, or z scores: • muac easy to understand. an excellent predictor of mortality. permits comparisons between age groups insofar as the low growth velocity of muac in the u5 age group makes data roughly comparable. may be used alone in "quick-and-dirty" convenience samples to estimate local prevalence of wasting. however, not used alone in authoritative anthropometric surveys, and is commonly part of a two stage screening process to determine eligibility for feeding programs. • overall whz gives higher prevalence of malnutrition than whm for the same population. this is most marked where there is low baseline prevalence of disease, and especially for adolescents (who get subsequently over-referred). whz is more statistically valid, but whm is better predictor of mortality and is used for admission to tfcs. weight-for-age is influenced by weight-for-height and height-for-age. it can be difficult to interpret. b. adults and adolescents (o10) anthropometrics: bmi = weight (kg) / height (m) 2 1. death rates-calculated incidence of death expressed per 10,000 p/d or per 1000 p/mo; data collected by retrospective surveys (eg 3 month period) to gauge severity of public health emergency particularly where sudden events lead to spike in mortality a. cdr-crude death rate b. asdr-age-specific death rate (eg u5dr or death rate of children 0-5 yr) during a studied time interval (written as 2. mortality rates-calculated probability of dying before a specified age expressed per 1000 live births; data collected by national health authorities in periodic (annual) demographic surveys to reflect ongoing health status a. cmr-calculated probability of mortality in given population for specific time b. imr-calculated probability of a live borne child dying before 1 yr c. u5mr-calculated probability of a live borne child dying before 5 yr nb mr ≠ dr. eg cmr ≠ cdr, u5mr ≠ u5dr. different rates measure different things and are not directly comparable. however, mrs may be converted into drs by the following: cdr or u5dr (deaths/10,000/d) = − ln(1−p/1000) × 5.47 where p = cmr or u5mr (deaths/1000 live births). however, this has little field utility. nb mmr-maternal mortality ratio has different units in numerators (maternal deaths) and denominators (live births), thus is a ratio, not a rate the application of study findings to an entire population from which the sample was drawn. if the survey was well-conducted, the results may be considered representative of the entire population. this is scientifically justified. however a confidence interval should accompany any parameter estimate of that population. extrapolation the extension of study findings to a population or period which was not represented in the sample. it works by association-if 2 populations appear to be experiencing similar conditions, the morbidity/mortality experience of one may be imputed to the other. this is not scientifically justified, but is often done where data are insufficient or impossible to collect. 6 s/sx think differential diagnosis (below). 2. severe muscle pain may be a symptom of sepsis even without fever. 3. elderly patients with sepsis may be afebrile. in elderly patients, fever is rarely caused by a viral infection. 4 . septic patients who are hypothermic have a worse prognosis than those with high fever. treat as a medical emergency. 5. fever in a postoperative patient is usually related to the surgical procedure (eg pneumonia, uti, wound, or deep infection). 6 . fever with jaundice is rarely due to viral hepatitis. think liver abscess, cholangitis, etc. 7. the rash of early meningococcal infection may resemble a viral rash. 8. generalized rashes involving the palms and soles may be due to drugs, viral infections, rickettsial infections, or syphilis. 9. all febrile travelers in or returned from a malaria infected area must have malaria excluded. 10. disseminated tb must be suspected in all elderly patients with fever and multisystem disease who have been in an area with endemic tb. 11. septic arthritis may be present even in a joint which is mobile. 12. back pain with fever may be caused by vertebral osteomyelitis or an epidural abscess. 13. a patient may have more than one infection requiring treatment (eg malaria and typhoid), especially if they are elderly, immunosuppressed, or have travelled. 14. always remember common infections, not just opportunistic infections, in aids patients with a fever. understand morbidity multipliers-measles, malnutrition, and tb/hiv. understand occult co-morbidities. for any undifferentiated illness, even in infants, think of hiv, tb, syphilis, and sarcoid. for any child, think of malaria, hookworm, and anemia. malarial anemia usually in pedes <3 year-old; hookworm anemia usually in pedes >3 year-old. for any icp, think of tb, vl, histoplasmosis, and strongyloides. must treat early. watch for clinical mimics-malaria presenting as pneumonia or diarrhea in pedes; vl presenting as malaria in adults; lepto presenting as mild df (esp in df endemic areas where the pt has mild onset of illness, worsening course, and no rash but jaundice). tx do basic things well, use equipment you understand, teach others, delegate. this annex profiles selected communicable diseases of epidemic potential whose incidence, management complexity, or mortality obliges particular attention. • if (+) agglutination to o1 antisera, then the strain is further tested for agglutination to antiserum of ogawa and inaba serotypes. • if (+) agglutination to o139 antisera, then the strain is not further subdivided (except as producer or non-producer of ct as noted below). • if (−) agglutination to o1 and o139 antisera, then the strain is known as non-o1, non-o139 v. cholerae. a strain is further identified as a producer or non-producer of cholera toxin (ct). ct production is a major determinant of disease development. strains lacking ct do not produce epidemics even if from the o1 or o139 serogroup. • serogroup o1 exists as 2 main biotypes-classical and el tor-though hybrids also exist. each biotype occurs as two serotypes-ogawa and inaba. classic biotype caused the 5th and 6th pandemics but little epidemic disease since the 1970s though it still causes cases in india. el tor biotype caused the 7th (current) pandemic and almost all recent outbreaks. el tor was first isolated in 1905 in el tor, egypt after importation by indonesian pilgrims travelling to mecca. it survives longer in the environment and produces ct similar to the classical biotype. presumably because of ct pathogenicity, the % of cholera patients with severe disease has doubled over the past 10 yrs. these patients tend to require iv fluid therapy. • serogroup o139 may have evolved from strains of o1 el tor as they share many properties though not agglutination. in spring of 2002 in dhaka, o139 cases exceeded o1 el tor cases for the first time, and it was postulated that o139 may become the cause of an 8th pandemic. however, since then, o1 has again become dominant. infective dose depends on individual susceptibility. relevant host factors include immunity produced by prior infection with serogroup o1 as well as stomach acidity. id 50 may be 100,000 orgs, so personal hygiene plays a lesser role than in shigellosis where the id 50 is much lower. shigella has 4 species. • s. dysenteriae type 1 (sd1 or shiga bacillus) causes the severest disease of all shigella sp because of its neurotoxin (shiga toxin), longer duration of illness, higher abx resistance, higher cfr thru invasive complications, and great epidemic potential. • s. flexneri is the most common, and is generally endemic, in developing countries • s. sonnei is the most common in industrial countries • s. boydii and s. sonnei give mild disease. some kinds of e. coli produce a shiga toxin. shiga toxin genes reside in a bacteriophage genome integrated into the bacterial chromosome. some abx, eg fluoroquinolones, induce expression of phage genes. the bacteria that make these toxins are variously called "shiga toxin-producing e. coli" (stec), "enterohemorrhagic e. coli" (ehec), or "verocytotoxic e. coli" (vtec). all terms refer to the same group of bacteria. • e. coli o157:h7 (often called "e. coli o157" or "o157") is the most commonly identified stec in north america, and it causes most e. coli outbreaks. approximately 5-10% of ehec infections result in hus. • non-o157 stec serogroups also cause disease. in the usa, serogroups o26, o111, and o103 are the most commonly identified e. coli pathogens overall. weather (esp weeks 15-20 in apr-may) creating increased biological activity; post-monsoon (esp weeks 30-40 in aug-sep) with contamination of water sources. pre-monsoon epidemics are generally worse than postmonsoon ones. dysentery has low level year-round incidence, but epidemics occur roughly each decade. epidemic strains display new, additive antibiotic resistance which probably triggers the epidemic. once resistant strains have become endemic, antibiotic susceptibility rarely reappears. sd1 acquires resistance quickly. sf acquires it more slowly, and that resistance may wane with decreasing abx pressure. at icddr, annual proportional incidence approximates the following: clean water and waste management especially for cholera. personal hygiene (hand washing with soap and clean towels) especially for shigella. water safe drinking water (boiled, chlorinated) nb sphere standards are not enough-you need increased quantities of chlorinated water at household level. san clean latrines for safe disposal of excreta hand washing with soap food safe food (cooked, stored) breast feeding fomites safe disposal of dead bodies with disinfection of clothing nb after outbreak of a fecal-oral pathogen, food hygiene and funereal practices may influence human-to-human transmission more than water quality. health education to affected population wash hands with soap: after using toilets/latrines. after disposing of children's feces. before preparing food. before eating. before feeding children. dukoral has been the main vaccine considered for use in high-risk populations. • morc-vax and shanchol-similar to dukoral except they do not contain the rbs, hence do not require a buffer, and are 1/3 the cost to produce. morc-vax, produced in vietnam, is derived from a vaccine administered to millions of people since 1997, but is not who pre-qualified, and is not expected to have international distribution. • shanchol, produced in india, has international distribution (eg used in the haiti cholera vaccination campaign of 2012), and is now the agent of choice for who. it confers immunity 10d p 2nd dose, effectiveness > 85% at 6 mo, and protection >50% at 5 yr. also confers short-term protection vs etec. dose: 1.5 cc vaccine followed by water ingestion but no fasting needed; 2 doses, 2 wks apart; cold chain required except for day of use. • orochol-bivalent formulation as in dukoral without rbs of ct. dose: single dose. no longer manufactured. who recommendations: "vaccination should not disrupt the provision of other high-priority health interventions to control or prevent cholera outbreaks. vaccines provide a short-term effect that can be implemented to bring about an immediate response while the longer term interventions of improving water and sanitation, which involve large investments, are put into place." [1] icddr recommendations: "because of limitations in terms of transport, formulation, and cost of the current dukoral vaccine, the cots program does not require the utilization of the vaccine during an outbreak; it is not necessary to vaccinate to overcome an outbreak. however, if dukoral is readily available and staff are properly trained in its use according to the guidelines that come with the vaccine, the cots program permits dukoral's use (ideally before an outbreak) in the following high-risk populations: refugee populations in which cholera is present, health care workers managing cholera cases, and communities in which the incidence rate is greater than 1 in 1000 annually." [2] epidemiological surveillance (specific to cholera) epidemiological assumptions (who, cots): estimated attack rates: 10-20% extremely vulnerable hosts and poor environmental health (who) 5% (refugee camps with malnutrition) (cots) 2% (rural communities of <5000 p) (cots) 1% (severe epidemic-good estimate of ultimate disease burden) (who) 0.6% (endemic areas with bad sanitation) (cots) 0.2% (endemic areas in open settings-suitable for initial calculations of early resource requirements) nb overall, 90% of cases are mild and difficult to distinguish from other types of d. nb asymptomatic carriers are very common (10x # of cases). referral rates for ivs 20% of cases (much higher-70% at icddr as it shortens recovery time) case fatality ratios 1% (with good care) the following catchment populations will yield 100 acute pts of whom 20 will be severely dehydrated: refugee camp of 2000 people (ar of 5% = 100 pts) open settings in endemic area with 50,000 people (ar 0.2% = 100 pts) a population of 100,000 infected individuals in an epidemic area will yield the following (who): population infected 100,000 clinical cases 1,000 (1% of infected population) cases needing early resources 200 (20% of cases) cases needing iv therapy 200 (20% of cases) anticipated deaths 10 (1% cfr) nb in non-endemic areas, ar adults > ar pedes because adults have higher exposure risks. in endemic areas, ar pedes > ar adults because adults have been exposed since childhood delivery of health services shigella are fragile and difficult to recover if transport time > 1 d. 5-10 isolates initially to confirm outbreak 30-50 isolates initially to create abx use policy (bacterial resistance renders cotrimoxazole, amp/amox, nalidixic acid, and tetracycline unusable) 20-30 isolates monthly from ipd and opd before abx therapy to assess evolving abx resistance 10-20 isolates periodically to reference laboratory to confirm abx resistance patterns and undertake molecular studies 20 isolates at end of the outbreak to confirm that new diarrheas are not epidemic pathogens nb systematic sampling is most representative-eg every 10th pt or all pts q 2 weeks adjusted as needed to collect the necessary specs. sensitivity > > important than specificity in rdt screening during an epidemic. pts from one geographic area are more likely to constitute a cluster involving a new pathogen. an area may be considered cholera-free after 2 incubation periods (total of 10 d) have passed without cholera disease. however, hospital monitoring should continue for a year due to tendency of enteric pathogens to re-emerge long after they are declared gone. cholera may be viable but nonculturable from the environment; environmental monitoring has many false negatives. consider improvements to existing diagnostic labs • hotline set up for reporting of rumor this often translates into a hastily conceived vaccination campaign that distracts from core principles of cholera management. for every symptomatic pt, there may be 90 asymptomatic carriers. in an established epidemic, the affected community is already extensively infected. cholera vaccination, under these circumstances, has little public health benefit for the resource investment. if undertaken, the following will apply: • vaccination campaign requires numerous staff. community mobilizers are key. clinical staff should not be poached from their clinical duties. supervisors must be free to move at will. • logistics is key-if the 1st day goes badly, the campaign goes badly. • mark the domiciles which are done. • hold after-action meetings each day. • last day, use mobilizers with mobile broadcasting to attract those who missed out. • second phase vaccination should include chws with multi-purpose messages on water and sanitation. avoid: press exaggeration abx prophylaxis reliance on ivf and insufficient ors lab investigation of cases once epidemic etiology is ascertained prolonged hospitalization hospital discharge criteria requiring multiple negative stool cultures enthusiasm for ocv during epidemic exaggerated water purification objectives concentration of technical competencies in moh at expense of districts failure to share information with stakeholders influenza viruses comprise 3 genera-influenza types a, b, and c-each with 1 species. • influenza type a is divided into subtypes based upon serological response to hemagglutinin (ha) and neuraminidase (na) glycoproteins. there are 16 different ha subtypes and 9 different na subtypes. h1n1, h2n2, and h3n2 are responsible for the major human pandemics in the last century. h2n2 virus circulated between 1957 and 1968 but currently does not. only influenza a subtypes infect birds, and all subtypes can do so. bird flu viruses do not usually infect humans. but, in 1997, an outbreak of h5n1 avian influenza in poultry in hong kong marked the first known direct human transmission of avian influenza virus from birds to humans. since then, h5, h7, and h9 avian influenza subtypes have been shown to infect humans. • influenza type b is morphologically similar to a and also creates seasonal and epidemic disease. • influenza type c is rare but can cause local epidemics. seasonal human influenza vaccine currently has 3 strains-h1n1/h3n2/b. influenza disease in humans has a short incubation period (1-3 d). early symptoms are non-specific. it is highly infectious, especially early in the course of the disease, with a large # of asymptomatic carriers. transmission potential (r 0 ) is a function of infectivity, period of contagiousness, daily contact rate, and host immunity. in general, the faster the transmission, the less feasible is interrupting transmission thru usual disease control tools of case finding, isolation, contact tracing, and ring vaccination. • specific groups of exposed or at risk in the community-most likely to work when there is limited disease transmission in the area, most cases can be traced to a specific contact or setting, and intervention is considered likely to slow the spread of disease eg quarantine of groups of people at known common source exposure (airplane, school, workplace, hospital, public gathering; ensure delivery of medical care, food, and social services to persons in quarantine with special attention to vulnerable groups) (useless once there is community-based spread) eg containment measures at specific sites or buildings of disease exposure (focused measures to > social distance) cancel public events (concerts, sports, movies) close buildings (recreational facilities, youth clubs) restrict access to certain sites or buildings • community-wide measures (affecting exposed and non-exposed)most likely to work where there is moderate to extensive disease transmission in the area, many cases cannot be traced, cases are increasing, and there is delay between sx onset and case isolation. infection control measures ari etiquette-cover nose/mouth during cough or sneeze, use tissues, wash hands avoidance of public gatherings by persons at high risk of complications nb use of masks by well persons is not recommended "snow" (stay-at-home) days and self-shielding (reverse quarantine) for initial 10 d period of community outbreak-may reduce transmission without explicit activity restrictions closure of schools, offices, large group gatherings, public transport (pedes more likely to transmit disease than adults) nb community quarantine (cordon sanitaire)-restriction of travel in and out of an area is unlikely to prevent introduction or spread of disease anopheles vector biology egg becomes adult mosquito in 9 d adult mosquito becomes infective in 12 d after bite on infected host susceptible human host becomes infective in 9 d after bite from infected mosquito :. earliest human clinical disease in 30 d after eggs are laid follow the 4-d rule: dusk and dawn stay indoors as much as possible with window screens in good repair dress in light colored long sleeve shirts and long pants when outside identify cause of the outbreak undertake vaccination campaign strengthen routine immunization and surveillance meningitis is a disease with significant mortality. meningococcus (neisseria meningitides) is renown for its rapid onset, rapid progression (death sometimes within hours), and high mortality (50% untreated). there are 13 serogroups of neisseria meningitides but only 6 (a, b, c, w, x, y) are known to cause epidemics. the bacteria spread from person to person via respiratory and nasal secretions. polysaccharide vaccines are available with 2 serotypes (a and c), 3 serotypes (a, c, and w) or 4 serotypes (a, c, w, and y). duration of immunity is approximately 3 years. meningococcal protein conjugate vaccines confer longer immunity but at higher cost than polysaccharide vaccines. monovalent conjugate vaccine against group c dates from 1999, and tetravalent (a, c, w, and y) conjugate vaccine dates from 2005. group b vaccine made from 4 bacterial proteins has been licensed since 2014 but is not readily available. meningococcal vaccines have a very low incidence of side effects. regular disease surveillance is necessary to detect outbreaks. the epidemic threshold is 10 suspected cases/ 100,000 population in any given week. two suspected cases of meningitis in the same settlement should trigger an outbreak investigation. nasopharyngeal carriage rates do not predict epidemics. 80-85% of meningococcal disease presents with meningitis. 80% of cases occur in patients <30 y/o. peak incidence in meningitis belt is ages 5-10 yrs. diagnosis is straightforward when patient presents with signs of meningitis-fever, headache, vomiting, changes in mental status. however, most patients have non-specific illness 1-3 days before onset of meningitis. cfr of untreated meningococcal meningitis can be 50%. cfr of properly treated meningococcal meningitis is <1%. 15-20% of meningococcal disease presents with septicemia unaccompanied by meningitis or other focal features. it is a dramatic illness which affects previously healthy children and young adults. it presents with acute fever leading to purpura fulminans (hemorrhagic or purpuric rash), shock, and waterhouse-friderichsen syndrome (acute adrenal failure). etiologic diagnosis can be easily missed. cfr of meningococcal septicemia is 50% and may be 25% even with proper treatment. diagnosis may be confirmed by agglutination tests, polymerase chain reaction, culture and sensitivity testing of spinal fluid and blood. in many situations, these tests are not available. throat swabs may be helpful on occasions. do not delay treatment for tests or test results. minutes count. it is more important to have a live patient without a confirmed diagnosis than a dead one with a diagnosis. differential diagnosis in a tropical patient with fever and altered mental status, but without purpura or shock, includes cerebral malaria. co-infection may occur. standardized case management of bacterial meningitis in developed countries involves 7-10 days of parenteral antibiotic therapy. drug of choice in adults and older children is ceftriaxone which also rapidly eliminates the carrier state. alternate drugs include ampicillin and benzylpenicillin which do not eliminate the carrier state. in developing countries, 4 days of parenteral antibiotic therapy are empirically shown to be effective. in large epidemics in resource-poor settings, a single im dose of chloramphenicol in oil is the drug of choice. for patients who do not improve in 48 hours, a repeat dose may be given. viral meningitis is rarely serious and requires only supportive care, recovery is usually complete. patient isolation and disinfection of the room, clothing, or bedding are not necessary. respiratory precautions are advised particularly early in the course of treatment. chemoprophylaxis of contacts is available in some settings but rarely in the disaster setting. vigilance and education of close contacts is mandatory. epidemic preparedness and early detection of outbreaks are key. vaccines against n. meningitides serogroups a, c, y and w135 are very effective in controlling epidemics. in epidemic settings, children 2-10 are the priority target with serogroups a and c typically the priority antigens. rapid mass vaccination campaigns can contain outbreaks in 2-3 weeks. for immunocompetent patients over 2 years, vaccine efficacy rate is 90% one week after injection. however, duration of immunity may be as little as 2 years in younger children. in some countries, vaccine may also be used with close contacts of sporadic disease cases to prevent secondary cases. chemoprophylaxis of contacts is not recommended in epidemics, but community education and ready access to health care are essential. preventive medicine [1] source control/reduction/elimination undertake quarantine and culling of sick reservoir animals and known disease carrier species. avoid unnecessary contact with or consumption of dead reservoir animals or known disease carrier species. avoid unnecessary contact with suspected reservoir animals and known disease carrier species (eg primates). avoid direct or close contact with symptomatic patients. establish appropriate communicable disease controls for burial of the dead. administrative controls (improve people's work practices) environmental and engineering controls (isolate people from the hazard) avoid needle stick exposure to blood specimens thru automated machine handling. ppe (protect people with ppe) use standard precautions-gloves, masks, and protective clothing-if handling infected animals or patients. wash hands after visiting sick patients. active surveillance and contact tracing (enhanced surveillance) through community-based mobile teams active case finding (screening and triage) and contact tracing dedicated isolation facility food provision to isolated patients so they are not dependent on family case definition treatment protocols emphasizing supportive care and treatment of complications essential drugs referral guidelines secondary prevention barrier nursing strictly enforced family and community education ministerial task force to address policy local health authority task force to address procedures national level task forces to comprise if a lab is not available, then you need a sampling strategy that addresses specimen acquisition, preparation, and transportation in compliance with international regulations on the transport of infectious substances. guidance note on using the cluster approach to strengthen humanitarian response international conference on primary health care selective primary health care-an interim strategy for disease control in developing countries water and excreta-related diseases: unitary environmental classification infections related to water and excreta: the health dimension of the decade world health organization. cholera vaccines: who position paper available from: international centre for diarrhoeal disease research history and epidemiology of global smallpox eradication available from: us department of health and human services communicable disease control in emergencies-a field manual. geneva: world health organization ebola: technical guidance documents for medical staff world health organization. manual for the care and management of patients in ebola care units/community care centers-interim emergency guidance. who/ evd/manual/ecu/15.1. geneva: world health organization what tests does it perform? is there transport to and from the laboratory? who prepares transport media? who provides specimen collection material and supplies? how can these supplies be obtained? who provides cool packs, transport boxes, car, driver …? • refrigerate other vials for cytology, chemistry (4 °c) leak-proof specimen container wrapped with enough absorbent material to absorb the entire content of the 1st container 2. leak-proof secondary container usually plastic or metal 3. outer shipping container whose smallest dimension is 10 mm diagnostic specimens use iata packing instruction 650 without biohazard label. infectious materials use iata packing instruction 602 with biohazard label. what to send with the sample? lab request form with: • sender's name and contact info • patient name, age, sex • sample date, time • suspected clinical diagnosis with main signs and symptoms • sample macroscopic description • context-outbreak confirmation, ongoing verification, outbreak end, etc • epidemiological or demographic data where to send the sample? • reference lab • contact person what and when to expect results? source: world health organization world health organization department of communicable disease surveillance and response. highlights of specimen collection in emergency situations. undated 4 . designate a lead official in the lcc. 5. anticipate roles for partner agencies (eg inter-agency and team coordination, disease surveillance, field epidemiological investigation, laboratory identification, case management guideline development, outbreak logistics, public information, and social mobilization). 6. identify sources of funds. 7. intensify disease surveillance. 8 . identify reference lab(s) for communicable diseases of epidemic potential. 9. ensure mechanism for specimen transport. a. initial response to suspected outbreak 1. form an emergency team to investigate and manage the outbreak a. identify key roles on the outbreak investigation team(s) (1) epidemiology and surveillance (2) case management (3) water and sanitation (4) laboratory services (5) communication b. staff those roles (1) epidemiologist-to monitor proper data collection and surveillance procedures (2) physician-to confirm clinical s/sx and train health workers in case management (3) water and sanitation expert-to develop a plan for reducing sources of contamination (4) microbiologist-to take environmental/biological samples for laboratory confirmation, train health workers in proper sampling techniques, and confirm use of appropriate methods in the diagnostic laboratory (5) key: cord-119626-qb6fea06 authors: cruz-aponte, mayte'e; caraballo-cueto, jos'e title: balancing fiscal and mortality impact of sars-cov-2 mitigation measurements date: 2020-06-02 journal: nan doi: nan sha: doc_id: 119626 cord_uid: qb6fea06 an epidemic carries human and fiscal costs. in the case of imported pandemics, the first-best solution is to restrict national borders to identify and isolate infected individuals. however, when that opportunity is not fully seized and there is no preventative intervention available, second-best options must be chosen. in this article we develop a system of differential equations that simulate both the fiscal and human costs associated to different mitigation measurements. after simulating several scenarios, we conclude that herd immunity (or unleashing the pandemic) is the worst policy in terms of both human and fiscal cost. we found that the second-best policy would be a strict policy (e.g. physical distancing with massive testing) established under the first 20 days after the pandemic, that lowers the probability of infection by 80%. in the case of the us, this strict policy would save more than 239 thousands lives and almost $170.8 billion to taxpayers when compared to the herd immunity case. during the covid-19 pandemic, many policymakers are usually facing two separated sources of information: economic models that usually predict an economic collapse [15] and epidemic models that focus on death counts [12] . however, both the economic and mortality figures are key policy variables during a pandemic but few articles integrate both approaches [9, 16] . in particular, no research (to our knowledge) has analyzed both the fiscal and mortality impact of different mitigation measurements. in this article we strive to fill that gap by approximating the impact of physical distancing and patient care on the death toll and government budget, in a attempt to find the optimal conditions to balance it all. vaccination or therapeutics can eradicate epidemics from the population, like the case of smallpox [2, 3] but when a newly discovered virus hits the population, the entire world is at risk because everyone is susceptible as in the case of the novel sars-cov-2 that is impacting us in 2020 [13] . in the case of an imported infection (i.e. not an endemic epidemic), the first-best strategy would be to control borders, identify, treat and isolate infected individuals. this occurred in the u.s. with the ebola virus, which never became an epidemic [7] . but when a virus is already circulating in a territory and there is no antidote or massive testing and contact tracing available, social or physical distancing is an alternative to mitigate a pandemic and provide the scientific community time to research and find alternative measures such as an effective treatment or a vaccine. also, physical distancing measure gives fragile healthcare systems the leverage to take care of chronically ill patients without saturation of existing capacity. what are the fiscal and human costs of all these measurements in the short and long run? thus, two research questions drive this study: what is the optimal physical distancing policies in a country and what are the implications of these policies for both the government budget and loss-of-life? we constructed an enhanced mathematical sir (susceptible, infected, recovered) epidemic model [5] to simulate the covid-19 epidemic in the us in an attempt to estimate the fiscal impact and the optimal conditions to mitigate this ongoing pandemic. we found that a policy of no physical distancing or a race towards herd immunity is not the optimal policy choice when both human and fiscal costs are considered. in section 2 we lay out our methodology. in section 3 we show the dynamics associated to our calibrated system of differential equations. in section 4 we discuss our results and in section 5 we conclude and recommend public policies. we first describe a simple economy with three sectors; businesses, government, and a household sector with two actors. in the second part of this section we describe our epidemic model. in this economy, the household sector is mobile within the country and is composed of l workers and u individuals that are not working. thus, employment is less than full. this characterization allows us to consider the supply shocks associated to the covid-19 pandemic [10] , where laborers are impeded to work fully because of lock-downs or infections affecting members of the household sector. firms produce goods and services i, which require x amount of l. a fixed amount of total output y is predetermined to be produced in period t=0 and is given by, y = x i * l i . however, firms are able to adjust its output when external changes hit the labor stock. the total output that considers the impact of such external changes is observed in, y t = yh t where h t = dl/dt. we hold the following assumptions over h: • if physical distancing is implemented at t=1, h t = −0.3 during the physical distancing. when the physical distancing ends in t=n, and h t=n = 0.1 this setting let us capture the v-shape growth that is being projected [11] in the post-covid-19 period. • if no physical distancing is implemented, the pandemic ends in t=n+j, h t=n+j+1 = 0, and h t<j = −0.1. this decline is lower than the lower bound estimate of fourteen percent decline in output constructed by jp morgan for the u.s., a country that did not declare a general lock-down [15] . in our case, n+j+1 equals 500 days. • if physical distancing is implemented late at t=i, with j<i, h t = −0.3 during the physical distancing, h t=n = 0.1 when the physical distancing gets relaxed, the pandemic ends in t=n+i. in our case, n+i equals 600 on the other hand, the government sector has a fixed level of expenditures predetermined in period t=0, that includes money transfers to low-income u. tax revenues c are governed by, where τ is the tax rate. if there is a pandemic, the government spends m v in the treatment of each infected individuals. in order to assess the fiscal impact that a pandemic such as covid-19 will convey by the epidemic dynamics we construct an epidemic model, with the addition of a fiscal impact differential equation based on the previous section that quantifies the impact in the budget of the country. the epidemic model is a typical sir-type model with the epidemiological classes needed for the evolution of covid-19 within the population. there are six epidemiological classes defined susceptible (s) individuals that progress to incubate the virus in the exposed (e) class when they have an effective contact with an infected symptomatic individual (i) with probability βsi n using mass action or if a susceptible individual had contact with an asymptomatic (a) individual with a lower probability of infection than a contact with a symptomatic governed by the factor βsµa n . treated individuals (t) are assumed to be quarantined, hence, they are not considered to be able to transmit the diseases in the context of this modelling approach. after 14 days of incubating the virus a proportion of individuals becomes symptomatic and the rest are asymptomatic (estimated here at 35% [1] ). infected individuals that are asymptomatic recover without further complications, symptomatic individuals that get critically ill seek treatment while the mild cases recover. following the related literature cited in table 1 below, symptomatic and treated individuals have a probability of dying due to health complications estimated at 3%. in table 1 below there is a description of the parameters used. the system of differential equations for covid-19, is now presented in equations 1 to 6. in particular, the system is described as: next couple of sections. pandemic. the parameter values in our table were taken from a recollection of the events developing on covid-19 and the literature. perturbations around the means showed in table 1 may affect the quantitative magnitude of our figures, but results are qualitatively similar after such perturbations. in other words, our findings for the second-best scenarios are robust to deviations of our parameters: such deviations will largely scale up or down the quantitative aspects of each scenario. the parameters of the model were fitted or estimated to maintain a basic reproductive number relatively close to 3.8. for comparison purposes lets note that the 0 of seasonal influenza ranges approximately within 1.7 to 2.1 [21] . for the 2009 influenza a-h1n1 the 0 was estimated to be between 1.2 to 1.6. in particular, the basic reproductive number 0 is the number of secondary cases a single infectious individual generates during the period of infectivity on a completely susceptible population. we assume that the entire population is susceptible such that s ≈ n and that the epidemic has not started in t=0. the individuals that can potentially infect the population in our model are infected individuals that are either symptomatic or asymptomatic. treated individuals are assumed to be quarantined. following the related literature [22] , we use the next generation operator to compute the 0 . let the vector f be the rate of new infections flowing to the latent compartment and the vector v to be the rate of transfer of individuals out of the compartment that are able to transmit the disease. then, using our sir system of equations we define in order to compute 0 , let the gradient of f be defined as f = ∂f ∂e , ∂f ∂a , ∂f ∂i and let the gradient of v be define as v = ∂v ∂e , ∂v ∂a , ∂v ∂i then we get: then 0 is the spectral radius of the second generation operator ρ(fv −1 ) also know as the dominant eigenvalue of the matrix fv −1 . hence, , which means that the basic reproductive number is: our intention in this article is to study the impact of the covid-19 epidemic in an attempt to estimate the optimal conditions to mitigate the fiscal and mortality impact associated to this pandemic. thus, the stability of the system and equilibrium points will not be addressed, only the 0 was computed. we focused our efforts on simulating scenarios, which are presented in the next set of sections. in order to go through the methodology of our simulations we lay out simple in the long run, herd immunity has a higher death toll and implies more government expenditures than the alternated intensity of physical distancing, as shown in table 2 . in particular, with no physical distancing would die 10,689 more individuals than in the alternated scenario and the government would lose $1.16 trillion more than in the varying physical distancing. note that under the alternated scenario scientists have approximately 400 days to find an antidote with a very low number of victims, vis-a-vis 150 days under the no distancing case. figure 2 : varying physical distancing starting two days after the epidemic: lowering the infection rate 20% for four-week intervals and increasing it between 50% to 75% for eight weeks intervals as described on table 2 . figure 1 , 2, and 3 on figure 2 , we illustrate the simulated figures that are obtained when we shorten the length of the cycles showed previously. here we are also holding the assumption that the pandemic started in day two. the variation in the probability of infection is shown in the gray line, which goes first to an extreme reduction of 80% for four weeks, then is relaxed to 50% for eight weeks, then two cycles of extreme measurement for four weeks followed by a relaxation of table 2 . we observed that the strict alternated scenario of figure 1 would save the federal government $142.9 billion more than in the relaxed scenario of figure 2 , while mortality is virtually the same. in other words, the distancing cycles shown in table 2 appear to be less optimal than in the more restrictive case of table 2 . table 3 . since there is no infection curve in the restrictive and permanent case, the budget of the government is mostly affected by the decline in tax revenues caused by lowered economic output. however, when infection rate is lowered just by 40%, the government budget becomes highly impacted after day 400: once infection starts to increase, the public sector is affected by both spending more in treatment and by the reduction in tax revenues caused by the economic collapse. in the case of no physical distancing, even though economic output decreased, the federal budget is highly reduced by the treatment cost. at the end, if there is an opportunity to implement a persistent and early public health policy, it would be better to do it intensely (e.g. by combining a strong physical distance with massive testing): otherwise, the relatively human and fiscal cost would increase significantly. how would the figures change if the persistent measure is implemented late? the evolution of infection and mortality is similar, but there are differences in the long run for the associated fiscal costs. when the infection is reduced by 40% or 80%, the associated cost of the pandemic to the government decreased by $51.4 billion than when the public health policy is implemented earlier like in figure 4 . this difference arises because the economy was not halted during the first weeks of the pandemic. that is, even in the case of these late decisions the government is better off in implementing the strict measure: death toll does not change while fiscal costs perform much either in day 2 or day 20 after the pandemic (see appendix b). in figure 5 we show the effects of lowering the probability of infection within the whole u.s. population for different time intervals. when infection is reduced by 90% for eight weeks, mortality is 10,713 lower than in the herd immunity case and the fiscal cost of the pandemic is the minimum with respect to any of the scenarios presented in this article. that is, this short and strong distancing policy would have a lower death toll (24 deaths) than in the alternated scenario of figure 1 , but with a lowered fiscal cost ($180.3 billion less). figure 5 if the infection rate is reduced by 50% for twenty four weeks, the death toll is a bit higher than in the case of a strong policy for eight continuous weeks. in either case, the pandemic would kill at least 10,705 fewer individuals than in the herd immunity case. in terms, of budget, the longer period under distancing induces a larger fiscal decline than in the case of the eight-week distancing. however, the herd immunity is again the most costly option in terms of both death and government budget. these results reveal that if a policymaker is going to implement distancing measures, overall better results would be found with a strict measure for a short period than with a weak measure for a long period. in fact, in terms of human and fiscal costs, we can state that this strong policy for eight continuous weeks is the preferred option after the case of figure 6 . we acknowledge that our model may not be exported to developing countries where the lock-down can also result in deaths of individuals from starvation: given the low safety nets and salaries in many poor countries, lack of employment can severely reduce dietary intake resulting in other serious health-related issues or death. in that case, we recommend adding a death variable associated to forced unemployment. such an approach exceeds the scope of this paper. we like to thanks dr. ricardo gonzález-méndez from the university of puerto rico school of medicine as well as dr. ricardo j. cordero-soto from california baptist university for their insights and suggestions. the usual disclaimers apply. • both authors contributed equally in the designed, codification and preparation of the article. all authors have approved the final article. • this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. • declarations of interest: none vaccine or effective treatment is not implemented before the physical measures are lifted, the epidemic will raise again. a policymaker would like to find an easier mechanism than a cyclical regime to combat the pandemic. in figure b .8 we show the case where just one restrictive policy that declines the infection rate by 80% is applied for 250 days. the probability of infection is then kept by a permanent reduction of 25% to account for people being more careful and taking measures to avoid being infected. in this case the peak of the distancing curve arrives much later than before at approximately day 600. as expected, more lives (26 persons) are saved than in the alternated cycles of figure 2 but now the federal budget is almost halved. note that in any of these scenarios, no physical distance or herd immunity is an optimal solution, either in terms of human or fiscal cost. figure b .8 cdc estimates that 35% of coronavirus patients don't have symptoms the eradication of smallpox the smallpox story: life and death of an old disease revenue statistics 2019 -the united states compartmental models in epidemiology -2016 ebola outbreak in west africa no evidence yet that recovered covid-19 patients are immune, who says the macroeconomics of epidemics. (w26882) macroeconomic implications of covid-19: can negative supply shocks cause demand shortages? the great lockdown real-time estimation of the risk of death from novel coronavirus (covid-19) infection: inference using exported cases and corona virus disease-2019 (covid-19): the epidemic and the challenges estimated cost of treating the uninsured hospitalized with covid-19, (kff assessing the fallout from the coronavirus pandemic adaptive cyclic exit strategies from lockdown to suppress covid-19 and allow economic activity the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study federal spending on the elderly and children risk factors for 2009 pandemic influenza a (h1n1)-related hospitalization and death among racial/ethnic groups in new mexico impact of h1n1 on socially disadvantaged populations: systematic review quantifying the transmissibility of human influenza and its seasonal variation in temperate regions reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission in this example we start physical distancing measures on day 20 (i.e. t pdon = 20 after the start of the epidemic) and lower the transmission rate 50% for two weeks (i.e. t pdof f = 34 two weeks after implemented + t pdon = 20). then, measures are relaxed for two weeks. at this time the infection rate is decreased by 10%, assuming people are a little bit more careful by washing their hands or using cloth masks. after these two weeks, when cases start to increase again, the physical distancing measures are taken more strictly, thereby decreasing the infection rate by 70% for six weeks. thus, we found the famous "flatten the curve" scenario in which we all desire to maintain our health system unsaturated. if a key: cord-000721-leedutqo authors: nawaz, sameena; allen, david j.; aladin, farah; gallimore, christopher; iturriza-gómara, miren title: human bocaviruses are not significantly associated with gastroenteritis: results of retesting archive dna from a case control study in the uk date: 2012-07-24 journal: plos one doi: 10.1371/journal.pone.0041346 sha: doc_id: 721 cord_uid: leedutqo gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. despite improvements in detection methods, a significant diagnostic gap still remains. human bocavirus (hbov)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between hbovs, and in particular type-2 hbovs, and gastroenteritis has previously been made. the aim of this study was to determine the role of hbovs in gastroenteritis, using archived dna samples from the case-control infectious intestinal disease study (iid). dna extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of hbov dna. all samples were screened in a real time pcr pan-hbov assay, and positive samples were then tested in genotype 1 to 3-specific assays. hbov was detected in 7.4% but no significantly different prevalence was observed between cases and controls. in the genotype-specific assays 106 of the 324 hbov-positive samples were genotyped, with hbov-1 predominantly found in controls whilst hbov-2 was more frequently associated with cases of gastroenteritis (p<0.01). a significant proportion of hbov positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. however, the distribution of the untyped hbov strains was no different between cases and controls. in conclusion, hbovs, including hbov-2 do not appear to be a significant cause of gastroenteritis in the uk population. in 2005 a novel parvovirus was discovered in respiratory secretions of young children and was termed human bocavirus (hbov-1) [1] . other important members of the parvoviridae family include b19 which causes fith disease and human parvovirus 4 (parv 4) which has not yet been associated with a disease [2] . parvoviruses in animals are generally associated with systemic disease but also with respiratory and enteric symptoms [3, 4] . since the discovery of hbov-1 three other hbov genotypes have been described, hbov-2, hbov-3 and hbov-4. the association between hbov-1 and respiratory disease has previously been well established [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19] . although all hbovs have also been detected in stool samples with prevalences ranging from ,1% to 20%, only hbov-2 has been reported to be associated with symptoms of gastroenteritis [20] . nevertheless, the role of hbov2 as an aetiological agent of gastroenteritis has not been clearly confirmed, furthermore, to date, no clear association between the presence of hbov-3 and hbov-4 and disease has been established [21, 22] . recent seroepidemiological studies indicate that exposure to hbovs occurs early in life and 90% of the population are seropositive by the age of 5, although differences were reported in the seroprevalence of type-specific antibodies to the different hbovs, which suggested that hbov-1 infections are more prevalent [23] . the infectious intestinal disease study (iid study) was a large case control study of gastroenteritis carried out in the uk between 1993-1996 [24] with the aim to determine the burden and aetiology of sporadic cases iid in the uk population. initially, the use of classical microbiology diagnostic methods and electron microscopy (for virus detection) failed to detect a potential aetiological agent or toxin in 49% of the cases [25] . retesting of the archived samples from this study using molecular methods for the detection of enteric viruses, bacteria and protozoa revealed viruses to be the most common aetiological agents of gastroenteritis,the diagnostic gap for iid was reduced to 25% from 49% [26] . the aim of the present study was to evaluate the role of hbovs in iid in the uk population, using archived dna samples from the matched case-control iid-1 study [26] . in addition, the presence specifically of hbov-1, 2 or 3 was investigated in order to determine any possible associations between specific hbov genotypes and iid. a total of 4,380 archived dna from the iid study [26, 27] were tested for the presence of hbov dna. this archive comprised dna extracted from stool samples from 2,256 cases and 2,124 controls. the qpcr assay targeted the ns1 gene (ratcliff et al., unpublished method, personal communication) and was performed using an abi taqman7500. oligonucleotide primer and probe sequences and positions are described in table 1. the reaction consisted of 0.1 m ddt (invitrogen), 1x platinum quantitative pcr supermix-udg (invitrogen), pan-hbov-f and pan-hbov-r primers each at a concentration of 100 mm, pan-hbov-ns1 probe at 10 mm concentration, rox 25 mm (invitrogen) 2.5 ml of template dna and rnase free water to a final reaction volume of 25 ml. the amplification consisted of an initial denaturation at 95uc for 10 min, followed by 40 cycles with denaturation at 95uc for 15 sec, annealing at 55uc for 30 sec and extension at 60uc for 45 sec. hbov-1, 2 and 3-specific primer pair and probes were designed in house through alignment of sequence data available in genbank. the reaction conditions are as follows; 1x platinum quantitative pcr supermix-udg (invitrogen), hbov-ns1-1f, hbov-1r, hbov2-r and hbov3r primers each at a concentration of 20 mm, the hbov1,2 and 3 probe at 10 mm concentration, rox 25 mm (invitrogen), 2.5 ml of template and rnase free water was added to a final reaction volume of 25 ml. the amplification conditions for the typing assay are the same as those described in the hbov ns1 detection assay above. plasmids containing a 1773 bp and a 1737 bp region of the ns1 encoding gene of hbov-1 and hbov-3 respectively were used for assay optimisation and as controls. control material was kindly provided by r. ratcliff, adelaide, australia. the controls were also used in order to generate a standard curve for use with the pan-hbov assay in order to allow for normalisation of the data generated including the comparison of relative sensitivities of the different assays and for quantitation of dna present in each of the positive samples. the standard curve was generated using the plasmid containing a genome segment of the hbov-1 and consisted of a series of 10 fold dilution containing from 300,000 copies/ml down to 3 copies/ml. inter-and intraassay reproducibility was analysed by performing replicate testing of the standards in a single run (x11) and repeated runs (x2), and the standard curve was also included in each assay run for quality control and normalisation of results. a subset of 17 samples positive in the pan-hbov assay but which failed to amplify in the type-specific assays were confirmed using an alternative method published elsewhere [28, 29] , and 6 were further confirmed though direct sequencing of the amplicons obtained after purification either from solution or agarose gels using agencourt ampure (beckman coulter, usa) and geneclean spin kit (qbiogene), respectively, following manufactures protocols. the chi-squared test was used in order to evaluate the significance of differences observed between groups. for comparison of median values (analysis of ct values) the mann witney utest was used. prevalence odds ratio (por = pcases/(1-pcases)/ pcontrols/(1-pcontrols)) was calculated in the total cohorts and by age group. table 1 . hbov-specific oligonucleotide primers and probes (all located at the ns gene). sequence ( a total of 7.4% of the samples tested were positive for hbov. no statistically significant differences were seen in the prevalence of hbov between cases and asymptomatic controls, por = 0.79 (table 2) . peak hbov infection was observed in children under the age of 5, both in cases and controls, with significantly higher hbov incidence in children between 1 and 4 in asymptomatic controls than in the cases of gastroenteritis (por = 0.6; p,0.02). the number of hbov positives in older age groups was too small for meaningful statistical analysis. the average ct values were 34.5 and 34.8, and the median ct values were 36.3 and 37.4 in cases and controls respectively (see distribution in figure 1 ). the majority of hbov-positives in both cases and controls had copy numbers ranging between 30 and 299 copies/reaction (or between 4.5610 3 and 4.5610 4 copies/ml of feaces). the distribution of hbov viral loads between cases and controls was comparable and the median ct values between cases and controls were not significantly different (u-test; z = 0.458139, p.0.05). hbov dna was found in 149 (46%) samples in the absence of other co-pathogens (table 3) . no statistically significant differences were observed in the proportion of cases or controls in which hbov was found as a single organism or in the presence of one or more pathogens in the cohort as a whole, however in the 1-4 years of age group, hbov in the absence of any other enteric pathogens was found in 29% of the cases, but in 56% of the controls (p,0.05). hbov infections were detected year round although a peak was observed in the spring/early summer months, between april and june 1994 (figure 2 ). hbov dna was found in 48.8% and 45.7% of female cases and controls, respectively. the distribution of hbov among females and males was not significantly different from the distribution of females and males in the entire cohort which was 53% and 47%, respectively. a total of 106 (32.7%) hbov positives were genotyped, whilst 218 (67.3%) remained untyped after testing in the hbov types 1, 2 or 3 specific assays (table 4 ). hbov-1 detection was found predominantly in controls, (p,0.001) and hbov-2 was predominantly associated with cases (p,0.01). the prevalence of hbov-3 was not significantly different between cases and controls. hbov-1 and -3 were predominantly found in children (table 4) . hbov-2 in the absence of any other pathogen was detected in 17 (81.3%) of the cases, compared to 9 (47.4%) of the controls. in cases, hbov-2 was found across the age groups, although more frequently in children ,5, whereas in controls they were found predominantly in children ,5 with only 1 example in an adult ( table 4 ). the prevalence of hbov-2 in children ,5 years old was however not significantly different between cases and controls, 2.8% and 2.6%, respectively. a subset of hbov that were negative in the type 1,2 or 3specific assays were confirmed in an alternative pan-hbov pcr, and sequencing of a small number confirmed them as types 1, 2 or 3. the majority of the untyped samples (70%) had a ct value of .37 in the screening pan-hbov pcr, indicative of low viral loads being present in the samples. this represents the largest study to date investigating the role and distribution of hbovs infections in community acquired sporadic gastroenteritis and in asymptomatic controls. the prevalence of hbov infection in the uk population was found to be 7.4% across all ages, with a higher percentage of the infections occurring in children ,5 years of age (19%). however, the prevalence of hbov infections was comparable in cases of gastroenteritis and in age-matched asymptomatic controls. although the presence of enteric pathogens, eg norovirus or rotavirus, in asymptomatic individuals is well documented, a significantly higher prevalence of the pathogen is seen in cases than in the controls [26] . therefore, our data suggests that hbov are not causally associated with gastrointestinal disease in the uk population as a whole, nor in children. the prevalence of detection of hbov in stool samples in previous studies varies widely (see summary in table 5 ), but most coincide in reporting the highest prevalence in children. hbov infections were detected all year round in the uk although a tentative peak was observed in the spring/early summer months in 1994 (between april and june). different seasonal patterns in the peak prevalence of hbov have been reported in different countries (see table 5 ), of the 324 hbov positive samples, 106 (32.7%) were genotyped in the type-specific assays. hbov-1 was found predominantly in controls (p,0.001) and the prevalence of hbov-3 was similar in cases and controls. both hbov-1 and -3 were predominantly found in children. hbov-2 was predominantly associated with gastroenteritis cases (p,0.01). the overall prevalence in cases was 1.4% and 0.8% in controls, however, in children ,5 year of age, the prevalence in cases and controls was similar, 2.8% and 2.6%, respectively. the prevalence of hbov-2 in children in the uk was significantly lower than that reported in a study in australia, in which hbov-2 was detected in 17.2% and 8.1% of the cases and controls, respectively [22] . the findings of the study in australia lead to the proposal of hbov-2 as an important aetiological agent of infantile gastroenteritis. it is noteworthy however, that in the australian study, the association of hbov-2 with gastroenteritis was only significant when cases with a bacterial co-pathogen were included in the analysis. although in the present study hbov-2 in the absence of other enteric pathogens was found more frequently in cases than in controls, the small numbers found in such large study suggest that the role of these viruses in iid, if any, is likely to be small. a lack of correlation between hbovs or hbov-2 and paediatric gastroenteritis was also reported in several smaller studies published elsewhere [30, 31, 32] . a total of 67% of the hbov-positive samples could not be genotyped using the genotype-specific pcr assays. the majority of these untyped samples (70%) had ct values .37. this suggests that failure to type may be associated with low viral loads and differences in the relative sensitivities of the genotyping assays compared to the detection assay. although under experimental conditions and using plasmid controls the sensitivities of all assays were comparable, it is likely that when applied to true clinical samples the sensitivity of the type-specific assays was inferior, possibly due to as yet not identified strain variability within genotypes. also, a hbov type 4-specific assay was not included in this study, therefore, any possible hbov4 infections would not have been typed. of the panel of samples that were tested in an alternative pan-hbov pcr, the strains typed through sequencing were hbov-1 (2 samples), hbov-2 (1 sample) and hbov-3 (3 samples). furthermore, the distribution of untyped hbovs was not significantly different in cases and controls. hbovs in the absence of other enteric pathogens were seen in 46% of the hbov-positive samples, and more frequently in the controls, 50.3% vs 40.9% in cases. no significant difference in hbov load was observed between cases and controls, or between the samples positive for hbov alone or in the presence of other pathogens. previous studies have investigated the relationships between viral load and disease severity [33, 34, 35, 36, 37] . in respiratory infections significantly higher hbov loads were seen in samples collected from children positive for hbov alone than in those from children with co-infections. in respiratory infections also, viral loads .10 4 were associated with disease, whereas loads ,10 4 were associated with asymptomatic children [33, 38] . this lead to the suggestion that higher viral loads are indicative of a causative role of hbov in respiratory infections [33, 38] . however, brieu et al [38] found no significant correlation between viral load and clinical symptoms or disease severity. in conclusion, the results obtained from investigating for the presence of hbov dna in archived dna samples from a large and previously well described case-control study of iid suggest that hbov, including hbov-2,do not appear to be a significant cause of gastroenteritis in the uk population, and particularly in the paediatric population. although hbovs are relatively frequent across all ages, and in particular in preschool age children, they are found just as frequently among children and adults without symptoms of gastroenteritis. cloning of a human parvovirus by molecular screening of respiratory tract samples new dna viruses identified in patients with acute viral infection syndrome epidemiological studies of parvovirus infections in calves on endemically infected properties minute virus of canines (mvc, canine parvovirus type-1): pathogenicity for pups and seroprevalence estimate frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in canadian children in a 1-year study complete coding sequences and phylogenetic 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respiratory infections human bocavirus infection, people's republic of china human bocavirus in hospitalized children frequent detection of bocavirus dna in german children with respiratory tract infections a newly identified bocavirus species in human stool human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections a novel bocavirus associated with acute gastroenteritis in australian children seroepidemiology of human bocaviruses 1-4 a report of the study of infectious intestinal disease in england.: food standard agency. the stationery office a study of infectious intestinal disease in england: microbiological findings in cases and controls detection by pcr of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the english case-control infectious intestinal disease study (1993-1996) detection of viral, bacterial, and parasitological rna or dna of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study: assessment of the stability of target nucleic acid human bocavirus in children hospitalized for acute gastroenteritis: a case-control study evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus 2 in children detection of human bocavirus-2 in children with acute gastroenteritis in south korea high prevalence of human bocavirus 2 and its role in childhood acute gastroenteritis in china human bocavirus and acute wheezing in children human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants quantitation of group a rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in south india diagnosing rotavirus a associated iid: using elisa to identify a cut-off for real time rt-pcr diagnosing norovirus-associated infectious intestinal disease using viral load human bocavirus infection in children with respiratory tract disease this study was conducted using existing material and data form the iid-1 study, which was funded by the food standards agency. we would like to thank the iid study executive committee for their support in approving the use of the archive dna material for this study. key: cord-247554-535cpe5x authors: moustakas, aristides title: ranking the explanatory power of factors associated with worldwide new covid-19 cases date: 2020-05-29 journal: nan doi: nan sha: doc_id: 247554 cord_uid: 535cpe5x disease spread is a complex phenomenon requiring an interdisciplinary approach. covid-19 exhibited a global spatial spread in a very short time frame resulting in a global pandemic. data of new covid-19 cases per million were analysed worldwide at the spatial scale of a country and time replicated from the end of december 2019 to late may 2020. data driven analysis of epidemiological, economic, public health, and governmental intervention variables was performed in order to select the optimal variables in explaining new covid-19 cases across all countries in time. sequentially, hierarchical variance partitioning of the optimal variables was performed in order to quantify the independent contribution of each variable in the total variance of new covid-19 cases per million. results indicated that from the variables available new tests per thousand explained the vast majority of the total variance in new cases (51.6%) followed by the governmental stringency index (15.2%). availability of hospital beds per 100k inhabitants explained 9% extreme poverty explained 8.8%, hand washing facilities 5.3%, the fraction of the population aged 65 or older explained 3.9%, and other disease prevalence (cardiovascular diseases plus diabetes) explained 2.9%. the percentage of smokers within the population explained 2.6% of the total variance, while population density explained 0.6%. patterns of infectious diseases across spatial and temporal scales are fundamental for understanding their dynamics and for designing eradication strategies [1, 2] . disease spread is a complex phenomenon and requires an interdisciplinary approach spanning from medicine to statistics and social sciences [3] . covid-19 exhibited a global spatial spread in a relatively very short time frame resulting in being characterized as a pandemic by the world health organisation [4] . to date there is no known anti-viral treatment or vaccine against covid-19 [5] . therefore the available options against the virus are the characteristics of the immune system and health status of each individual, the health system of the country that the individual has access to, the social behaviour of the other individuals forming the society, public interventions of movement or public campaigns, and testing [6] [7] [8] . thus, there are relatively few options to be employed in order to diminish the spread of the disease. this scarcity of options makes quantifying the factors associated with disease spread more important and ranking the relative contribution of each factor on covid-19 spread may facilitate diminishing it [9] . analysis so far has often been dominated either on a one-at-a-time factor analysis (e.g. new cases per testing effort or total cases per age structure) or on a country basis analysis (e.g doubling time in one country in comparison to other countries). while such comparisons are straightforward and comprehensive, from a statistical perspective they are examining one factor at a time and masking underlying characteristics within countries under a variable 'country' [10] . this results in a hidden burden of the underlying factors regarding disease spread and causality is often discussed in a speculative manner. admittedly the problem is complex due to the large differences across the potential underlying factors across countries. a small variance implies that the mean contains virtually all of the information, while a large variance implies that more information than the mean is present [11] . when examining several countries together and across time, quantifying the variance may be more informative than the mean [12] . variance is introduced both by space, there are large e.g. climatic differences between locations on the same date, as well as by temporal e.g. climatic or behavioural changes within locations in seasons [13] [14] [15] . the potential effect of economic, epidemiological and public health, or governmental interventions may become clearer when the contribution of these factors into new covid-19 cases are analysed accounting for the fact that they derive from different countries as well as in different time snapshots [16] but in a way that the effect of each factor can be quantified in conjunction with the effects of other factors. to that end methods that can account for both spatial and temporal autocorrelation [17] in the data of new covid-19 cases but can quantify the effect of each epidemiological, economic, public health, and governmental intervention are key to our understanding of how the disease spreads in populations worldwide [18, 19] . in this study spatio-temporal worldwide data of new covid-19 cases form the "our world in data" database [20] were analyzed using computational statistics. the spatial replicate of the dataset included over 150 countries time replicated for each country across a period of ≈five months. data driven analysis was performed in order to quantify the optimal variables in cases where several candidate variables were available. during the data driven variable selection, the fact that data derived from different countries and were time replicated was accounted for by nesting the variance of time within the variance of country and treated as random effects [21, 22] . the percentage of the total variance explained by each epidemiological, economic, public health, and governmental intervention variables associated with covid-19 new cases was quantified using hierarchical variance partitioning [23, 24] thereby ranking the relative contribution of the variance of each factor on new covid-19 cases worldwide. the objective was to quantify epidemiological, public health, economic, and governmental intervention factors associated with covid-19 spread worldwide. new covid-19 cases per million per country per time step were used a proxy of disease spread. new cases per million in each country was chosen instead of new cases per country as this estimator is less biased by the total population of each country -countries with higher populations are more likely to produce higher new cases or total but the number may be relatively low in comparison to the total population pool. data regarding new covid-19 cases per million from the "our world in data" database were analyzed. the dataset was last accessed on 25/05/2020 and the download location is https://github.com/owid/covid-19data/tree/master/public/data. the data derive from the european centre for disease prevention and control (ecdc), an eu agency with the aim to strengthen europe's defense against infectious diseases. the ecdc collects and aggregates data from countries around the world. the most up-to-date data for any particular country is therefore typically available earlier via the national health agencies than via the ecdc. this lag between nationally available data and the ecdc data is not very long as the ecdc publishes new data daily; typically this time lag is at the level of some hours and less than a day. the ecdc collects compiles and harmonizes data from around the world in a consistent way which allows us to compare what is happening in different countries. the spatial replicate of the dataset comprised of 160 countries while the temporal replicate spans from 31/12/2019 to (including) 25/05/2020. the variables included: from the available variables male and female smokers were averaged as 'smokers'. we employed generalised linear mixed effects models (lme; [26] ) with new covid-19 cases per million as the dependent variable. as the dataset contained several potential indexes of testing, population density, or age structure within each country, initial analysis was conducted in order to select the most informative index of each. we initially sought to quantify the most parsimonious data driven index of testing which included the fixed effects of (i) news tests (ii) total tests (iii) new tests per thousand (iv) total tests per thousand (v) new tests smoothed, (vi) new tests smoothed per thousand. this was achieved by fitting six lmes with new cases per million as the dependent variable and six lmes with i -vi as the single independent variable. the random effect structure of each lme included the nested variance of time within each country (random~country/time). doing so the fitted lmes accounted for both temporal and spatial autocorrelation in the time replicated data deriving from different geographic locations [21, 22] . lmes were fitted with maximum likelihood (ml) estimation to allow comparisons between models with different fixed effects and selecting the lme that exhibited the lowest akaike (aic) value [27, 28] . here and throughout the analysis, there were 19,709 data points in the analysis but there were variables with missing values at some time steps or at some countries. missing values were omitted from the statistical analysis. therefore aic values are compared between models fitted with different fixed effects but also with potentially different sample sizes. similarly, lmes with new cases per million as the dependent variable and the fixed effects of (i) population or (ii) population density, and the nested random effects of time within country were fitted with ml and compared against aic values to select the optimal data driven population index. regarding age structure of the population within each country, the available variables were (i) median age of the population, (ii) the percentage of the population aged 65 or older, and (iii) the percentage of the population aged 70 or older. the analysis proceeded by selecting the ml fitted lme with the lowest aic between the three available age population structure variables. all three fitted lmes contained the random effects of time nested within country. regarding economic status of the population within each country, the available variables were (i) gdp per capita, and (ii) percentage of the population under extreme poverty. the analysis proceeded by selecting the ml fitted lme with the lowest aic between the two available economy status variables. the two fitted lmes contained the random effects of time nested within country. having selected the optimal index of testing, population density and age structures the analysis proceeded with the following variables: (1) population density, (2) new tests per thousand, (3) governmental stringency index, (4) percentage of the population aged > 65, (5) percentage of the population under extreme poverty, (6) cvd death rate, (7) diabetes prevalence, (8) percentage of smokers, (9) percentage of the population with access to hand washing facilities, and (10) hospital beds per 100k inhabitants within each country as independent variables. hierarchical variance partitioning (hvp) statistical modelling was implemented to account for the contribution of each data driven epidemiological, economic, public health, and governmental intervention explanatory variable to the total variance of new covid-19 per million cases [29, 30] . hvp is a statistical framework that is capable of handling correlated independent variables, whilst providing a reliable ranking of predictor importance of each variable [29] . variance partitioning is calculated from the akaike (aic) weights of each explanatory variable and it is based upon the number of times that a variable was significant in all possible combinations of the explanatory variables. the hvp function produces a minor rounding error for hierarchies constructed from more than nine variables [31] -the available data driven variables were 10. to check if this error affects the inference from an analysis, the analysis was repeated several times with the variables entered in a different order [31] . the analysis resulted in changes in the derived results when the order of the variables was changed. the analysis proceeded by creating a new variable that merged together other disease related variables: other diseases variable= (cvd_death_rate + diabetes_prevalence) plus the other remaining eight variables resulting in a total of nine variables. there is no known statistical bias in hvp when 9 or fewer variables are used [31] . the optimal data driven index for explaining new cases per million from the ones available regarding testing was new cases per thousand as fitted by lmes and selected against the lowest aic value (table 2a) . new tests per thousand smoothed could not be fitted as the lme did not converge (table 2a) . the optimal data driven population index for new cases per million was population density (table 2b ). the optimal data driven index for age structure within the population was the percentage of the population within each country aged 65 or older (table 2c ). the optimal aic selected lme regarding the economic status of the population within each country in relation to new covid-19 cases per million was extreme poverty (table 2d) . results from hvp indicated that total tests per thousand explained 51.6% of the total variance of new cases per million, while governmental stringency index explained 15.2% (fig. 1, table 3 ). availability of hospital bed per 100k inhabitants explained 9% (fig. 1, table 3 ). extreme poverty explained 8.8% of the total variance of new cases per million, hand washing facilities 5.3%, the fraction of the population aged 65 or older explained 3.9%, other disease prevalence (cardiovascular diseases plus diabetes) explained 2.9% (fig. 1, table 3 ). the percentage of smokers within the population explained 2.6% of the total variance of new covid-19 cases per million, while population density explained 0.6% (fig. 1, table 3 ). the best model fit regarding new covid-19 cases per million and the economic status of the country where the new cases are recorded indicated that extreme poverty was a better predictor of new cases than gdp per capita. it is thus the poorest individuals within each country impacted rather than poor countries. from the data available, the fraction of the population aged 65 or older explained optimally new cases per million and not median population age. total tests per thousand and not new tests or new tests smoothed or other available indexes is a better predictor of new cases per million, perhaps unsurprisingly as the number of new cases is already normalized by the population and thus the number of tests also normalized by the population explains better the pattern. the latter also applies for population density instead of total population as the best available predictor of new cases per million. summing up, from the data-driven analysis it is evident that new covid-19 cases per million are best explained by extreme poverty prevalence within each country as well as by the fraction of the population older than 65, thereby indicating association of the spread of the disease with the poor and older. results from variance partitioning of the data-driven selected 9 epidemiological, public health, economic, and governmental intervention variables explaining covid-19 new cases per million across countries through time, indicated that the vast majority of new cases per million are explained by the number of tests conducted. the number of new tests per thousand explains over 50% of the total variance through time and countries and thus the message regarding the efficacy of testing against covid-19 spread is strong, at least form the results derived here. the efficacy of testing has been highlighted as the best strategy against other diseases too across humans, agriculture, and wildlife [1, 24, 32, 33] . it therefore seems that the optimal strategies against covid-19 spread should include high number of tests both to suspicious cases as well as random population testing. would increasing the number of tests result in detecting more covid-19 cases? lost covid-19 cases are not uncommon [34, 35] . from a statistical perspective, variance partitioning does not provide information on the sign of the effects (positive or negative) it simply shows in how many cases this variable could not be excluded from the final optimal statistical model in explaining new covid-19 cases. the slope between new tests per thousand inhabitants and new cases per million vary between countries (fig. 2a) . indeed there are countries where the slope between new cases per million and new tests per thousand are positive indicating that testing more would actually result into identifying more cases (fig. 2a) ; [32] . however, there are also countries with a negative slope between new tests -new cases' indicating that testing frequency is saturated (fig. 2a) . overall, using data from all countries and time steps, the relationship between testing and new cases is positive indicating that worldwide more tests would result in identifying higher number of new cases (fig. 2b) . therefore the efficacy of testing has not been saturated. the second best variable in explaining new covid-19 cases per million was the governmental stringency index. the governmental stringency index contains several measures taken by governments including school closures, national and international movement restrictions, public gathering and public events restrictions, exiting home restrictions as well as testing policies and financial measures [25] . to that end testing policy is in part contained in the stringency index as a weighted percentage of the overall index. however the relationship between stringency index and new tests per thousand is very weak with both r 2 and slope close to zero: linear regression (new_tests_per_thousand) = 0.1959 + 0.002479 (stringency_index), s = 0.597, r 2 = 1.1%, p<0.001. therefore of the available governmental measures summarized in the stringency index, testing frequency can be treated independently. in general, countries increased their level of stringency as their number of confirmed covid-19 cases raised, however there is significant variation in the rate and timing of this relationship [25] . another study indicated that in the early and accelerating stages of the pandemic, many citizens across 58 countries viewed their governments' response as insufficient [36] . in general the status of the infection spread and policy implementation influence restrictions uniformly across every countries [37] . given the overall large effect of testing on new cases, it has been investigated whether there exists a testing frequency for covid-19 such that the shutdown could have been avoided [38] . the study concluded that indeed there is an optimal testing frequency such that lockdown and thus governmental stringency may not be deemed necessary [38] . the test against covid-19 is known to be imperfect but not precisely known [39] and testing strategies to surmount this problem have been proposed [40] . the availability of hospital beds per 100k inhabitants, hand washing facilities, the effect of covid-19 in the older people as well as prevalence of other diseases and smokers have been highlighted [19, 41, 42] and this study confirms their importance. environmental factors have also been reported to play an important role in covid-19 dynamics [43] however this study did not explore their relative contribution. table 2 . data driven variable selection in cases where several candidate variables were available. variable selection was conducted by fitting lmes with new covid-19 cases per million as dependent variable and the candidate explanatory variables as single fixed effect variables and the variance introduced time nested within the variance of countries as random effects. models were fitted with ml to allow comparisons between lmes fitted with different fixed effects. the optimal model is bolded and italicized in each case. abrupt events and population synchrony in the dynamics of bovine tuberculosis synchrony, waves, and spatial hierarchies in the spread of influenza interdisciplinary public health reasoning and epidemic modelling: the case of black death: the case of black death world health organization. director-general's opening remarks at the media briefing on covid-19-11 no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection ethical and legal considerations in mitigating pandemic 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birds species in cyprus estimating and modelling bias of the hierarchical partitioning public-domain software: implications in environmental management and conservation regional and temporal characteristics of bovine tuberculosis of cattle in great britain. stochastic environmental research and risk assessment covid-19 epidemic in switzerland: on the importance of testing, contact tracing and isolation. swiss medical weekly internationally lost covid-19 cases quantifying undetected covid-19 cases and effects of containment measures in italy global behaviors and perceptions in the covid-19 pandemic international comparison of behavior changes with social distancing policies in response to covid-19 how often should people be the accuracy of covid-19 tests opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients. medrxiv covid-19: what has been learned and to be learned about the novel coronavirus disease the role of environmental factors on transmission rates of the covid-19 outbreak: an initial assessment in two spatial scales. available at ssrn 3552677 key: cord-254340-e1x0z3rh authors: cruz, christian joy pattawi; ganly, rachel; li, zilin; gietel-basten, stuart title: exploring the young demographic profile of covid-19 cases in hong kong: evidence from migration and travel history data date: 2020-06-26 journal: plos one doi: 10.1371/journal.pone.0235306 sha: doc_id: 254340 cord_uid: e1x0z3rh this paper investigates the profile of covid-19 cases in hong kong, highlighting the unique age structure of confirmed cases compared to other territories. while the majority of cases in most territories around the world have fitted an older age profile, our analysis shows that positive cases in hong kong have been concentrated among younger age groups, with the largest incidence of cases reported in the 15–24 age group. this is despite the population’s rapidly aging structure and extremely high levels of population density. using detailed case data from hong kong’s centre for health department and immigration department, we analyze the sex and age distribution of the confirmed cases along with their recent travel histories and immigration flows for the period january to april 2020. our analysis highlights hong kong’s high proportion of imported cases and large overseas student population in developing covid-19 hotspot areas such as the united kingdom. combined with community action and targeted and aggressive early policy measures taken to contain the virus, these factors may have contributed to the uniquely younger age structure of covid-19 cases in the city. consequently, this young profile of confirmed cases may have prevented fatalities in the territory. recent research has highlighted the importance of a demographic approach to understanding covid-19 transmission and fatality rates. the experience in hong kong shows that while an older population age structure may be important for understanding covid-19 fatality, it is not a given. from a social science perspective at least, there is ‘no easy answer’ to why one area should experience covid-19 differently from another. the hong kong special administrative region of the people's republic of china (hereafter hong kong) is a city and special administrative region of china in the eastern pearl river delta by the south china sea. in 2019, hong kong had roughly 7.5 million people in a 1,104-square-kilometre (426 square miles) territory while in kowloon, where more than three in ten residents reside, population density is at 48,930 persons per square kilometer. this makes it one of the most densely populated territories in the world [1] . apart from being a rapidly aging society [2, 3] , hong kong is also a migration destination. for the period 2015 to 2020, the net migration number was 147,000 or a net of four in-migrants per 1,000 of the population [1] . hong kong faces the challenge of infectious diseases as a consequence of various factors, including high population density, increasing environmental pollution, high migration inflows and outflows, the emergence of new infections as well as the changing lifestyle and behavior of its residents [4, 5] . the most recent significant outbreak was the severe acute respiratory syndrome (sars) outbreak which reached hong kong in march 2003 [5] . based on world health organization (who) data until july 11, 2003 , a total of 1,755 sars cases had been identified in the territory, of which 298 people died of the disease [6] . at that point, it was largely believed that hong kong was unprepared to be one of the epicenters of the sars epidemic. from this recent experience, did hong kong learn from the hard lessons of the past? the who officially declared the novel coronavirus infections (hereafter covid-19) outbreak as a pandemic on march 11, 2020, after it had spread to more than 100 countries and resulted in tens of thousands of cases within a few months. in hong kong, however, the first case was reported on january 23, 2020. in early february, the government was strongly criticized for policy responses related to a variety of issues, including the legality of, and access to face masks; border closure; medical fees and quarantine policy [7] . during this early period, unfavorable comparisons were made with other regional governments (especially macau and singapore) who appeared to be managing the crisis more effectively [8, 9] . however, by the time of writing in june 2020, new cases being reported in hong kong-especially local transmissions-are very rare [10] . that this has occurred without the general lockdown policies seen in other parts of the world is even more remarkable. our study includes an examination of the age and sex distribution of the covid-19 confirmed cases in hong kong and an exploration of how the different measures to combat this outbreak resulted in a relatively low number of cases and deaths. specifically, as demographers, we wished to explore the extent to which insights from demographic science could assist in explaining the nature of the hong kong experience of covid-19. in this paper, we highlight the potential impact of the young profile of the confirmed cases on the total number of mortalities and the effect of early, aggressive policy measures including travel bans, enforced quarantines and contact-tracing imposed by the hong kong government as early as january 27, 2020 in containing the spread of the covid-19. data on confirmed covid-19 cases were taken from the centre for health protection (chp) of the hong kong department of health. we assessed the age and sex distribution of the confirmed cases, discharge status (discharged, hospitalized or died) and type of transmission. we obtained the data regarding cumulative cases by age group from january 23 to april 16, 2020, which allowed us to determine the age group that registers the highest number of confirmed cases over time. daily migrant inflows and outflows data was retrieved from the hong kong immigration department showing arrivals at each border checkpoint into hong kong broken down by citizenship status. in addition, we retrieved detailed archived datasets pertaining to the travel histories of confirmed covid-19 cases, which are updated on an almost daily basis by the chp. we excluded travel histories pertaining to domestic travel (buses, trains and ferries) and a small number of journeys pertaining to outbound from hong kong. we linked travel histories to confirmed case identifications (ids) in order to examine the age structure and timing of cases where the apparent source of infection was not in hong kong (i.e. not a community infection). these include cases with a travel history from countries with widespread infection or where infection from a confirmed case who traveled occurred. by doing so, we highlight the importance of returnee hong kong residents from overseas hotspots on the relatively young age structure of confirmed cases during the second wave in march 2020. we also gathered data on policy measures implemented by the hong kong government in order to highlight the possible impact of major border closures and quarantine arrangements imposed by the hong kong government in reducing further numbers of imported covid-19 infections. we also utilized secondary data for comparative demographic analyses from the hong kong census and statistics department, united nations population division and the chinese center for disease control and prevention. compared with the aging hong kong population [1] , the covid-19 confirmed cases have an entirely different distribution. fig 1 shows [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . less than a tenth (7.9%) are aged 65 and over. this age distribution of the local covid-19 confirmed cases does not fit the general profile of other territories wherein the infections are more concentrated among those in the older age groups. although, of course, this may be related to testing patterns by age, rather than true differences in incidence. the who coronavirus disease 2019 situation report 89 shows that as of april 13, 2020, there were a total of 716,570 confirmed cases with reported information on age and sex coming from 113 countries, territories and areas; of these confirmed cases, the median age for males is 52 (interquartile range, iqr: 35-64) years and for females 50 (iqr 35-64) years [11] . examples of territories with an older profile of confirmed cases are mainland china where the majority of confirmed cases (54.0%) as of february 11, 2020 belong to the older age groups, at least 50 years old [12] , and the philippines wherein as of march 25, 2020, the majority of the confirmed cases are in the age group 50 and older [13] . there is also an imbalanced sex ratio among the covid-19 confirmed cases in hong kong with a higher proportion of male cases, representing 54% of the total. the number of male cases outnumbered females in almost all age groups, except in the 25-34 age group where they made up only 47% of cases. for all other age groups, male cases were between 50 to 60% of the total; an exception was for those aged under 15 of which 68% of cases were male, however, the total number of cases at this age was extremely small (25 out of the total 1,017 cases). this sex profile with a higher prevalence of male cases is similar to the outbreaks in other territories [12, 13] the youngest confirmed cases in hong kong were two males under one-year-old while the oldest confirmed case is a 96-year-old female. nearly half of the 1,017 confirmed cases (48.0%) had already been discharged from the hospital as of april 16, 2020. in short, covid-19 confirmed cases in hong kong were found primarily among the working and school-age groups, and to a certain degree, among men. by april 16, 2020, almost three months after the first reported case on january 23, 2020, four deaths among confirmed covid-19 cases were reported in hong kong. three such deaths were over 70 years old (two males and one female) while the fourth was the case of a 39-year old male. it is possible that this young age structure of confirmed cases in hong kong contributed to the extremely low numbers of fatalities compared to other territories. a large proportion of hong kong's confirmed cases were 'imported'. three in five confirmed cases (61.0%) have travel history from countries with widespread infection or were directly infected by a confirmed case who travelled; hence, they are considered cases with imported transmission (see fig 2) . two-fifths of these imported confirmed cases (41.1%) are young adults in the age group 15-24 years old. the proportion of imported versus local cases differs significantly based on age group. nine in ten of the confirmed cases in the 15-24 age group (91.4%), the group with the largest incidence of covid-19, were in fact imported. in contrast, 59.7% of cases aged 55-64 and less than half of those in other age groups are considered imported. most of hong kong's cases in the younger age groups occurred in a second wave of infections during march. before march 18, 2020, the age group with the highest frequency of covid-19 cases was 55-64 followed closely by the 65 and over age group (see fig 3) . however, after march 18, 2020 the number of new cases in hong kong sharply increased, most of these being imported cases in younger age groups. this timing of these imported cases is similar to that experienced by china wherein the imported cases more than doubled starting from march 18, 2020, mostly coming from countries with high outbreak of covid-19 infections like the united kingdom (uk), united states of america (usa) and spain [14] . however, age-specific data about imported cases is not available in china. analysis of the travel histories of hong kong cases suggests that the majority of imported cases in the 15-24 age group may be hong kong residents who are studying or working abroad. the travel history data collected and archived by the chp over the study period was matched to case numbers from the case data to study the travel history and age profile of all known imported cases. the data collected by the chp contains the travel histories of all confirmed covid-19 cases up to fourteen days before the case was reported and formed part of attempts of the government to track and trace the movements of all newly reported cases. this is done in order to contact and isolate anyone with whom they had close contact in the days before the onset of symptoms, including those whom they may have sat close to on flights. of the 508 covid-19 cases with an overseas travel history tracked by the chp, 47.4% were imported from the uk, 9.1% from the usa and 3.9% each from qatar, canada and switzerland. by far the largest age group with a travel history was those aged 15-24 with 205 cases, of which the majority were from the uk (62.4%), 8.3% from the usa, and 4.4% to 5.4% each from switzerland, qatar and the netherlands. the next largest group with travel histories was those aged 25-34 with 86 cases, nearly half of these coming from the uk (48.8%). we are not able to ascertain the employment or other characteristics of these cases. hong kong has a large population of mobile residents who hold citizenship or permanent residency but work, study or are retired overseas. according to the 2016 by-census, just under 220,000 permanent residents of hong kong were deemed 'mobile', meaning they had spent between one to three months in the city of the previous six [15] . in 2016, the largest population of mobile residents was those aged 15-24, with a total of 47,938 most of whom were registered as students. we were not able to obtain the country of temporary overseas residency from the census data. the next largest population of mobile residents were those aged 65 and over (42, 299 ) who were mostly registered as retired, while a further 40,926 were aged 55 to 64 mostly employees or retirees. in addition, according to the 2016 by-census hong kong is home to a sizable number of non-permanent residents; many of these residents may return to their home countries on a regular basis for study, work, or family visits. the largest groups of non-chinese nationals were filipinos (186,000) and indonesians (159,901), most of whom work as domestic workers on temporary foreign-worker permits. hong kong was also home to 35,069 british citizens, almost 15,000 americans and 66,690 citizens from south asian countries (india, nepal and pakistan), while a further 121,775 hong kong chinese were registered as being domiciled overseas in 2016 [15] . the large increases in newly confirmed cases in the younger age groups, the majority of which were imported, occurred from march 10, 2020 onward (fig 4) . this was in tandem with a large increase in confirmed cases and deaths throughout multiple countries in europe, usa and canada. on march 13, 2020, the hong kong security bureau had announced a 'red outbound travel alert' ('red ota') for the schengen area, announcing a mandatory 14-day home quarantine for all arrivals from the schengen area. this was followed by a 'red ota' for ireland, united kingdom and the usa on march 15, 2020 with the announcement of home quarantine arrangements for all travelers from the three states to begin four days later (see s1 table for a full timeline of policy events). fig 4 shows the arrivals into hong kong over the period from january 24 to april 16, 2020. most arrivals were hong kong residents travelling via the airport. on february 4, 2020, all land and sea border points were closed except for two control points-the shenzhen bay control point and hong kong-zhuhai-macao bridge. daily arrivals into the city fell dramatically after the end of the chinese lunar new year holidays at the end of january 2020, and took another dramatic fall after the home quarantine arrangements for all arrivals from china were put into place on february 8, 2020. arrival numbers followed a relatively steady pattern at under 25,000 per day until dropping off sharply from march 19, 2020, when the new 14-day home quarantine arrangements were put in place for all arrivals into the city, regardless of whether or not they were residents. from march 25, 2020, all non-residents were barred from entering the city except for nationals of macau, taiwan or mainland china. these border closures and sharply lower inbound-travel movements together with hong kong's aggressive policy of testing, contact-tracing and quarantine of confirmed cases and their close contacts (see s1 table) undoubtedly contributed to the sharp decline in newly confirmed cases during the month of april [16] . on april 20, 2020, hong kong recorded its first day without a confirmed case, from a peak of more than 60 cases per day in late march. social distancing and rapid population behavioral changes also likely played a role, with measures such as wearing masks, working from home and school closures leading to an estimated 44.0% reduction in seasonal influenza incidence [16] . the containment of a severe local outbreak of covid-19 in hong kong thus far, and the very high incidence of confirmed cases in younger age groups mostly among hong kong residents returning to the city from overseas hotspot areas, have surely contributed to the very low number of fatalities in the city-state, with only four deaths reported by april 23, 2020 out of 1,030 confirmed cases. one death occurred in the 80 years and over age group, out of twelve total cases. in italy, china and south korea, vastly higher case fatality rates were recorded for those in the older age groups; as at march 31, 2020 case fatality rates for those above 80 years old were at 27.7% in italy and 18.3% in south korea [17, 18] . this paper takes a social scientific approach to illustrate the particularities of covid-19 outcomes in hong kong up until now. the paper is a simple, descriptive analysis of the available data on hand. a recent paper highlighted the importance of a demographic approach to understanding covid-19 transmission and fatality rates [18] . the paper suggested that 'the age structure of a population may help explain differences in fatality rates across countries and how transmission unfolds.' fig 5 shows the full population pyramid for hong kong in 2016. observing the discrepancy in the shapes of the pyramids in figs 5 and 1, it is clear the experience from hong kong suggests that age structure alone is not a universal factor in shaping transmission and fatality rates. despite having more than 18.0% of the population 65 and over and an extremely high population density, a package of policies designed to contain the virus spreading from younger imported cases and becoming a sustained local outbreak ensured that case and mortality numbers stayed low. a further difference between hong kong and other settings characterized by higher rates of infection (and fatality) is the lower levels of distribution of residential care and support among older persons. there is strong evidence that people living in residential/nursing homes are particularly vulnerable to not only infection and its rapid spread, but to severe covid-19 infection and fatality [19] [20] [21] ; so much so that the who referred to care home infection and fatality rates as an "unimaginable human tragedy" [22] . in a study of official data in ten countries, deaths in care homes account for between 19-72% of all deaths [21] -although international comparison is difficult because of differences in cause of death reporting as testing procedures. in common with other parts of the world, the hong kong government has issued guidelines to support residential care homes in preventing infection [23] , as well as offering other support in terms of provision of personal protective equipment (ppes) and infection protection services [24] and a switch to online care support for those who would ordinarily visit day care centers [25] . there may thus be institutional reasons for the low levels of transmission among older people. the size and percentage of the older population resident in care homes differs widely around the world [26] . in the uk, where a high percentage of infections and fatalities have occurred in care homes [19, 20] , it is estimated that around 5.3% of the population aged over 70 is resident in care homes [27, 28] . in addition, a further 6.9% of that population receive care support in their own homes, including from carers making multiple home visits in a day-potentially another area of risk for infection [27, 28] . in contemporary hong kong, meanwhile, the proportion of over 70s in residential/nursing homes is estimated to be around 3.6% [1, 29] . these lower rates of care home residence may have contributed to lower overall transmission and fatality numbers among the elderly by creating lower opportunities for sustained local spread within the elderly population. low-income migrant workers, an already neglected group in terms of health and other support mechanisms during this pandemic [30] , represent a further group often characterized by communal living. highly elevated transmission rates have been seen in some settings where such migrant workers often live in cramped, unsanitary conditions [31] . in singapore, for example, over half of the purpose-built and factory-converted dormitories have been affected [32] ; a factor held primarily responsible for the 'second wave' of infections in the city-state. it has been estimated that some 80% of all cases have been linked to such dormitories. compared to singapore, such 'dormitories' are rare in hong kong. perhaps the primary, related housing issue in hong kong is 'subdivided housing'; home to up to 209,000 poor, urban individuals [33] and sometimes referred to as 'coffin houses' because of their very small sizes [34] . while often characterized by poor hygiene, low environmental [35] and safety standards [36] , it appears that policy measures in hong kong, which succeeded in stemming local transmission chains, meant these quasi-communal units were not left exposed to rapid covid-19 transmission. a final possible factor mentioned by dowd et al. [18] concerns the possibility that 'intergenerational interactions, co-residence, and commuting may have accelerated the outbreak in italy through social networks that increased the proximity of elderly to initial cases.' this would be derived from a mechanism whereby the younger population most susceptible to initial infection [37] transmit to the elder population through such contact. in hong kong, multi-generational residence is common [38] , where around 50% of those aged 65 and over live with their adult children [39] -much higher than in italy (or, indeed, any other setting in europe or north america characterized by high transmission rates) [40] . the extremely high population density of hong kong coupled with short distances and highly efficient transport systems means that there is a high degree of residential proximity as well regular contact between older parents and their children. a further dimension of intergenerational, intrahousehold interaction involves migrant domestic workers, who increasingly operate a major means of care support within the household. there is little evidence in hong kong that such workers were responsible for transmission within the household. as such, the hypothesis suggested for italy appears inconsistent with the hong kong case at least. the general quality of population health data in hong kong is generally accepted to be high, not least because of the centralized healthcare system in the territory. furthermore, in this case a very high degree of health surveillance and monitoring was in force throughout the period. this analysis relied heavily on secondary data for both the information on the confirmed cases and travel histories. given this, there are possible concerns for under diagnosis and under reporting especially during the start of the outbreak until before its first peak as have also been observed in other countries [41] . the unique age and sex distribution of the cases in hong kong may also be affected by testing patterns. there may, of course, have been unreported and/or asymptomatic cases in hong kong which will have escaped our analysis. however, there is no current consensus on such rates of asymptomatic infection and, therefore, how one might either estimate or correct in the hong kong case [42] . finally, the challenge of ascertaining primary cause of death and the role played by covid-19 interacting with other factors [43] [44] [45] is not a significant issue in hong kong given the very small number of deaths. a major limitation is that the spread of covid19 has not yet ended. any observations of a 'case in progress' are prone to future, unexpected changes which will change the narrative already described. of course, by the time this paper is published it is eminently possible that events have taken a turn for the worse and a third wave of infections occur. despite this, hong kong has clearly demonstrated a capacity to control both the transmission and fatality of covid-19 until this point. as of june 11, 2020 there have been no covid-19 ascribed deaths since march 14, 2020. furthermore, as of april 29, 2020, hong kong has reported no new cases for the sixth time in ten days [10] , and there were just five cases of local transmission in the month of may. from the start of may, public facilities began to reopen, some border restrictions were lifted, and civil servants returned to work in their offices. as of mid-june, public facilities such as beaches, libraries, museums, swimming pools have largely reopened, as have the last group of commercial leisure/entertainment facilities (karaoke lounges, nightclubs, bathhouses and party venues). clearly, much more research is required to concretely establish both the epidemiological and social factors contributing to the hong kong experience. the data which will allow such analysis will only come on stream in the future. at this point more complex statistical analysis will be able to be performed. furthermore, it will then become possible to link data on transmission and fatality through to other clinical and vital records (including the planned census in 2021). such data will also need to be complimented by survey data and qualitative research to provide a broader sense of the hong kong context. policy interventions, institutional systems, household and living arrangements each played a role in a complex, interwoven way. however, we must not overlook the role played by the community itself who, through behavioral change and increased vigilance, appear to have been equally instrumental in shaping hong kong's covid-19 experience. it may be in this way that the greatest lessons of sars have been learned [5] . supporting information s1 table. timeline world population prospects: the 2019 revision the population problem in pacific asia remeasuring ageing in hong kong sar; or "keeping the demographic window open prevention and control of communicable diseases in hong kong. government printer the sars epidemic in hong kong: what lessons have we learned? who. cumulative number of reported probable cases of sars. in: who [internet 7 reasons hongkongers are angry about the gov't response to the coronavirus macau's last covid-19 patient recovers, with no new cases for a month why did singapore have more coronavirus cases than hong kong? in: south china morning post another day of no new covid-19 cases in hong kong situation report-89. world health organization the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china demographic research and development foundation, inc. (drdf). covid-19 and the older filipino population: how many are at risk? uppi/ drdf research brief weekly assessment of the covid-19 pandemic and risk of importation-china population by-census impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study. lancet public health the epidemiological characteristics of 2019 novel coronavirus diseases (covid-19 demographic science aids in understanding the spread and fatality rates of covid-19 death toll in uk care homes from coronavirus may be 6,000, study estimates. financial times number of deaths in care homes notified to the care quality commission, england. in: office for national statistics mortality associated with covid-19 outbreaks in care homes: early international evidence. international longterm care policy network unimaginable human tragedy" in europe's care homes, says who. japan times guidelines for residential care homes for the elderly or persons with disabilities for the prevention of coronavirus disease (covid-19) (interim) online day care keeping elderly hongkongers active during isolation nursing homes in 10 nations: a comparison between countries and settings how big is the problem in care homes? bbc office for national statistics kong ngo shows care homes they can stop keeping elderly in restraints. south china morning post the neglected health of international migrant workers in the covid-19 epidemic migrant workers in cramped gulf dorms fear infection current situation on migrant workers in dormitories-home thematic report: persons living in subdivided units tiny affordable housing in hong kong air and hygiene quality in crowded housing environments-a case study of subdivided units in hong kong a brief discussion on fire safety issues of subdivided housing units in hong kong social contacts and mixing patterns relevant to the spread of infectious diseases solidarity, ambivalence and multigenerational co-residence in hong kong. contemporary grandparenting: changing family relationships in global contexts living arrangements and older people's labor force participation in hong kong intergenerational co-residence during later life in europe and china level of underreporting including underdiagnosis before the first peak of covid-19 in various countries: preliminary retrospective results based on wavelets and estimating the extent of asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis. medrxiv pathological findings of covid-19 associated with acute respiratory distress syndrome autopsy in suspected covid-19 cases case-fatality rate and characteristics of patients dying in relation to covid-19 in italy we would like to thank the academic editor and reviewers for their helpful and constructive suggestions. we would also like to thank professor stéphane helleringer for his comments on an earlier draft. the authors have declared that no competing interests exist. key: cord-253851-27nt0op8 authors: koh, david; lim, meng‐kin; chia, sin‐eng title: sars: health care work can be hazardous to health date: 2003-06-17 journal: occup med (lond) doi: 10.1093/occmed/kqg090 sha: doc_id: 253851 cord_uid: 27nt0op8 nan reported 7548 probable sars cases and 573 deaths. the case fatality ratio varies from 0 to 50%, depending on the age of the patient, with an overall estimate of 14-15% [2]. these figures will undoubtedly change with time as more cases emerge. the aetiological agent is a novel coronavirus (sars-cov), with patterns of spread suggesting droplet or contact transmission [3, 4] . clinical features are those of atypical pneumonia, with the common presenting symptoms being fever and dry cough. sars patients are classified as either 'suspect' or 'probable' cases [5] . a suspect case is a patient who presents with a history of high fever (>38°c) and a cough or breathing difficulty. in addition, there must be one or more of the following exposures within 10 days of the onset of symptoms: either a close contact with a person who is a suspect or probable case of sars, or a history of travel to a sars-affected area. a suspect case is upgraded to 'probable' with the appearance of radiological changes consistent with pneumonia or respiratory distress syndrome (rds), or in the event of death, autopsy findings consistent with the pathology of rds without an identifiable cause. at the time of writing, there is no validated, widely and consistently available laboratory test for infection with the sars-cov. however, from 1 may 2003, the who amended the definition of a probable case to include a suspect case of sars who has positive laboratory tests for sars-cov, under conditions drawn up by the who. empirical therapy for sars has included corticosteroids, a broad spectrum antiviral agent and antibacterial cover [6] . health care workers (hcws) are a high-risk group for sars-cov infection. according to the who, they constitute the biggest, single group of probable sars patients worldwide. as at 4 may, 41% of 203 sars patients in singapore and 22% of 1629 cases in hong kong [7] were hcws. the majority of cases in canada (74.4%) have been attributed to exposure in a hospital or health care setting [8] . as at april 25, more than 100 hospital workers at three greater toronto area hospitals have become ill with sars [9] . unfortunately, a number of deaths have occurred among hcws. an early casualty was dr carlo urbani, the who expert working in hanoi who was among the first to identify the clinical disease, and in whose honour it has been proposed that the causative agent bear his name. the index case (and first reported death) of the hong kong outbreak was an elderly chinese physician who had treated sars patients in guangdong. three doctors, two nurses and a health care attendant in singapore have also succumbed to sars. the vulnerability of hcws can be explained by their close contact with patients. the innocuous, 'flu-like' clinical presentation of sars does not help to raise the index of suspicion. in the early stages of the outbreak, there was also not the same degree of vigilance with regards to potentially high exposure situations such as aerosol-generating procedures. these included aerosolized medication treatments (i.e. nebulizers), the use of high-flow venturi masks and non-invasive positive pressure ventilation for sars patients, airway suctioning and endotracheal intubations. as a poignant illustration, in singapore, a cluster of 41 probable and 21 suspected cases was traced to a single sars patient who was initially undiagnosed for 10 days and treated for gastrointestinal bleeding, chronic kidney disease and diabetes [10]. the cluster included 26 hospital staff working as doctors, nurses, radiographers and housekeepers. an occupational health audit, which included a walk-through of the hospital 'hot spots' carried out by the authors, revealed a small number of deficiencies which could well be weak links in an otherwise strong preventive chain. that the cluster of cases included housekeepers is also significant-preventive measures need to target much broader groups of hcws than just the doctors and nurses in direct contact with patients. frontline hcws like counter clerks, porters and ambulance drivers are also at risk, and must be educated and protected. the encouraging news is that with the institution of stringent infection control measures and personal protection, the situation appears to have improved somewhat. this was the case in a singapore hospital [11] , where the experience was reported as: 'we did not see any further transmission from this index patient after we implemented strict infection control measures involving use of n95 masks, gown, gloves, and handwashing before and after patient contact'. doctors in hong kong [12] are also 'hopeful that further cases among our staff will be prevented' after rigid application of infection control measures. substantial risks remain. until we know more, precautions should include placement of patients in isolation rooms with negative pressure relative to the surrounding area and the use of n 95 respirators for people entering the room. precautions to avoid droplet contact should include the use of gowns, gloves and goggles as a minimum for contact with patients or their environment. higher-risk procedures with the potential for splattering or spraying of sputum or other body fluids require the use of full-face shields, and improved respiratory protection such as the use of powered airpurifying respirators. perhaps the most important personal hygiene measure that all hcws can adopt is regular handwashing. excellent guidelines on infection control and exposure management are available at various websites, such as those of the us centers for disease control and prevention [13] , health canada on-line [14] and the who [15] . the adoption of extra-stringent control measures involving the donning of personal protection equipment is often a new experience for hospitals, clinics and their staff. the adoption of these measures on such a large scale, involving all hcws with contact to patients, is unprecedented in sars-affected countries. an audit of these newly implemented safety measures might reveal lapses. even if adequate measures, including the provision of facemasks, gloves and gowns, and proper waste decontamination procedures are in place, their observance may be lax because of lowered vigilance or simply human error. for example, the tight seal around the wearer's face may be compromised if respirator fit testing is not properly carried out; and improper handling of contaminated biohazardous waste might result in serious consequences. training of staff and close supervision by trained professionals knowledgeable in occupational health and safety methods are therefore vital. the psychological well-being of hcws must also not be overlooked. hospital staff tend to be on physical and emotional overdrive in an already overstretched system. the precautionary need to home-quarantine entire health care teams upon discovery of a new cluster of sars cases further strains resources, and may even result in hospital shut-downs. forcibly quarantining hcws in hospitals, as has happened in some countries, raises further questions concerning the hcws' individual human rights and their societal 'duty to care'. there is bound to be significant disruption in home life as well. staff anxiety under these abnormal conditions naturally runs high, especially when there is fear of contagion, and of infecting family, friends and colleagues [16] . it is therefore important to implement proactive, remedial actions to avoid burnout among overworked and highly stressed hcws. appropriate psychosocial support and scheduled rest periods should be in place before people run themselves into the ground. sars is a new occupational disease which we are only beginning to come to grips with. it is also a grim reminder that health care work is potentially hazardous. in the battle against sars, the safety and well-being of the 'troops'-hcws all over the world-must be accorded the priority it deserves. department of community, occupational and family medicine, faculty of medicine, national university of singapore, singapore severe acute respiratory syndrome (sars)-multi-country outbreak a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome case definitions for surveillance of severe acute respiratory syndrome (sars). revised outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report the war against an unknown pathogen: rising to the sars challenge sars among ontario health care workers severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts severe acute respiratory syndrome (sars): infection control infection control guidance for health care workers in 242 occupational medicine health care facilities and other institutional settings. severe acute respiratory syndrome (sars) the immediate psychological and occupational impact of the 2003 sars outbreak in a teaching hospital key: cord-010513-7p07efxo authors: daniels, norman title: resource allocation and priority setting date: 2015-08-31 journal: public health ethics: cases spanning the globe doi: 10.1007/978-3-319-23847-0_3 sha: doc_id: 10513 cord_uid: 7p07efxo there has been much discussion of resource allocation in medical systems, in the united states and elsewhere. in large part, the discussion is driven by rising costs and the resulting budget pressures felt by publicly funded systems and by both public and private components of mixed health systems. in some publicly funded systems, resource allocation is a pressing issue because resources expended on one disease or person cannot be spent on another disease or person. some of the same concern arises in mixed medical systems with multiple funding sources. risks matters, not just the aggregate impact. resource allocation in public health thus focuses on deciding what risks to reduce-which depends in part on their seriousness as population factors and who faces them-and how to reduce risks. the cases in this chapter that discuss resource allocation force us to contemplate decisions about priorities in public health as opposed to the more frequently discussed medical issues about health care priorities. later we suggest that making decisions about these issues should be part of a deliberative process that emphasizes transparency, stakeholder participation , and clear, relevant reasoning. collectively, these resource allocation cases bring out several important points. separately, they raise other central issues. it is worth noting these general issues before commenting on the more specifi c problems raised by each case. the fi rst point the cases collectively make is that effi ciency has ethical and not just economic importance (daniels et al. 1996 ) . if one health system is more effi cient than another, it can meet more health needs per dollar spent than the less effi cient one. if we want systems to meet more health needs, and we should, then we prefer more effi cient health systems. specifi cally, if we think we have obligations to meet more health needs, or if we think meeting more "does more good," and we ought to do as much good as we can with the resources we have, then we have an ethical basis for seeking more effi cient health systems. the economic pursuit of effi ciency should not, then, be dismissed as something that has no ethical rationale. a second point the cases collectively make is that effi ciency is not the only goal of health policy , for we have concerns about how health benefi ts are distributed as well as how they add up. health policy is not only concerned with improving population health as a whole, but also with aiming to distribute that health fairly (daniels 2008 ) . that means many resource allocation decisions involve competing health policy goals. the point about competing goals is illustrated by a problem often encountered in policy decisions: should we always favor getting the best outcome from the use of a resource, or should we give people "fair" chances to get a benefi t if it is at least signifi cant (brock 1988 ) ? for example, during an i nfl uenza pandemic, should we allocate ventilators to those with the best chance of survival, or should we give signifi cant but lesser chances to a broader group? reasonable people often disagree about when the difference in expected benefi ts means we should favor best outcomes over fair chances, or even about what counts as a fair chance. hence, a third point emerges from the cases taken collectively: reasonable people often disagree about the choice, and it is not possible to simply dismiss one side as irrational or insensitive to evidence and argument (daniels and sabin 2008 ) . indeed, reasonable people will disagree about how much priority to give to the sickest (or worst off) patients. they may think we have to weigh the seriousness of an illness against the potential benefi t that we know how to deliver, they may disagree about how to trade off those considerations, or they may disagree about when modest benefi ts to larger numbers of people outweigh greater benefi ts delivered to fewer people. together these "unsolved rationing" problems-the best outcome versus fair chances problem (when to prefer best outcomes to fair chances), the priorities problem (how much priority to give to those who are worst off), and the aggregation problem (when do modest benefi ts to more people outweigh significant benefi ts to fewer people)-mean that there is pervasive ethical disagreement underlying many resou rce allocation problems (daniels 1993 ) . there are other common sources of disagreement. one of the most common sources of controversy in resource allocation decisions arises when a particular intervention is seen as the last chance to extend life by some-a necessity if we are to act compassionately-and when it is seen primarily as an unproven intervention by others that we have no obligation to provide it. denials of such interventions in last-chance cases have been considered the "third rail" of resource allocation decisions (daniels and sabin 2008 ) . here we have two competing public value s-compassion and stewardship-and most public offi cials would prefer to be seen by the public as committed to saving lives rather than as ha rd-nosed stewards of collective resources. the cases taken collectively bring out one fi nal point: our main analytic tools for aiding resource allocation decision making are limited in several ways, particularly by insensitivity to various ethical issues, especially issues of distribution. in short, these tools may take the fi rst point, about the importance of effi ciency , seriously, yet fail to help us with the second and third lessons the cases collectively bring out, that we are also interested in distributing effi ciently produced health fairly, and that reasonable people disagree about how to do that. to see this, consider two widely used tools: comparative effectiveness research (cer) , which has been given prominence as a r esearch focus in the patient protection and affordable care act of 2010 , and cost-effectiveness analysis (cea) . both help to answer policy-making questions. for example, a typical use of cer compares the effectiveness of two interventions (drugs, procedures, or even two methods of delivery), and policy makers may want to know if a new technology is more effective than older technologies. of course, they may also want to know if the new technology provides additional effectiveness at a reasonable cost , which points to a shortcoming of much cer in the united state s, where considerations of cost are generally avoided. similarly, if there is only one effective treatment for a condition, cer tells us nothing useful. it also tells us nothing about whether a more effective intervention is worth its extra cost. and, cer cannot help us compare intervention outcomes across different disease conditions, since it uses no measure of health that permits a comparison of effectiveness. indeed, decision makers face many resource allocation questions that cannot be answered by cer, even if cer can help avoid wasteful investments in interventions that do not work or that offer no improvement ov er others. in germany, however, cer is combined with an economic analysis that takes cost into account and that allows the calculation of " effi ciency frontiers " for different classes of drugs (caro et al. 2010 ) . presumably, this method could be extended to different classes of public health interventions if they are grouped appropriately. to calculate an effi ciency frontier, the effect of each drug in a class in producing some health outcome is plotted against its cost , and the curve is the effi ciency frontier for that class of drugs. it is then possible to calculate if a new intervention in that drug class improves effectiveness at a price more or less effi cient than what is projected from the existing efficiency frontier. this use of cer allows german decision makers to negotiate the price of treatments with manufacturers, rejecting payments that yield ineffi cient improvements. german policy makers can then cover every effective intervention sold at a price that makes it reasonably effi cient. still, because german use of cer cannot make comparisons across diseases, it allows vast differences in effi ciency across conditions. cea aims for greater scope than cer. it deploys a common unit for measuring health outcomes , either a disability-adjusted life year (daly) or a quality-adjusted life year (qaly) . this unit purports to combine duration with quality, permitting us to compare health states across a range of disease conditions. with this measure of health effects, we can construct a ratio (the incremental cost-effectiveness ratio, or ic er) of the change in costs that results from the new intervention with the change in health effects (as measured by qalys or dalys). we can then calculate the cost per qaly (or dal y) and arrive at an effi ciency measure for a range of interventions that apply to different condi tions. critics have noted p roblematic ethical assumptions in the construction of the health-adjusted life-year measures and in the use of cea (nord 1999 ; brock 2004 ) . to see some of these problems, consider the following table: rationing problem cea fairness priorities no priority to worst off some priority to worst off aggregation any agg regation is ok some aggregations ok best outcomes/fair ch ances best outcome s fair chances cea systematically departs from judgments many people will make about what is fair. the priorities problem asks how much priority we should give to people who are worse off. by constructing a unit of health effectiveness , such as the qaly, cea assumes this unit has the same value , regardless of who gets it or wherever it goes in a life ("a qaly is a qaly" is the slogan). but intuitively, many people think that a unit of health is worth more if someone who is relatively worse off (sicker) gets it rather than someone who is better off (less sick) (brock 2002 ) . at the same time, people generally do not think we should give complete priority to those who are worse off. we may be able to do little for them, so giving them priority means we would have to forego doing more good for others. few would defend creating a bottomless pit out of those unfortunate enough to be the worst off. similarly, cea assumes that we should aggregate even small benefi ts. then, if enough people get small benefi ts, it outweighs giving large benefi ts to a few. but intuitively, most people think some benefi ts are trivial goods that should not be aggregated to outweigh larger benefi ts to a few (kamm 1993 ) . curing many people's colds, for example, does not outwei gh saving a single life. finally, cea favors putting resources where we get a best outcome, whereas people intuitively favor giving people a fair (if not equal) chance at a benefi t. locating an hiv/aids treatment clinic in an urban area may save more lives than placing a clinic in a rural area, but in doing so, we may deny many people a fair chance at a signifi cant benefi t (daniels 2004 ) . in all three of these examples of rationing problems, cea favors a maximizing strategy, whereas people making judgments about fairness are generally willing to sacrifi ce some aggregate population health to treat people fairly. in each example, whether it is giving some priority to those who are worse off, viewing some benefi ts as not worth aggregating, or giving people fair chances at some benefi t, fairness deviates from the health maximization that cea favors. yet we lack agreement on principle s that tell us how to trade off goals of maximization and fairness in these cases. people disagree about what trades they are willing to make, and this ethical disagreement is pervasive. determining priorities primarily by seeing whether an intervention achieves some cost/qaly standard is adopting a health maximization approach. this approach departs from widely held judgments about fairness, even where people differ in these judgments. thus, the national institute of clinical and health excellence (nice) in the united kingdom has had to modify its more rigid practice of approving new interventions only if they met a cost/ qaly standard in the face of recommendations from its citizens council. this council, intended to refl ect representative social and ethical judgments among british citizens, has proposed relaxing nice's threshold in various cases where judgments about fairness differed from concerns about health maximization. the judgments of the citizens council in this regard agree with what the social science literature suggests are widely held views in a range of cultures and contexts (dolan et al. 2005 ; menzel et al. 1999 ; nord 1999 ; ubel et al. 1999 ubel et al. , 2001 . there are, of course, those who criticize departures from the nice threshold of the sort that the citizens council recommended. compromising the maximization of health that cea promotes may be seen as a moral error, perhaps the result of elevating the rescue of an "identifi ed" victim (say, a cancer patient whose life might be extended modestly by a new drug) ove r benefi ts to "statistical" lives (using the resources to provide greater benefi ts to others). the reasonable disagreement about how to proceed suggests that we should view cea as an input into a discussion about reso urce allocation, not as an algorithm for making decisions. this "aid to decision making" role was proposed by the public health service in its recommendations about the use of cea (gold et al. 1996 ) . in short, controversial ethical positions are embedded in cea, and using cea uncritically commits one to these views, even though many disagree with them. we have already noted that the effi ciency of a health system has ethical consequences. but what should we count as effi ciency ? should we use our resources to generate more revenues for a unit of the health system-say, a hospital? doing so would defi ne effi ciency the way most businesses do: other things being equal, an allocation that produces a greater return on investment is a more effi cient use of stockholder or owner resources. alternatively, we might narrow the range of effects to health effects on the covered population . then we have greater effi ciency when an allocation produces more positive health effects in that population than an alternative allocation. the case guzmán brings from colombia raises this issue forcefully. should hospitals, or a specifi c health plan, allocate resources favoring services (certain treatment s) that raise more revenues than an alternative allocation (certain preventive measures)? perhaps the gains from the treatments will involve fewer population health gains over time than those obtained by the preventive or health promotional measures, even if they show their improvement more quickly and so look better sooner. which plan should the policy maker adopt? this issue examines our purpose in designing a health system. is it to meet the health needs of a population or is it to provide a good return on investment for those who invest in health services? we might think that this question is easier to answer in a system where health care delivery is seen largely as a public undertaking aimed at improving population health. in such a system, it might seem that there is only one purpose behind the health care system. return on investment for the taxpayer funding such a system should be measured by how effi ciently the system improves population health. in systems where resources are owned privately (and there are many of these), however, it seems we must consider at least two goals. even if the private sector must in part seek to improve population health , which may be a requirement of state-imposed health care regulation or, in some people's opinions, a social responsibility of corporations, private health-care organizations still must deliver a reasonable return on investment for owners. thus, policy makers within private health-care organizations have a dual task. balancing return on investment with improvement in populatio n health thus becomes the central issue in the colombian case study. the chilean case written by gómez and luco raises a similar issue, but this case focuses on measurable differences in the cost effectiveness of certain services and in the severity of two conditions. if we consider only cost effectiveness, we view effi ciency in one way-the best health outcomes in the aggregate for the population for an investment in health. if we take severity of condition into account, we might view this as an equity demand-in which case, we have an effi ciency-equity confl ict and must make a trade-off. or, we might think of effi ciency as a ranking of needs by severity of condition. in the latter, the resource allocation case turns on how we defi ne effi ciency. specifi cally, the chilean category of guaranteed health interventions could include cataract surgery (the leading cause of blindness in the chilean population), but not multiple sclerosis (ms) treatment s, which might be viewed as maximizing effi ciency in a standard sense. or, the guaranteed health interventions scheme could include the less cost-effective treatment of ms but not cataract surgery, since ms is viewed as a more severe condition (because it can be life threatening and lead to premature death), even if it is far less prevalent than cataracts. if this were the case, the more effi cient system, in this nonstandard view, would rank treating more severe conditions as more effi cient than treating less severe conditions. if budget limitations mean only one should be included in the guaranteed health interventions program, either m s or cataract surgery, which should it be? the cataract surgery intervention delivers a signifi cant benefi t in terms of qalys to a larger part of the population than does the intervention package for ms, but the greater severity of premature death seems to be an important reason for favoring ms. if this reason is given priority over cost effectiveness and over the standard view of effi ciency , then are less effective treatments for more severe conditions supposed to have priority over more effective and cost-effective treatments for less severe conditions? if so, what kind of a health system does that produce if all needs can not be met given resource limits? alternatively, do we want a system that always we ighs cost effectiveness more highly than the severity of a condition that some people have? that too seems problem atic. suppose we think improving population health is a worthwhile and defensible goal of a health system, we favor improving population health over increasing revenues for the private sector (in the guzmán case), and we also favor giving priority to cost effectiveness over severity of a condition (in the gómez and luco case). a confl ict still remains between health maximization in the aggregate and concerns about equity , as illustrated in the blacksher and goold case (and arguably in the case about triage in pand emics by smith and viens). in the case that blacksher and goold describe, the task is to decide whether to reallocate resources from a program focused on maternal-child health and reduction of b lack-white in fant mortality dis parities to a program that may get more health per dollar spent through other interventions. infant mortality among blacks and whites has declined rapidly in the united state s; and in absolute terms, the decline has been more rapid for blacks. still, the ratio of black infant mortality to white infant mortality has increased. because the public health department is in a highly segregated city, this shift in program focus might seem to require viewing the remaining bl ackwhite health d isparities as morally a cceptable (especially given the high rate of improvement that past programs gave to black infant mortality rates). when should we view health disparities as morally acceptable? when should we weigh reducing health disparities as more important than some aggregate gains in health that we know how to produce in a population ? if public health has two goals-improving population health and distributing that health fairly-how should we weigh the goals when they confl ict? one important feature of the blacksher and goold case, namely the opinions within the community whose inequalities are at issue, is really a feature to which nearly all cases warrant attending. people affected by a policy ought to have some infl uence in determining that policy. some people might believe this is what democracy requires. a diffi culty this view of democracy faces, however, is that those who speak for the community may not appropriately represent the community affected by the decision. nevertheless, the opinions of a broader range of stakeh olders may improve deliberation (depending on how those opinions are managed). it may also improve the acceptance of the decisions, which arguably enhances the legitimacy of the decision-making process . resistance to including a broader range of stakeholder s in decision making about health priorities may come from a concern that they bring with them "partiality." this resistance may come from the view that greater impartiality leads to better deliberation. arguably, this concern about partiality ignores the positive gains that partiality often brings to deliberation, especially if we know how to manage such deliberation so that we minimize the risk s that partiality sometimes brings. we need such management skills in any case since partiality is unavoidable in most contexts. rather than banning what cannot be eliminated, managing partiality in deliberations is the best way to improve decision making in contexts of reasonable disagreement. the confl ict between improving population health and treating people fairly can arise in other contexts. arguably, the problem raised by smith and viens about the principle that should govern triage in pande mics can be viewed as a confl ict between health maximization, in this case, saving the most lives, versus recognizing the claims that the sickest people have on us for assistance. ordinarily, health systems give some priority to those who are sickest, but should that priority disappear in favor of saving lives when scarce resources, such as ventilators, are allocated in pandemic conditions? if we allocate our ventilators to the sickest patients, we may save fewer lives than if we allocate them to those whose lives we can better expect to save. even if we think we should give priority to those worst off, do we ordinarily think that concern for them should govern triage policy in pandemics? if we believe saving the most lives trumps concerns about helping those who are sickest in pandemics, can we justify why the priority we give to the sickest should be revised in pandemics? suppose we have an acceptable way of measuring the burden of disease in a population , and according to this measure, mental illness is not given the priority it ought to have. that is, it contributes more to the burden of disease than is normally recognized in standard health systems, which provide too few services to meet mental health needs. this is the problem upon which rentmeester et al.'s case focuses. specifi cally, some mental health conditions require signifi cant resources for what medicaid terms as "behavioral management," which is seen as a social support service not a medical treatment . as a result, these services, to the extent they are provided, fall to state-funded social service budgets. the services place a burden on state fi nances that would be diminished if they were instead included in medicaid bud gets (50 % of which are fi nanced by each state). arguably, the stigma that attaches to mental health issues is one important reason for this underprovision of social supports for people with mental health issues. in nebraska, the political opposition to expanded medicaid coverage through the affordable care act ad ds to the burden on state budgets and the potential under-servicing of these mental-health induced needs. it takes resources to meet public health needs. suppose we can increase the resources to meet some of those needs by accepting a pu blic-private partnership that improves a compromised private partner's image? should we meet health n eeds at this price? that is the issue posed by the hernández-aguado case from spain . specifi cally, should public health authorities put their stamp of approval, in the form of their logo, on fl u epidemic notices printed on soft drink labels? the inclusion of the logo is a requirement of the private entities that are willing to donate space on the labels of their products. obviously, this provides a form of public support for soft drinks that arguably contribute to obesity in a population and thus to the prevalence of noncommunicable diseases associated with obesity. but in view of the low budgets available for fl u warnings, is this a price worth paying? what would the decision maker have to know about the effects of such labels to decide this case, or is the decision something that can be made independently of the specifi c payoffs of implementing the warning system? is there a way to consider the cost and assess whether the outcome of the warning is worth this price? is this simply an effi ciency calculation about the cost effectiveness of reducing a disease burden in this way? one fi nal crosscutting issue lurks behind all the cases in the resource allocation chapter (perhaps all the cases in the volume)-namely, the nature of the decisionmaking process that addresses the issues they raise. public health decisions about resource allocation-judging from the cases on that topic in this volume-face reasonable ethical disagreement. that is because the tradeoffs involved in the two main goals of public health policy -improving population health and distributing health fairly-are trade-offs about which people often reasonably disagree. how can public health decisions be made in real time, given these ethical disagreements, in ways that enhance their legitimacy and are arguably fair to all parties? one approach to the problem is to construct a fair process for making those decisions and to rely on the outcomes of such a process. people will judge the outcomes of a fair process to be fair (daniels and sabin 2008 ) . what conditions should such a decisionmaking process meet if it is to be considered fair? four conditions are arguably necessary (even if some may think they are not suffi cient and want to add others): (1) the decisions and the rationales for them should be made public. (2) they should be based on reasons all think are relevant. (3) they should be revisable in light of new evidence and arguments. and (4) , these conditions should be enforced so that the public can see that they obtain. some explanation is needed for these conditions. the publicity condition is widely embraced, even if it is fairly strong. it calls for the grounds for decisions-not just the content of the decisions-to be transparent. people have a right to know why decisions that affect their health are made the way they are. moreover, making the reasoning for such decisions public is a way of exposing them to scrutiny so errors in reasoning or evidence can be detected and decisions improved. even though we may not be able to be explicit in advance about all criteria we use to decide such cases, that is, we may work out our reasons through deliberation, we can explain on what we base our decisions. and that gives people affected by our decisions the knowledge they have a right to possess. the search for reasons that all consider relevant to making a reasonable public health decision about resource allocation can narrow disagreement considerably. even if people can agree on what reasons they think are relevant-in the spirit of fi nding mutually justifi able grounds for their decisions-they may not agree about the weight they give these reasons. one way to test the relevance of such reasons is to subject them to scrutiny by an appropriate range of stakeholder s. what counts as appropriate may vary with the case. who should be heard in deliberations is itself worthy of deliberation. stakeholders raise different arguments that should be heard, and including their voices improves buy-in to decisions. since stakeholders may not in many instances be elected representatives, we may be skeptical about whether the democratic process is improved by including them, but, if the deliberation is well managed, the quality of the discussion may improve greatly. the revisability condition , requiring that decisions be modifi able in light of new evidence and argument, is also widely embraced and not considered controversial. decisions are made on the basis of evidence and arguments, and better evidence and arguments may emerge that require revisiting some decisions. some decisions can then be modifi ed, though it may be too late for others, and our consolation is that we made the best choices we could, given the evidence and arguments. the intent of the enforcement condition is to ensure that the other, more substantive, conditions are met. sometimes enforcement is a matter of state regulation . sometimes it can be the result of vol untary conformance with a process. since ethical disagreements abound in resource allocation decisions , we need a process that enhances legitimacy. but can we claim that a decision-making process that is fair yields fair outcomes? one view is that we may ultimately become persuaded by a good argument that fairness requires a different decision than one that emerged from a fair process. we can in this way defeat the fairness we might ordinarily attribute to the outcome of a fair process. does the prospect of defeating the fairness of a decision emerging from a fair process mean that we should not attribute fairness to the outcomes? alternatively, we can admit that the fai rness that comes from a deliberation is only "defeasible" fairness, but it is the fairest conclusion we ca n reach at the time. during the 1990s, many latin american countries began reforming their health systems according to a neoliberal development model that emphasizes free markets (homedes and ugalde 2005 ; stocker et al. 1999 ) . approved in 1993, health reform in colombia was supposed to overcome problems such as low coverage, inequality in access and use of health care services, and ineffi ciency in the allocation and distribution of resources. but the reform also hoped to encourage more focus on illness prevention and health promotion and more community participation in health decision-making processes. the reformers advocated predominantly for neoliberal value s like effi ciency, free choice, universality, and quality. although they were also committed to the communitarian values of solidarity , equity , and social participation . the colombian health reform was one of the fi rst examples of implementing managed competition in the developing world (plaza et al. 2001 ) . to stimulate competition among insurers and health service providers, both public and private, health reformers applied the theory of managed competition (enthoven 1993 ) . according to this theory, competition achieves effi ciency and reduces cost , making health care services responsive to consumer needs (londoño and frenk 1997 ) . hospitals become responsive when they are able to sell services and become fi nancially sustainable. to achieve sustainability, supply subsidies (direct transfers from the state to hospitals) had to replace demand subsidies (transfers directed to the poor through a subsided s ecurity plan). the colombian reform established a general social security system in health that featured two insurance plans: (1) the contributory plan, fi nanced by mandatory contributions (formal employees and employers from the public and private sectors). (2) the subsidized plan, funded by resources from the contributory plan and from taxes and other sources, which covered people unable to pay (vargas et al. 2010 ) . the actors of the system are the insurance companies, the health service providers, and the state regulatory organizations. insurance companies contract with health service providers, and the regulatory organizations control compliance with the defi ned basic health packages. to optimize resources, the reform placed controls on medical practitioners and established explicit priority criteria based on clinical guidelines that defi ned benefi t packages. from 1993, some adjustments to the reform have been introduced, such as the creation, in 2012, of the institute for health technology assessment to provide a n evidence base for health decisions. the institute recommends which medical technologies should be paid with public resources on the basis of which technologies optimally improve the quality and cost effectiveness of medical care. to determine these technologies, it conducts health outcomes research that guides technology development, evaluation, and use (giedion et al. 2012 ) . nevertheless, 20 years later, the promise of reform lies unfulfi lled and many patients still experience high out-of-pocket costs, long wait times, or denial of services. to access health services, frustrated citizens are turning to the legal system as a last resort and, by so doing, congesting the courts (defensoría del pueblo 2012 ). physicians are responding to economic incentives and penalties by restricting hospitalization time and decreasing the use of expensive diagnostic tests and specialist referrals (abadía and oviedo 2009 ) . to further reduce labor costs, service providers have increased the workload of health profession als and the number of patients seen per day, whi le reducing the time spent with each patient (defensoría del pueblo 2007 ). insurance companies often take a long time to pay health service providers, and they also contract their own service network (a process known as vertical integration), so many public hospitals are in serious fi nancial diffi culties. meanwhile, hospital workers frequently disrupt the normal operation of hospitals as they strike to improve work conditions and have their paychecks issued more promptly. should hospitals fail-40 % of the 968 public hospitals in colombia are classifi ed as being at medium or high fi nancial risk -nearly ten million people could be left without health service (ministerio de salud y protección social 2012 ; quintana 2002 ) . add to that, the reforms have increased inequity, as more affl uent patients can more easily access quality health care services than can low-income patients (vargas et al. 2010 ) . the described problems refl ect a complex situation that requires profound structural reform . as one way to address the immediate problems of effi ciency and quality, colombia in 2012 instituted public hospital accreditation. accreditation requires hospital directors to reach goals in service delivery related to fi nancial viability, quality, and effi ciency. hospital boards can now fi re directors who fail to meet these g oals within a specifi ed period (rodríguez 2012 ) . given the imbalances between budgets, service demands, and ongoing costs, hospital directors face enormous challenges and ethical dilemmas in formulating and executing their mana gem ent plans. you are a director of a public hospital that focuses on health promotion and prevention activities, such as general practice, dentistry, clinical laboratory, hospitalization, and emergency care. in developing your management plan, you must make decisions about which services to prioritize . if you prioritize services that represent higher revenues and lower costs as a way of conserving resources, you may have to reduce priority for some services. to guide your decision making, you conducted a retrospective study of service billing in the past 2 years and learned that the clinical laboratory and external medical consultation yielded higher incomes. the lowest yielding programs in the short term-vaccination , educational programs to improve lifestyles , and provision of micronutrient supplements to children and pregnant women-were associated with the best long-term health results. taking seriously your fi duciary responsibilities, you try to guarantee fi nancial sustainability by containing labor costs, restricting consultation times, and shortening hospital stays. your challenge is to do these things without diminishing the quality of patient care. but because you compete with other institutions, you must also assure suffi cient reserves to maintain and update medical equipment that will improve the "sale of services." knowing that every management decision you make will affect the population you serve, you begin to refl ect on the factors affecting your h ospital man agement plan. 1. who are the major stakeholder s in this case and what are their interests, value s, and moral claims? between which of them are there ethical confl ict s or tensions? 2. which of these interests, values, and moral claims should be prioritized? how would you justify your priorities? 3. would you prioritize programs that in the short term brought in needed revenues or those programs that had highest impact long term? 4. how can tensions between the goals of effi ciency, fi nancial viability, and quality be resolved? what weight should be assigned to each goal by the hospital board when evaluating your performance? 5. at least in the short run, the new reforms seem to be prioritizing effi ciency, viability, and quality over equity . should a health system attain the former goals before tackling the problem of equity, or should it insist on equity from the start? 6. can equity in health care be achieved without doing something about wealth inequity and other social determinants of health? 7. should you justify your decisions by emphasizing solidarity with other hospital directors and seeking community support? 8. how could collaborations between public health, communities and the health care system begin to address neoliberal concern s with effi ciency, viability, and quality? the global burden of disease (gbd) compares disease burdens based on epidemiological measures of prevalence, mortality, disability, and associated cost s. the gbd for mental illness amounts to 14 % of the world's total disease burden (world health organization 2005 ). i n the united state s alone, every fi fth child suffers from a mental disorder (perou et al. 2013 ) . although mental illness clearly causes disabilities (prince et al. 2007 ), underservice to those with mental illness is commonplace. lack of access to mental health services counts as the fi rst of many hurdles facing families who have a child with a mental illness. stigma and the lack of parity in health coverage for physical and mental illness are other hurdles for these families. not surprisingly, these hurdles can critically affect the development of children with mental illness. lack of access to mental and behavioral health services for children 5 years and younger especially threatens their development. rapid brain growth occurs in the fi rst 5 years of life, which lays the foundation for cognitive, emotional, and moral development. exposure to chronic stress can prompt the release of hormones in the brain that can have enduring consequences for how the adult brain is organized and how it functions (shonkoff and phillips 2000 ) . because poor health can show up in children as developmental delay, access to mental and behavioral health services is critical. longitudinal studies demonstrate positive and long-acting effects of early childhood interventions, such as environmental enrichment programs, on a range of cognitive and noncognitive skills, social behaviors, academic achievement, and adult job performance (heckman 2008 ) . the esti mated annual rate of return on investment from targeted early childhood development programs is 7 %, and early intervention reduces the predictable need for higher, more costly levels of care in later life (heckman et al. 2010 ) . in the united state s, medicaid is a government-funded program that provides health coverage to people with certain disabilities and to low-income adults and their children. the federal medicaid act (fma) requires states participating in medicaid programs to provide medically necessary treatment to eligible children. under federal medicaid law , states must provide "early and periodic screening , diagnostics, and treatment," also known as epsdt services, to eligible medicaid recipients under age 21 (u.s.c. § 1396d(a)(4)(b)). the defi nition of epsdt includes necessary health care , diagnostic services, treatment, and other measures described in the medical assistance subchapter for the united states code (42 u.s.c. § 1396d (a)) (2012) that correct or ameliorate defects and physical and mental illnesses and conditions discovered by the screening services, regardless of whether such services are covered under the state plan (42 u.s.c. § 1396d (r)(5)) (2013). the medical necessity standard , which is based on clinical standards of care, refers to interventions that may be justifi ed as reasonable, necessary, or appropriate. states must comply with the fma standard to cover all treatments for a medicaideligible child's physical or mental condition, even if service coverage is optional for adults covered by medicaid. fma also bars states from arbitrarily denying or reducing the amount, duration, or scope of a required service to an otherwise eligible recipient solely because of the diagnosis, illness, or condition (nebraska legislature 2012 ). despite the provisions of fma, the u.s. department of health and human services, which oversees the medicaid program, excludes certain behavioral health treatments for children with developmental disabilities and autism (national health law program 2012 ; autism society of nebraska 2012 ). in addition, some states' medicaid contracts allow insurers more freedom than other states to deny payment for services. states also vary in who-the claimant or the insurer-must prove whether coverage provisions are adequate or fall short of federal medicaid legal standards (rosenbaum and teitelbaum 1998 ) . differences among states in approval of payment for specifi c treatments, including mental and behavioral health treatment, illustrate the need for more consistency in medicaid coverage provisions and the lack of parity between mental and physical health coverage. mental health benefi ts must be offered at parity with medical services to newly eligible recipients as part of the 2010 patient protection and affordable care act (aca), and medicaid expansion controversy is clear evidence that parity is a work in progress (mental health america 2013 ; u.s. department of labor 2008 ). because of inadequate coverage for mental and behavioral health services for medicaid-eligible children , some parents have no option other than to surrender their child to the child welfare system so that the child will receive full coverage for necessary mental and behavioral health care services. this results in signifi cant cost-shifting from medicaid to the state's child welfare system. that is, when a state provides federally mandated services to medicaid-eligible children, it receives a fi nancial match from the federal government to pay the cost s. when a state denies federally mandated medicaid services and a family surrenders a child to state custody so the child can receive care, the state pays the expense of the previously denied medicaid costs plus the expense of entitlements the child acquires as a ward of the state. the aca medicaid expansion offers a window of opportunity to increase coverage for behavioral health treatment for children with mental illnesses. although the federal government will bear the primary fi nancial burden of medicaid expansion, some states have elected, for political reasons, not to participate in this expansion. for participating states, aca medicaid expansion will replace state and local mental health services funds with federal medicaid money that will cover a wider range of home and community-based services for mental illness treatment (bazelon center for mental health law 2012 ). public health agencies and leaders often provide input for the medicaid system, helping to develop protocols, criteria, and rules about which treatments are defi ned as medically necessary . such decisions about medical necessity affect clinicians, patients, and families because they determine which treatments get recommended at the clinical level and infl uence which treatments insurers cover. you are the medicaid director of a state with the country's highest percentage of children in the child welfare system. twenty-fi ve percent of children in the state's foster care system are there not because of abuse or neglect, but because of behavioral problems and mental illnesses. as a state offi cial, you are aware that this results in signifi cant cost-shifting from medicaid to the state's child welfare system. recently, the case of 4-year-old sam has come to your attention. sam's family cannot afford mental and behavioral health care for sam, although he is medicaideligible and insured through magiscare (a private company with a state contract to administer medicaid for mental and behavioral health services). sam's parent s are considering surrendering their boy to become a state ward to get him the mental health services he needs. sam, you learn, eats random objects and dirt, throws tantrums, bangs his head on the ground, hits and bites himself and others, and often runs away. recently diagnosed by his physician as having autism, sam was referred to a psychologist who recommended outpatient behavioral therapy. both the physician and the psychologist expect this therapy to be covered through the family's magiscare plan. magiscare denied the psychologist's requests for payment on the grounds that, for children of sam's age, behavioral management is not covered under state law because it is not "medically necessary." magiscare substantiated their denial of payment because sam's behaviors primarily refl ect developmental disabilities related to autism, which are not covered under their contract with the state. when you ask the magiscare executive director about this case, she suggests that sam's parent s could attend therapy sessions to help them cope with their son's behaviors, but she reasserts that behavioral management is not covered for children as young as sam under state law because it is not medically necessary. members of the state legislature and child mental health advocacy groups are trying to expand access to home-based and community-based mental health services. they have asked you to support their efforts. you also consider that your governor, who is your boss, has publically stated his fi rm opposition to aca medicaid expansion, thus denying the state the opportunity to expand coverage for children's mental and behavioral health treatment through the aca. at present, you know that your state is offering limited mental and behavioral health service s and that narrow defi nitions of medical necessity are used to limit access to those services. as the state medicaid director, which steps should you take? 1. who are the main stakeholder s in this case, and what are their primary interests? 2. "passing" the expense of coverage denied by medicaid to other components of public service, such as the child welfare system, has fi scal and social implications. (a) what are some of these implications? (b) how should prevalence, mortality, disability, and cost be factored into thinking about ways to balance short-and long-term risk s and benefi ts to individuals and to the public in this case? 3. suppose a policy advisor warns that expanding behavioral health care for children will strain the medicaid budget and require cuts in services for adults or reduce their eligibility. (a) how should you respond? (b) which considerations or priorities would guide your funding allocations? 4. what role should ethical principle s such as stewardship , public health leadership , and moral courage play in this case? 5. medical necessity implies an acute care model of health service delivery and refl ects a clinical perspective. how well does this idea apply to a public health prevention model of health service delivery? are there better alternatives? 6. parity in insurance coverage for mental health is federally mandated for private insurers, which covers most citizens, but has proven to be an elusive goal for people who do not have private insurance or do not have enough coverage. medicaid is a public ( government funded) insurance program, not a private one. although medicaid benefi ciaries receive coverage for medically necessary mental health services, e ach state defi nes medical necessity uniquely. (a) should a federal mandate defi ne medical necessity for mental and behavioral services? (b) what fi nancial implications would such a mandate have from a state perspective and from an overall perspective? 7. the term principle-policy gap can be used to characterize situations in which most people support health coverage in principle ; but in practice, they are unable to pay for coverage or unwilling to take the political , social, cultural, or fi scal risk s necessary to enable such coverage. what do such gaps tell us about which value s the majority favors, and how might the term principle-policy gap help us understand the dynamics in this case? what roles should public health leaders play in responding to principle-policy gaps? public health systems are usually underfunded in comparison with health care systems. in fact, the organisation for economic co-operation and development (oecd) countries allocate on average only 3 % of their health spending to pub lic health and prevention activities (oecd 2011 ) . this low funding of public health programs hinders the capacity to implement effective public health policies (robert wood johnson foundation 2011 ). population health challenges, such as infl uenza pandemics, are increasingly complex, and tackling them involves urgently executing a wide array of public health measures to prevent disease transmission. in the case of infl uenza pandemics, measures can vary from border quarantine, social distancing, provision of antivirals and vaccine s, and personal hygiene strategies. recommendations often need to be made quickly even when knowledge about the seriousness and potential health and social effects are incomplete. the target for preventive interventions is the entire population. however, resources for intense and sustained health campaigns through mass communications are expensive. in addition, the social determinants of the disease must be understood and considered (crowcroft and rosella 2012 ) . this typically involves the need for policies that engage the health and non-health sectors, such as educational policies and social or economic factors (savoia et al. 2012 ). this complexity, together with decreasing funds and other factors, has contributed to increasing private sector involvement in health care. according to the world health organization (who), a public-private partnership gathers a set of actors for the common goal of improving population health through agreed roles and principle s. this may also be described as public sector programs with private sector participation (who 2013 ). who has described several types of partnerships, including philanthropic, transactional, and transformational. sponsorship is a form of a public-private partnership defi ned as "any form of monetary or in-kind payment or contribution to an event, activity, or individual that directly or indirectly promotes a company's name, brand, products, or services" (kraak et al. 2012 ) . in this sense, sponsorship is a commercial transaction, not type of philanthropy. public-private partnerships have become increasingly common for public health campaigns. some transnational companies and their corporate foundations collaborate with public institutions, such as united nation s agencies and government s, to tackle complex public health problems, such as treatment of diarrhea in developing countries (torjesen 2011 ) , tuberculosis , and malaria (ridley et al. 2001 ) . these collaboration s have been encouraged by international institutions and experts as a way to mobilize resources and expertise, which could complement the public sector. who has also encouraged using public-private partnerships to deliver health services for a range of health problems, including hiv infection, malaria , tuberculosis , trachoma, and vaccine-preventable diseases (buse and walt 2000a , b ) . however, corporations' increasing role in public health has been criticized as jeopardizing the mission of public health and its commitment to population health (hastings 2012 ; ludwig and nestle 2008 ) . some corporations have used tactics that discredit public health actions, such as distorting scientifi c information and using fi nancial tactics and political infl uence to avoid unfavorable regulations (wiist 2011 ) . public health profession als, public health agencies, and governments often must decide whether to collaborate with the private sector to improve population health. these decisions are increasingly frequent as health department budgets shrink and public-private partnerships are seen as a way to secure funds for core public health programs. ethical considerations can help us decide whether and when to form such partnerships. however, the available public health ethics frameworks (e.g., public health leadership society 2002 ; nuffi eld council on bioethics 2007 ; kass 2001 ) do not specifi cally discuss public-private partnerships. only the public health leadership society provides guidance for such collaborations. principle 10 proposes that, "public health institutions and their employees should engage in collaborations and affi liations in ways that build the public's trust and the institution's effectiveness." continued discussion about the ethical implications of private-public partnerships is needed. top health offi cials in an industrialized country have declared a public health emergency due to an infl uenza pandemic. the head of the country's health department receives a call from the president of a multinational company that produces sugary, high-calorie drinks. the company president expresses his concern about the pandemic and wants to collaborate with the government to prevent the spread of fl u. the company offers the health department a considerable amount of space, onethird of each can, on its star product (a soft drink) free of charge, to include messages on fl u prevention . the company insists that the health department logo be included on the can along with the preventive messages. for them, the association between the health department (through the logo) and their product is essential for the collaboration as it would be an acknowledgement by the health department of the company's social responsibility. the head of the health department arranges a meeting with several health authorities and offi cials to consider the offer. on one side, some members of the group support the proposal because of the need to carry out far-reaching public health campaigns to limit the impact of pandemic fl u. at that stage, the incidence of pandemic fl u is increasing quickly and the number of new outbreaks in schools is worrying the health authorities and the population . there have been recent budget cuts to the health department, and some offi cials argue the company's contribution may be the best option to ensure a far-reaching campaign on prevention measures to benefi t the population. they see sponsorship as a form of social responsibility because the company does not have any apparent economic interest in fl u-related activities. they also note that there are no other companies offering a similar collaboration. but other offi cials say the company's soft drink products contribute to the obesity and diabetes epidemic and that the company's use of the health department logo would label it a pro-health industry with the backing of the highest health authority in the country. they also raise concerns about risking the independence of the health department in future regulatory action on sugar-rich beverages. as the hea d of the health department, you must decide if you should collaborate with the company. 1. what considerations should the health department director weigh when deciding whether to collaborate with the beverage company? 2. who are the major stakeholder s the health department should consider, and what value s might each of these stakeholders bring to this decision? 3. in making your decision, what values should be prioritized? 4. what positive or negative impacts would displaying the health department logo on the soft drink cans have on health department operations? 5. how might sponsorship by a company that produces sugary beverages affect public trust in the health department and the institution's effectiveness? 6. would the decision be different if the company produced healthy foods and the department's logo was placed on a healthy product? 7. would community involvement facilitate decision making and the consideration of the ethical questions? what ethical criteria or guidance should be established to accept or reject a future donations or sponsorship of a public health program by a company? preterm births, the leading cause of infant mortality, are increasing annually worldwide (world health organization 2012 ). the united state s shares company with nigeria, india, and brazil among the top ten countri es with the highest numbers of preterm births and ranks 31st among organisation for economic co-operation and development ( in 1900, about 100 infants died per 1000 live births. by 2000, that number fell to 6.89. during the last half of the twentieth century, the rate of black infant mortality dropped dramatically. in 1950, black infant mortality was 43.9 deaths per 1000 live births compared with 26.8 deaths per 1000 live births among whites (mechanic 2002 ) . but by 1998 black infant mortality fell to 13.8 deaths per 1000 live births compared with 6.0 deaths per 1000 live births among whites. as these numbers show, both groups made signifi cant absolute gains, with blacks gaining more in absolute terms-a reduction of 30.1 for blacks and 20.8 for whites. yet, black infant mortality still remained about twice that of whites. these disparities have persisted in the twenty-fi rst century. in 2006, non-hispanic black women experienced the highest rate of infant mortality, with 13.4 infant deaths per 1000 live births, while non-hispanic white women had a considerably lower rate, with 5.6 infant deaths per 1000 live births. citing a 2006 report from the national healthy start association, macdorman and mathews ( 2009 ) report that programmatic efforts to reduce disparities in black-white infant mortality have had some successes at local levels, but eliminating the disparities is diffi cult. the u.s. centers for disease control and prevention and the u.s. department of health and human services have prioritized both the elimination of health disparities and improvement in overall population health. these twin goals-one distributive, the other aggregative-are separate and sometimes confl ict (anand 2004 ) . increases in health disparities often accompany advances in aggregate gains in population health (mechanic 2007 ) . although this case is specifi c to the united state s, the dilemma is not. data show that signifi cant progress on child mortality has been made in many countries but that this overall success is often coupled with increased inequalities between advantaged and disadvantaged groups (chopra et al. 2012 ) . in china and india, for example, disparities in mortality persist between boys and girls younger than 5 years, a function of entrenched gender discrimination (you et al. 2010 ) . these examples raise challenging questions about how ethically to assess such cases and set priorities for the allocation of scarce public health resources. you serve as the director for the local health department in a racially segregated urban city in the midwest with one of the greatest concentrations of african americans in the united states. the city has a long history of civil rights activism that led to protests and marches that ultimately empowered and mobilized black communities and organizations. your health department has a history of prioritizing maternal-child health and the elimination of black-white disparities in infant mortality in its programs, an investment of resources affi rmed by the city residents through the department's community outreach program and planning processes. chronic underfunding of public health, made worse by the economic downturn, has resulted in drastic and unprecedented reductions in the public health budget. in consultation with your staff and community board of health, you have raised the possibility of redirecting resources from maternal-child health into other programs based on a number of practical and ethical considerations. as with national statistics, the city has seen signifi cant declines in black infant mortality, even as blackwhite disparities remain. you note that although the maternal-child health programs are cost-effective, their impact on reducing black-white disparities seems to have stalled. other programs appear to meet targets more consistently. to help support these other programs, you note that allocating resources to more effective programs provides more "health" per dollar, thus meeting the utilitarian demand to maximize overall health, which many view as the primary goal of public health and health policy (powers and faden 2006 ) . in addition, although black-white disparities in infant mortality persist, blacks have made signifi cant gains, declining more than whites in some decades. you note that remaining inequalities could be deemed ethically acceptable by some standard s of equity , such as the "maximin" principle . although this distributive principle is subject to interpretation (van parijs 2003 ) , it is generally understood to require that social and economic inequalities work to benefi t society's least advantaged groups. thus, inequalities (even signifi cant ones) are morally acceptable as long as the least advantaged have signifi cantly benefi ted (powers and faden 2006 ) . the director of community outreach proposes that the health department not make this decision unilaterally, but instead listen to community opinions on these questions of priorities and fairness. he suggests that the health department collaborate with community partners to host a series of public forums. he insists that a topic of such historic and contemporary concern to the community must be subject to public deliberation. despite having a history of supporting community discussions, you are concerned about the cost of community forums, noting that they will drain resources from an already slim budget. the chilean sy stem of guarantees in health-created by law in 2004-aims to establish guaranteed health care interventions in health promotion, disease and injury prevention , diagnosis and treatment , rehabilitation and palliative care (ministerio de salud 2004 ) . the law mandates that public and private insurers provide the resources needed to protect the public against excessive health-related spending and guarantee timely and universal access to authorized interventions based on standard s of care. 1 national health objectives, established by the ministry of health, determine the list of guaranteed interventions. this list, however, is reviewed every 3 years and amended as new scientifi c and health information emerges. as of 2013, the system o f guarantees in health included interventions for 80 health-related conditions (ministerio de salud 2013 ), accounting for almost 60 % of the chilean burden of disease. the system of guarantees in health is a priority system based on acknowledged criteria, namely scientifi c evidence and socially shared value s. for the system to be effective, the criteria must be transparent, publicly accepted, and open to review and modifi cation. the law that created the system of guarantees in health also mandated a procedure for selecting the guaranteed interventions (ministerio de salud 2004 ) . the procedure factors in public opinion research to identify social consensus on health priorities, studies to identify effective interventions that prolong and improve quality of life, and assessments of interventions' cost effectiveness (burrows 2008 ). the procedure determines priorities with an algorithm that includes these factors and information on disease burden and health system capacity (missoni and solimano 2010 ) . after choosing the health interventions, the health ministry elaborates on a package of interventions related to specifi c health conditions and develops clinical guide lines for such interventions. you direct a team within the ministry of health that is responsible for recommending priorities for guaranteed health interventions. the priority ranking system emphasizes the selection of cost-effective interventions for conditions with the greatest burden. however, the health ministry also has authorized including expensive interventions that are less effective or treating health conditions with low prevalence, if that condition or those interventions signifi cantly impact health. because of budget reductions, a number of interventions are under review. your team has been asked to recommend funding interventions for two health conditions-cataract (a common condition with highly effective treatment ) and multiple sclerosis (a less prevalent condition but one with signifi cant health and social impact). cataract, the main cause of blindness, primarily affects people over 40. this health problem has a high impact as measured by quality-adjusted life years (qalys) (ministerio de salud 2007 ) . its surgical treatment is effective for 80-95 % of patients. the package of guaranteed interventions includes diagnostic confi rmation within 180 days after suspected diagnosis and surgical treatment 90 days after confi rmation. in 2013, it was expected that 48,424 cataract surgeries would be performed in chilean public hospitals and 416 in private institutions. multiple sclerosis , an autoimmune infl ammatory disease leading to demyelination in the central nervous system, produces a progressive deterioration of health and quality of life. it represents a minimal disease burden at the population level, mainly due to premature death. in chile, it is estimated that 385 patients are treated for multiple sclerosis each year. the package of guaranteed interventions includes diagnostic confi rmation within 60 days; confi rmed cases must receive treatment within 30 days. treatment includes pharmacological therapy and p hysiotherapy. contingency arrangements. to make the best use of resources and personnel (even in the absence of a pandemic), patients are triaged-evaluated to determine the type and priority of care to be received. while medical information informs the development of triage criteria, ethical considerations about triage goals-whether explicit or implicit-also play a role. for public health emergencies that overwhelm capacity, some propose adjusting critical care triage criteria to emphasize certain public health goals, like saving the most lives possible (christian et al. 2006 ; silva et al. 2010 ) . some contend that utilitarian reasoning should predominate in critical care triage, based on the intuition that, when resources are scarce, allocation decisions should produce the greatest good for the greatest number (charlesworth 1993 ; childress 2004 ) . critics of utilitariani sm reply that it requires coercion or covertness to succeed, because the public will not voluntarily sacrifi ce their lives or their loved ones for the greater good (baker and strosberg 1992 ). utilitarian triage may be unpalatable to the public on the further ground that it quantifi es and judges the value of one life over another, which could disproportionally impact particular population groups (hoffman 2009 ). others therefore would base triage decisions on egalitarian considerations, for instance, by giving everyone an equal chance at obtaining a scarce good, an approach for which historical precedent exists (baker and strosberg 1992 ) . whatever approach is adopted, prior arrangements between policy makers, practitioners, and the public based on thoughtful, transparent deliberation about the most ethical approach to ccu triage usually will improve the legitimacy of d ecisions. those who promote an approach based on fairness and equity need to consider that, during public health emergencies, the goal of saving lives may force a retreat to utilitarian ethics (kirkwood 2010 ; veatch 2005 ) . while not necessarily unethical in itself, a retreat that overturns prior arrangements lays itself open to charges of illegitimacy. variability in the frameworks used to allocate public health resources illustrates the importance of refl ecting upon the value s that undergird policy decisions and individual practices, like critical care triage. appealing spontaneously in the heat of the moment to values that have not been adequately refl ected upon or discussed in a transparent and deliberative manner may lead to undesirable outcomes and accusations of unethical practices. while discussions of ccu triage criteria ultimately concern institutional clinical policy and practice, they refl ect a larger discussion about the overarching public health goals in the face of large-scale, widespread public health emergencies, like pandemics. an outbreak of a novel infl uenza virus has progressed to the point that the world health organization has declared a pandemic. in the pandemic's fi rst wave, hospital capacities were suffi cient to handle the infl ux of pandemic infl uenza patients, whose morbidity and mortality rates mirrored rates for seasonal infl uenza. however, despite a vaccination campaign and other measures, such as ensuring surge capacity, rates of morbidity and mortality associated with the virus have increased drastically during the pandemic's second wave. the resulting increased number of patients needing hospital beds has overwhelmed even the surge capacity of the ccus of a metropolitan city's tertiary care hospitals. to meet this challenge, a teleconference has been scheduled between several members of the hospitals' administration, the ccu directors from each hospital, and public health offi cials involved in leading the jurisdiction's pandemic response. as a public health offi cial who played a central role in developing the pandemic plan for your jurisdiction, you have been included on the call to provide guidance for the pandemic response. during the meeting, a number of ccu directors report that their physicians and nurses are concerned about the type of patients bein g admitted into the ccu. some of the directors see a trend that they suggest is ultimately undermining the effi ciency of the pandemic response. they argue that, as the severity of the pandemic continues to increase, their triage criteria should be modifi ed so as to use ccu resources to save the most lives possible. they worry that admitting those who present with the most need is preventing treatment of those who will benefi t most from ccu admission. "so long as our triage scheme saves the most lives, it is ethically justifiable" a number of them declare. the group takes up the proposal of a ccu director to triage according to sequential organ failure assessment (sofa) scores-which are derived using a tool that determines a patient's organ function and failure rate to predict outcomes (vincent et al. 2000 ) . were the pandemic's severity to increase, the group suggests that, in addition to the ccu director's proposal to use sofa criteria, even more inclusion, exclusion, and priority criteria could be added with the goal of saving as many lives as possible. they've proposed exclusion criteria for ccu admittance that include patients with a poor prognosis, patients with other known health issues, and some mention of age cut-offs, to name a few. others involved in the teleconference question whether this is the right approach to take. they argue that, by aiming to save the most lives possible, those who may benefi t less from ccu admission, like older adults or individuals with disabilities, will be unfairly affected. they say, "we should not just aim to save lives, but rather save lives fairly ." as you and your public health colleagues are leading the pandemic response, the hospital administrators and ccu di rectors look to you for a recommendation or decision about how to proceed. 1. ensuring that the ccu has surge capacity is a common strategy to accommodate an infl ux of patients who have been infected with pandemic infl uenza. (a) does surge capability require alternative critical care triage criteria? (b) if the population's health needs exceed contingency arrangements, should alternative critical care triage criteria be used? (c) how should these decisions be made? (d) what principles, value s, or processes should infl uence these decisions? 2. what considerations might exist during a pandemic that do not exist in everyday critical care and critical care triage that do or do not support the modifi cation of triage criteria? if pandemic critical care triage requires a unique conceptual framework, what principles ought to be valued in such a framework (e.g. need, equality, utility, effi ciency)? 3. would the severity of a pandemic ever warrant the use of a utilitarian scheme for critical care triage, given that the public generally fi nds it unpalatable and carrying out such a plan could require coercion? how could an adverse public reaction to coercive or covert measures be mitigated? 4. in a pandemic, the most seriously ill patients with the lowest probability of being saved might be left untreated because their care would require too many resources with little prospect of recovery. this illustrates a confl ict between the common good and the best interests of individual patients. what other confl icts might arise when triaging in a pandemic? 5. triage can be used to maximize the number of lives saved with available resources. should we aim to maximize the number of lives or, alternatively, the number of life years saved? this can also give rise to questions about the quality of those lives and years lived. is it ever appropriate to make allocation decisions based on quality of life or life years? ethical issues in recipient selection for organ transplantation priority to the worst off in health care resource prioritization ethical issues in the use of cost effectiveness analysis for the prioritization of health care resources the effi ciency frontier approach to economic evaluation of health-care interventions rationing fairly: programmatic considerations how to achieve fair distribution of arts in "3 by 5": fair process and legitimacy in patient selection just health: meeting health needs fairly setting limits fairly: learning to share resources for health benchmarks of fairness for health care reform qaly maximization and people's preferences: a methodological review of the literature cost-effectiveness in health and medicine morality, mortality: death and whom to save from it toward a broader view of values in cost-effectiveness analysis in health care cost value analysis in health care: making sense out of qlays bureaucratic itineraries in colombia: a theoretical and methodological tool to assess managed-care health care systems autonomía médica y su relación con la prestación de los servicios ?option=com_content&view=article&id=1335:la-tutela-y-el-derecho-a-la-salud-2011&cat id=58:libros&itemid=104 . accessed the history and principles of managed competition serie de notas técnicas sobre procesos de priorización de salud: introducción a la serie de priorización explicita en salud why neoliberal health reforms have failed in latin america structured pluralism: towards an innovative model for 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cdc's offi ce of minority health & health equity (omhhe) understanding the determinants of the complex interplay between cost-effectiveness and equitable impact in maternal and child mortality reduction infant deaths-united states the challenge of infant mortality: have we reached a plateau? disadvantage, inequality, and social policy population health: challenges for science and society oecd health data: infant mortality social justice: the moral foundations of public health and health policy hhs action plan to reduce racial and ethnic health disparities difference principles born too soon: the global action report on preterm birth levels and trends in child mortality priority setting in healt h care: ethical issues m. inés gómez and lorna luco centro de bioética, facultad de medicina clínica alemana-universidad del desarrollo what are some of the ethical, scientifi c, and social considerations that should be weighed in deciding if interventions for both cataract and multiple sclerosis should be covered by the system of guarantees in health? is there an obligation for health systems to cover all health problems affecting a population? are there limits? how should health problems be prioritized and who should have the authority to make these decisions? which criteria should receive the most weight in ranking priorities? how should resources be distributed among health conditions affecting many people versus health conditions affecting few people? how should resources be distributed among procedures that are preventive versus treatments for existing conditions? how does taking a public health perspective versus a clinical medicine perspective affect your thinking about including these two conditions in the system of guarantees in health? what role should transparency play in the selection procedure? references burrows establece un régimen general de garantías en salud estudio carga enfermedad y carga atribuible decreto supremo no. 4. aprueba garantías 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scoring systems for assessing organ dysfunction and survival addressing ethical issues in pandemic infl uenza planning: discussion papers the authors thank student chelsea williams for her assistance in assembling the facts of the case. we also thank creighton university's center for health policy & ethics. acknowledgements we thank mr. jonathan whitehead for language editing. open access this chapter is distributed under the terms of the creative commons attribution-noncommercial 2.5 license ( http://creativecommons.org/licenses/by-nc/2.5/ ) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. the images or other third party material in this chapter are included in the work's creative commons license, unless indicated otherwise in the credit line; if such material is not included in the work's creative commons license and the respective action is not permitted by statutory regulation, users will need to obtain permission from the license holder to duplicate, adapt or reproduce the material. this case is presented for instructional purposes only. the ideas and opinions expressed are the authors' own. the case is not meant to refl ect the offi cial position, views, or policies of the editors, the editors' host institutions, or the authors' host institutions. infectious diseases such as pandemic infl uenza and severe acute respiratory syndrome (sars) have attuned the attention of policy makers and health practitioners to the importance of protecting and promoting the public's health in the face of increased care needs and extreme resource scarcity. in particular, acute care needs for the critically ill and discussions of treatment priorities have been the subject of much debate in pandemic planning (hick et al. 2007 ; melnychuk and kenny 2006 ; uscher-pines et al. 2006 ). this is not surprising, as it has been estimated that more than 700,000 americans may require mechanical ventilation during a pandemic, far outnumbering available ventilators (rubinson et al. 2010 ; u.s. department of health and human services 2005 ) . additionally, shortages of hospital beds, personnel, and other equipment can be expected during a pandemic, which may limit the ability to meet an expected increase in patient volu me (world health organization 2008 ).prudentially planning for the public's increased care needs during a pandemic requires assessing surge capacity, especially in critical care units (ccu). however, as pandemics increase in severity, they can overwhelm critical care capacity and key: cord-016826-oatjcmy0 authors: arata, andrew a. title: old and new pestilences date: 2005 journal: understanding the global dimensions of health doi: 10.1007/0-387-24103-5_3 sha: doc_id: 16826 cord_uid: oatjcmy0 a. “any fatal epidemic disease, affecting man or beast, and destroying many victims.” the oxford universal dictionary, 3(rd) edition, 1955, oxford press, 2515 pp. b. “a contagious or infectious epidemic disease that is virulent and devastating.” webster’s seventh new collegiate dictionary, 1965. g. & c. merriam co., springfield, mass., 1221 pp. accordingly, a pestilence should be an infectious disease, devastating (killing) a large number of people (or animals). there has been much popular interest in, as well as technical concern over, newly emerging diseases, and there is a fear that heretofore unknown virulent pathogens will create new, global epidemics. at the time of this writing, two such pathogens are active, warranting such concern: a) cases of sars (severe acute respiratory syndrome, caused by a coronavirus) appeared in china in november, 2002, and has spread to western and central europe and north america; b) a strain of avian influenza virus (n5h1), first identified in hong kong in 1997, reemerged in 2002 in southeast asia. other avian flu strains found simultaneously in poultry in north america have underscored the concern of local and international health authorities. both sars and avian flu demonstrate high mortality rates, but, to date, the number of cases has been only in the hundreds. so, are these pestilences? what constitutes a pestilence? is the term synonymous with newly emerging diseases? two definitions of pestilence have near unanimity, but are not very specific: a) "any fatal epidemic disease, affecting man or beast, and destroying many victims." the oxford universal dictionary, 3 rd edition, 1955, oxford press, 2515 pp. b) "a contagious or infectious epidemic disease that is virulent and devastating." webster' s seventh new collegiate dictionary, 1965. g. & c. merriam co., springfield, mass., 1221 pp. accordingly, a pestilence should be an infectious disease, devastating (killing) a large number of people (or animals). the truly epidemic diseases are usually of viral or bacterial origin (although we will make a case for some other types of pestilences). the classification of that may have been bubonic plague continuing until a.d. 750). the "black death" of the fourteenth century, which continued to appear in chronic pockets of europe and the middle and near east for centuries thereafter, is by far the best-known plague, and the one that produced the greatest mortality and social impact on the affected populations. consider that as a conservative estimate, 25−33% of the european population died, and maybe more. this death rate reduced the available work force so that, for the first time, peasants and landless people could sell their labor, which introduced freedom of movement and resulted in economic changes that eventually contributed to the decline of the feudal system. the 3 rd pandemic of plague began in the 1850s and continues to this date, although reduced in more recent years. a major characteristic of this epidemic has been the dissemination of plague from its traditional homes in africa and asia into areas previously plague free, especially north and south america, by the inadvertent transport of rats and their fleas by boat. the infection is now well established in africa (gerbils); central asia (gerbils, ground squirrels or "susliks," and marmots); southeast asia (various rattus species); north america (ground squirrels and some native field mice); and south america (introduced rattus). the last major urban outbreak was in surat, india in 1994: more than 6,500 cases and 56 deaths were reported. however, the impact of this outbreak was also seen in the number of people who fled the plague zone, and the over two billion dollar loss that ensued. only a few outbreaks are reported to who each year. indochina and burma frequently report, as well as sites in africa (ovamboland on the frontier between namibia and angola); the united states has a vast infected area in the west of the country, but only 8-10 cases per year are reported, with 1-2 deaths on average every 10 years. many other sites of infection are known and should be monitored, as some rodent species are highly susceptible to serving as effective amplifying reservoirs, whereas others maintain low-level infections for long periods of time, allowing much time to pass between outbreaks. environmental measures (rat-proofing, rodent and flea control, etc.,) are the first measures of control. surveillance, prompt diagnosis, and treatment with antibiotics (e.g., streptomycin and tetracycline) are recommended. yellow fever is the best known of the arboviral (arthropod-borne virus) diseases. there are some 500 known arboviruses, of which about 100, produce disease in man. both the yellow fever virus and the primary mosquito vector, aedes aegypt, are of african origin -the species name, 'aegypti,' refers to classical africa in general, not, specifically, modern egypt. most cases in africa occur east to west along the transition zone (ecotone) between the savannas and the rain forests inhabited by numerous aedine vectors as well as a. aegypti. the disease has two cycles: a 'jungle' cycle involving various tree dwelling mosquitoes and nonhuman primates as reservoirs, and an 'urban' cycle, with a. aegypti as the vector and humans as reservoirs. the last reported major african outbreak vectored by a. aegypti occurred in nigeria and involved some 20,000 cases and over 4,000 deaths between 1986 and 1991. yellow fever was introduced into the americas one or more times most likely during the age of sail: the virus can be transmitted vertically (transovarian passage) in a. aegypti. the mosquito' s eggs can easily be laid in water barrels and withstand desiccation for months, only to hatch and develop when submersed at a later date. epidemics of yf raged throughout the caribbean and tropical america until the end of the 1800s, when the transmission cycle was elucidated by the team led by walter reed, confirming the role of a. aegypti which had been proposed by, but not confirmed by, carlos finlay. epidemics occurred as far north as philadelphia in the united states and the last epidemic in north america occurred in new orleans as late as 1905, with over 3,000 deaths. cases (with 25-50%) mortality continue to occur sporadically in brazil and in the foothills of several andean countries (bolivia, peru, ecuador, and colombia) . often the victims are young, indigenous males from the highlands who were temporarily working in the coca processing plants in the forests. these infected areas are only kilometers from large cities (with populations of more than 1 million people) such as santa cruz, bolivia, which are accessible by public transportation and are heavily infested with a. aegypti. although the yf vaccine is one of the oldest, safest, and most effective available, and immunological protection is rated for at least 10 years, vaccination coverage in many of the affected areas of africa and south america is low. the cholera pathogen, vibrio cholera, originally described by robert koch, was one of the first human pathogens (along with anthrax and tuberculosis) to be identified, in the late 1800s, shortly after pasteur' s publication of the "germ theory." koch and his students studied material they collected in alexandria, egypt, during an 1883 outbreak. it was difficult to determine the origin of cholera and/or to distinguish it historically from other diarrhetic diseases except by the severity and rapidity of onset. health historians such as mcneill suggest an origin on the indian sub-continent, associated with dense populations, poor hygiene, and certain religious practices such as communal bathing; thus the term "asiatic cholera," by which the disease became known in europe in the 1800s. the disease' s appearance in europe and the americas (london and new york in 1832, and again in 1854) were clearly associated with intercontinental traffic. it was during the 1854 epidemic in london that a physician, john snow, noted the clustering of cases and deaths in people using the same water source, and proposed what turned out to be the correct action to stop the epidemic ("take the handle off the broad street pump!!"), although he had no idea of the actual cause of the disease. however, it was such observations, along with structural, hygienic, and administrative changes in major cities, particularly in europe and north america, that established the public health measures that we tend to take for granted in this early part of the 21 st century. cholera is still with us: various serotypes of the vibrio have spread since the early 1960s, affecting over 25 countries in asia, 21 in the americas, and into the west pacific. in 1993, approximately 400,000 cases and 6,800 deaths from cholera were reported. in 1991, the el tor strain of cholera was reported in lima, peru; by 1994, almost a million cases had been reported in the western hemisphere. measles is one of the oldest known and most widespread infections of man: epidemics ascribed to measles appear in the oldest literature, although they are often confused with smallpox. however, in 622 a.d., ad ahrun, a christian priest living in alexandria, egypt, described the pox lesion, and in 910 a.d. the arab physician al-razi distinguished between the two diseases. prior to widespread immunization, measles was common in childhood-more than 90% of people were infected by age 20. although endemic in large communities, measles became epidemic every several years, with the severity of infection decreasing with the frequency of the epidemics. in his study of the history of plagues, mcneill makes mention of the importance of animal husbandry and zoonotic diseases in the area. measles, he claims is probably related to both rinderpest (in hoofed-mammals) and canine distemper. because dogs, sheep, and goats have been domesticated for at least 10,000 years, measles may have been among the first viral diseases to have "jumped the species barrier." as we will see, most, if not all, of the new pestilences are, or may be, derived from animal wild or domesticated reservoirs. measles was responsible for (or contributed to, along with smallpox) the decimation of the indigenous amerindian populations, first in central and south america at the time of the spanish conquest (1500s), and later (1700s and 1800s), in north america. amerindian populations lacked immunological protection from these and other imported infectious diseases. some attribute this immunological naïveté to the comparatively small number of domesticated animal species-dogs, ducks and turkeys, guinea pigs, and cameloids (llamas and relatives) in the andes, and few, if any, in large number prior to the european invasion. in any case, the attack and mortality rates were staggering. by one estimate, a pre-conquest amerindian population of perhaps thirty million by 1556 was reduced by 90%, down to only 3 million. this catastrophe occurred in less than 50 years after the spanish entered the american mainland. influenza is another viral disease that has many unstable varieties infecting a host of mammalian and avian species, both wild (sylvatic) and domestic. epidemics with symptoms similar to modern influenza were noted by hippocrates as early as 412 b.c., and later, in rome, by livy. various medieval and renaissance writings describe influenza-like illnesses. robert johnson of philadelphia is credited with the first "modern" description of an influenza epidemic, which occurred in that city in 1793. his description was applied to subsequent epidemics in 1833, 1837, 1847, 1889-90, and 1918. antigenic shifts in the structure of the influenza virus may change the virulence of the strains, increasing the likelihood of epidemics. the most severe flu epidemic ever recorded (1918) (1919) -also known as the spanish flu (although it did not originate there)-first struck world war i troops of all combatant nations while in northern france, and it continued on to become a global pandemic. conservative estimates of mortality range between twenty and forty million persons, and other estimates more than double these figures. the ease with which the various influenza strains infect domestic mammals, pigs, and poultry (chickens and ducks) producing huge reservoirs of potentially infectious material, often proximate to human habitations, is a major public health concern. especially worrisome are the conditions under which millions of such animals are raised and brought to market. the 'old' diseases examined above are only a few of those which might be used as examples of the old pestilences: others might prefer to include schistosomiasis, typhus (murine and/or louse-borne), and several of the classic childhood diseases (diptheria, pertussis, tetanus, rubella), as well as leprosy, yaws, the leishmaniases, and, certainly, smallpox. fortunately many of those mentioned here (schisto and others) are being controlled rather well in some areas by vaccines, specific drugs, and/or antibiotics when applicable, at least in the more developed countries. even polio, which had been a major epidemic threat for centuries, has been virtually eliminated as a threat in areas where the politics and health infrastructure allow the efficient application of this very effective vaccine. much of the fear engendered by specific diseases depends on the time, place, and severity of the local outbreaks, as well as the knowledge and perception of the community. for example, i was raised in new orleans, in the southeast of the united states, during the 1930s. although i and my brothers were normal, well nourished children, our parents were fearful of dogs (rabies), cuts on unshod feet (tetanus), and any summer colds or stiffness/weakness of the extremities (polio), and they preached cleanliness as a means to prevent anything bad happening. these diseases are old, but at present each has developed certain new characteristics that make their modern expression different from their historic ones, and decreases our ability to control them. in the last 50 years, malaria parasites have developed resistance to chloroquine, the most common, globally used anti-malarial drug; at the same time, the anopheline mosquito vectors of malaria have progressively developed a parallel resistance to the insecticides used to control them. dengue, and dengue hemorrhagic fever (dhf), have spread globally, infecting vast new areas, especially urban areas where the human living conditions are substandard, but readily suited for vector breeding. finally, tuberculosis, whose incidence was slowly reduced in the late 1800s and early 1900s by improved public health, housing conditions, and nutrition, has again surfaced as a secondary infection to immuno-compromised persons, especially those suffering from hiv infections. at the same time, the causative agent, mycobacterium tuberculosis, continues to develop resistance to the most economic and readily available antibiotics. malaria is caused by blood parasites of the genus plasmodium and vectored by anopheline mosquitoes. there are four species of human malaria parasites: p. falciparum, p. malariae, p. vivax, and p. ovale, as well as a number of related species infecting other mammals (non-human primates, rodents, etc.). historians note that malaria-like symptoms were discussed in the chinese canon of medicine (2700 b.c.) and malaria-like illnesses were described in 6 th -century b.c. cuneiform literature from nineveh (now part of iraq). hippocrates made a connection between stagnant water and fevers in the local population. it is estimated that there are still several hundred million unreported cases each year resulting in 1-2 million deaths per annum, mostly children. although malaria is still endemic in asia, latin america, and africa, 90% of the cases are found in africa, where p. falciparum is the most common malaria parasite. such huge figures mask the focal, and sometimes epidemic, nature of malaria, which may be brought about by natural or man-made environmental conditions. some of the human activities that may enhance malaria transmission may be development projects for agriculture (e.g., irrigation schemes), other water and land use projects (as in the amazon basin, converting forest areas through resource extraction such as mining and logging) into marginal livestock and farming areas. often such environmental changes bring about changes in malaria transmission from 'stable' (endemic) to 'unstable' (epidemic). in highly endemic areas, severe malaria and death is concentrated in the younger age groups, whereas in the areas of unstable (epidemic) transmission, severe malaria and death is more evenly distributed throughout all age groups. needless to say, prevention and /or case control strategies must be different for each transmission type. in many parts of the world the anopheline vectors of malaria have developed resistance to the insecticides used for their control. frequently, this is due to the use, often excessive, of the same or similar insecticides for control of agricultural pests in the same geographic areas. such resistance not only hinders control operations directly, but also indirectly, by increasing the need for greater quantities and/or more costly insecticides. broadscale usage of insecticides has also become limited on environmental grounds, because some donors have reduced funding insecticide purchases. by far the most serious setback to malaria control in recent decades has been the emergence and spread of chloroquine-resistant strains of p. falciparum, the causative agent of the most severe form of malaria, and the most common in africa. emerging in the 1950s in southeast asia and south america, resistance spread rapidly from these focal points. it was not noted in africa until 1979-80 but spread rapidly in the ensuing ten-fifteen years. chloroquine-resistant strains of p. vivax have been identified in some areas of southeast asia, new guinea, and indonesia. efforts to produce a malaria vaccine(s) have been under way for over 25 years. a number of candidate vaccines have been produced, but none are operational in humans as yet. like yellow fever, described earlier, dengue and dengue hemorrhagic fever are vector-borne diseases transmitted (primarily but not exclusively) by the mosquito aedes aegypti. "classical" dengue is caused by infection with one of the four serotypes of the dengue virus. dhf may occur following a subsequent infection with a different serotype. the following quotation is from an article written by the author in 1999 (r. lennox and a. arata, dengue fever: an environmental plague for the new millennium. capsule report, environmental health project/usaid. 8 pp.): with 2.5 billion people at risk and estimated cases in the tens of milllions, dengue is considered by many to be the second most important vector-borne disease in the world (surpassed only by malaria). classical dengue and its more lethal form, dengue hemorrhagic fever (dhf), now circle the world with endemic illness and continuing threats of epidemics. dengue is very much an environmental disease, affecting urban and periurban settlements in more than 100 countries. it is characterized by seasonal outbreaks of illness carried by mosquitoes that thrive in household containers which collect water (such as flowerpots and washtubs) and in the detritus of human consumption, such as bottles, tin cans, and old bottles. children, specially in asia, are most frequently and seriously affected by the severe form of the infection, dhf. mosquito control is the only effective approach to prevention, although effective case management will reduce mortality. insecticides targeted at larval mosquitoes are effective, but resistance of mosquitoes to affordable and environmentally safe chemicals as well as declining will and infrastructure have all but eliminated this approach in most countries. vaccines are in the pipeline, but a system which could deliver them to half the world' s population is probably at least a decade away. community action-to protect containers from becoming havens for mosquito breeding and to dispose of empty containers and trash, along with surveillance and personal protection-is the best hope for transmission risk reduction. tuberculosis is another ancient disease that has bridged the old to new definition: the tb bacillus, mycobacterium tuberculosis, was among the first to be scientifically identified and described (by robert koch, in 1882). the disease is transmitted by airborne droplets from people with pulmonary or laryngeal tuberculosis. this mode of transmission is most effective in dense populations, and hence tb became widespread with the development of urban centers in the middle ages (europe), and was very common from the 14 th century until recently in europe. with improvements in housing and nutrition tb rates continued to decline (except for periods of war) until the first half of the 20 th century. at that time, two conditions emerged: the development of multiple drug resistant tb (mdrtb) and the emergence and spread of acquired immune deficiency syndrome (aids) upon which tb is an opportunistic infection. prior to 1984, about 10% of tb bacilli isolated from patients in the u.s. were resistant to even one antibacterial drug: in 1984, 52% were resistant to at least one drug, and 32% were resistant to more than one drug. in the u.s. the cost of treatment of ten cases of mdrtb in texas in 1990 was us$ 950,443. who lists tb as one of the major causes of mortality in the world. a new major funding effort (who and world bank and various bilateral donor groups) is focusing on hiv/aids, tb, and malaria as the most serious, and intractable, causes of death. other forms of tb, including non-pulmonary cases and those associated with other species of mycobacterium sp. (e.g. m. bovis), are sporadic, but suggest the possible very early animal origin of the pathogen group. diseases such as chagas disease and schistosomiasis are examples of diseases that do not easily fit the epidemic definitions of a pestilence mentioned earlier in this chapter, but they do heavily impact the affected populations, not only through mortality rates, but especially through morbidity/disability. there are several forms of schistosomiasis caused by different species of schistosoma, a blood fluke (trematode)-this is an ancient illness, known from egyptian antiquity. infections occur in fresh water where people work and/or wash and children play. larval worms, known as cercaria, developed in a snail intermediate host, pass through the skin and penetrate diverse organs according to species. the most important effects are those that arise from chronic, and cumulative, infection. chagas disease has a very different etiology, mode of transmission, and pathology than does schistosomiasis. by definition it could be new because it was first described in 1907 by the brazilian carlos chagas, who subsequently described the pathogen, a flagellate protozoan, trypanosoma cruzi, and the vectors, bloodfeeding triatomine bugs. the disease is also know as american trypanosomiasis, and occurs only in the western hemisphere, from mexico to argentina-a few cases have been reported in north america. this form is very different from african trypanosomiasis (sleeping sickness). the initial (acute) phase of the disease usually occurs in children; there is then a long latent phase (∼20 years or more), culminating later in life in a chronic phase which may include irreversible cardiac and/or intestinal manifestations and shortened life spans in the victims. paho and who consider chagas disease to be the most serious parasitic disease in latin america and the main cause of heart disease in the region. there is no adequate medical intervention. the infection can be transmitted by vectors, congenitally, or by transfusion of blood or blood products. an estimated 100 million persons in the region are at risk, and in some countries (e.g., bolivia) 25% of the 8 million inhabitants have been shown to be seropositive. in addition, in bolivia, one study demonstrated that the burden of chagas disease, in terms of disability adjusted life years (dalys), was 4 million dalys, or estimated loss of 494 million bolivianos: equal to more than 100 million us dollars at the time of the report (1994). the purpose of this brief segment is to emphasize that pestilences need not carry with them only high mortality. very high morbidity and sustained disability with all the concurrent social and economic implications can be a tremendous burden on a population-or a nation. puerperal fever, a forgotten pestilence, is caused by a streptococcal infection and is an iatrogenic disease (induced by a physician) that was once the scourge of pregnant women, before physicians learned to wash their hands before examining pregnant women and/or assisting at childbirth. improved hygiene in hospitals was concurrent with the development of the germ theory and mortality rates dropped quickly. this disease, also called childbirth fever, was never reported as one of the great pestilences, however a few figures reveal the state of scientific knowledge regarding any infectious diseases, both endemic and epidemic. it is frightening that not only was the incidence of puerperal fever higher in the hospitals, but so was the associated mortality: 35 % of the patients died if the disease occurred after a home delivery, but 80-90 % died if the disease was contracted in a hospital. although we have no crystal ball to predict what, if any, new pestilences are in store for mankind in the future, several groups of zoonotic viruses include likely candidates (table 2) . also included is hiv/aids, truly a new pestilence that already, in a relatively brief period, has taken its place among the worst pestilences ever known to man. as mentioned above, there are over 500 arboviruses isolated and characterized-about 100 are capable of infecting humans, from nonapparent infections to very severe ones. two of these have already been mentioned above (dengue and dhf and yellow fever), but the arboviruses as a group represent the source of many potentially new diseases-or, put more correctly, existing zoonotic diseases that emerge when humans accidentally become involved in their cycles. a good example is the recent outbreak of west nile encephilitis in the u.s. in 1999 and 2000, the virus was isolated from/around new york city from large numbers of dead birds (especially crows and jays): 21 human cases and two deaths were confirmed. by 2001, the disease moved west toward the mississippi river, infecting 55 people and killing nine. in 2002, there were over 2,400 cases (117 fatal); by 2003, the virus, and human cases, were found in all 48 contiguous states (excepting alaska and hawaii). the virus has been found in mammals, birds, and mosquitoes throughout the u.s. but is this a new disease, or just a disease new to us? west nile virus has been found in over 50 countries since its discovery in 1937 in uganda, and has been most of the above are mosquito-borne, and the major mosquito vector genera, culex, aedes, and anopheles, have global representatives from which a competent vector might be found. the same is true of ticks, sandflies, and other potential vectors. rodents, or other local vertebrates, may serve as reservoir hosts while infected migratory birds may provide distribution of the infection. although many arboviral infections have broadly similar transmission cycles, the ecology and dynamics of each may differ widely. arboviruses do not belong to a single viral family, but rather, to several,which increases their diversification. although the potential for increased arboviral epizootics or epidemics is high, the most recent episodes have not been high on the pestilence scale; rather, the most severe arboviral epidemics have been yf and dengue/dhf, the oldest of the group. the arenaviruses were thought for years to be monotypic, a single species, lymphocytic choriomeningitis (lcm), occurring primarily in the house mouse/ laboratory mouse, mus musculus. the virus (first described in 1933) has been isolated in numerous locations, but human disease is known only from europe and the americas. a second arenavirus was isolated from a phyllostomatid (fruit-eating) bat from trinidad, but there was no associated human disease. severe hemorrhagic cases in argentina and later in bolivia in the 1950s and 1960s resulted in the discovery of new viruses and diseases in these countries-junin virus/argentine hemorrhagic fever (ahf) and machupo virus/bolivian hemorrhagic fever (bhf). more recently, additional arenaviruses found in brazil (sabia virus) and venezuela (guanarito virus) produce similar hemorrhagic symptoms. ahf is the most common, 200-4,000 recorded annually between 1958 and 1995-the others are only sporadic, but mortality rates are high in all these diseases. in each of these, transmission is by contact with infected rodent excreta, dust, and other substances associated with grain harvesting and storage. there are another five arenaviruses in the americas that are not known to cause any illness in humans or their rodent hosts. all of the rodents associated with these viruses belong to only one of the 13 rodent families currently inhabiting south america. these rodent genera (calomys, sigmodon, oryzomys, et al) , are very closely related, and share a common ancestry. paleontological evidence indicates that the isthmus of panama was a bridge connecting north and south america more than 2-3 million years ago, allowing a faunal interchange. the sigmodont rodent progenitors entered south america at that time, and rapidly evolved into the modern genera and species. presumably the "ancestor virus" tagged along, co-evolving into the situation that now exists. by far the most important arenaviral disease is lassa fever: discovered in nigeria in 1970, it is known from 15 african countries, mostly in west and central africa, but also zimbabwe and mozambique. the natural host of lassa virus is the multi-mammate rat, mastomys natalensis, one of the most common and widely distributed african field rats. like their south american counterparts, the ahf and bhf hosts, mastomys, is basically a grassland species, easily adapting to the man-made grasslands of maize, sorghum, millet, sugarcane, and other cultivated grasses. cases of lassa are generally associated with agricultural activities and food storage: transmission is by contact with excreta of infected rodents. without laboratory facilities for confirmation, it is difficult to distinguish lassa fever from ebola, yf, or even severe cases of malaria. there are an estimated 500,000 cases a year, with more than 15% mortality rate in hospitalized cases. the disease is more severe in pregnancy, with fetal mortality reported at more than 80%. in the early 1970s (and before ebola outbreaks occurred), lassa caused great consternation in europe and the americas over the possibility of introduction of this disease. these concerns still exist and have been heightened after the appearance of these other groups of viral hemorrhagic diseases. the hantaviruses are comprised of two large groups of viruses, all transmitted by rodents and producing a range of hemorrhagic, renal, and/or pulmonary complications. the old world hantaviruses are comprised of over 20 different viruses, several known for some time under a different classification (e.g., hanta virus is the cause of korean hemorrhagic fever with renal syndrome, an important military disease in the 1950s). most cases still occur in agrarian and military populations and occur in over 50 countries in asia, africa, and europe: each year approximately 200,000 cases occcur in eurasia, with more than 50% of these reported in china. case fatalities range from 0.1% to 10.0% depending on the virus. the 15 or so new world hantaviruses produce a pulmonary, rather than a renal, syndrome. since being described as a group in 1993, approximately 1,000 cases have been reported in the americas, with a high case fatality rate (45-50 %). the natural hosts/reservoirs for the hantavirus groups are mostly muroid rodents (old world group), and cricetid rodents (new world group). this is not surprising, as these two are amongst the largest and most widely distributed mammalian families. however, the manner and zones of transmission are similarrodent contamination of grain crops in the field and storage where people come in contact with rodent excreta. the two closely related filoviruses (marburg and ebola) are among the most virulent viruses yet described with an overall fatality rate of more than 75%, and higher in several outbreaks (possibly augmented by use of dirty syringes and needles to give injectable chloroquine (an anti-malarial drug) to the patient' s friends who carried him/her to the hospital. marburg virus was first described (1967) among monkeys sent from east africa to european laboratories, there killing laboratory technicians. subsequent outbreaks have occurred in africa. ebola virus appeared in 1976 in simultaneous outbreaks in zaire (democratic republic of congo). (barry, 2004) one ebola strain was implicated in an outbreak in an animal holding facility in reston, virginia, u.s.a. several humans seroconverted but showed no disease symptoms. the repeated outbreaks of ebola and marburg virus, mostly in central africa, have been described as commencing with "rapidity and devastation." during an epidemic, transmission is generally by contact with contaminated blood or other tissues from infected persons. most outbreaks have been in rather remote areas with poor health care facilities, so that patients are seen only with advanced symptoms. we have not been able to find reservoir organisms (there have been subsequent, better equipped expeditions than the one described in the footnote, but none have been successful), nor do we know the mechanism(s) of transmission in the wild. one distinct ebola virus strain from ivory coast was isolated from a chimpanzee: primates are hunted and eaten by humans in parts of africa and this may serve as the 'link' at which the virus(es) are able to "cross the species barrier" and enter the human population. (barry, 2004) the government of sudan requested who assistance, and the government of zaire requested the same from the u.s. government (cdc). representatives of who and cdc met in the next few days at the london school of hygiene and tropical medicine to work out details and coordination (who was represented by dr. paul bres and the author, and cdc by dr. karl johnson). we had all thought of lassa and marburg viral fevers, and were surprised when dr. johnson said it was neither: he then showed us electron photomicrographs of tissue taken from an early case-the stringlike "6 and 9" figures were just like marburg. but, he explained, this one was serologically distinct from marburg, and they proposed to name it after a river in the area, the 'ebola'. we agreed that i (aaa) and a virologist (dr. bruce johnson) from the lshtm would go to the site in sudan to sample potential reservoirs and/or vectors. bruce would bring the supplies needed for taking tissue samples and the liquid nitrogen containers needed to return the samples to the uk. i was to gather the animal collecting materials. who had no such equipment in geneva, of course, so i borrowed 'mist' nets for collecting bats from the british musum (natural history) and the museé d' histoire naturelle in geneva and borrowed sample rodent traps from the swiss agricultural research station in nyon, near geneva. we had the traps made in nzara, one of the sites of the outbreak in sudan. to autoclave the dissecting instruments we purchased two household 'pressure cookers' at the local super market (migros) in geneva. placed on stones over an open fire, they served well. an experimental ebola vaccine has been reported to be successful in trials with non-human primates. human trials will be conducted soon. two previously unknown and unrelated human viral infections, severe acute respiratory syndrome (sars) and an asian avian influenza (strain h5n1), originating in southeast asia, have received a great deal of popular attention and public health concern. in november 2002, cases of a respiratory illness, subsequently labeled sars, appeared in china. a delay in timely reporting of the initial cases allowed it to spread to other southeast asian countries, australia, the americas, and at least 10 european countries. reports of the actual number of persons infected varied, but cases numbered in the thousands, and mortality rates of up to 15% were indicated. surveys of wild animals captured for human consumption quickly showed that ferrets, civets (related to mongooses), and raccoon dogs (shaggy fox-like carnivores) were positive for harboring the virus, but it is not known if any of these are the true reservoir in nature. the who has reported that the chain of transmission may have been broken (no new cases reported in a period of time equal to two consecutive 10 day incubation periods). this is clearly a case of a virus "species jumping". in the world' s largest, most densely populated country this could spell disaster, especially if the reporting network is compromised. the asian avian influenza strain initially appeared in poultry in hong kong in 1997, when it jumped the species barrier and killed 6 out of 18 infected persons. this recent outbreak spread to korea (december, 2003) , then japan and vietnam (january 2004). hong kong reportedly slaughtered 1.4 million chickens and ducks, and as many as three million slaughtered through the southeast asia region, but other reports indicate that there are nonspecific wild variants of this strain in wild birds that serve as natural reservoirs. of major concern is that outbreaks of highly pathogenic avian influenza are increasing in frequency and severity. reportedly, in the 40 years from 1959 to 1998, there were only 17 outbreaks, but in the past six years, from 1997 to 2003, there have been six, not including the most recent incidents. if bubonic plague was the quintessential pestilence of the ancient and medieval worlds, acquired immunodeficiency syndrome, caused by the human immunodeficiency virus (aids/hiv) is the chief pestilence of the modern world; and it is still growing, not receding. there is also a vast literature that will not be reviewed here, but the following 2003 data points describe the severity of the pandemic pestilence: r aids is gaining a firmer foothold in the large populations of india and china; r world wide, 40 million people are infected with hiv; r 25-28 million of these infected people live in sub-saharan africa; r 5 million persons became infected this year, 700,000 are children; r 3 million persons died of aids this year, 500,000 of them less than 15 years old; r existence of simian immunodeficiency virus (siv) suggests animal origin. the social damage accompanying this pandemic is not reflected in the bare figures given above; especially the orphaned children, destroyed family structures, and so forth. it has been estimated that 10 billion dollars us, per annum, is required to provide the prevention and treatment facilities and services needed: to date, less than one-half ($4.7 billion per annum) has been made available. some of the old category diseases are still strongly with us (e.g., malaria, tb, influenza), and, by adapting traits such as drug-resistance and crossing or jumping species, they expand their reservoir-host base. as such, they could be considered new. some other old diseases are rather well controlled in the developed countries where the surveillance systems are efficient and vaccination and other preventive services are readily available and properly used. these would include smallpox (eradicated), polio (eradicated in some areas), and childhood illnesses such as pertussis, diptheria, tetanus, measles, and so on. even bubonic plague could be characterized as being under control-it is widespread, but also well understood, and with vector control and appropriate antibiotics, outbreaks are not severe and mortality is low. on the other hand, some of the new (most recently discovered) diseases like ebola and hiv/aids are hard to handle. we know little about the natural history of ebola, lassa, or the south american hemorrhagic fevers, and our knowledge of hiv/aids in the laboratory probably exceeds our understanding of the socioeconomic impacts it is having on whole cultures. when lassa virus "jumped" from the field rat, mastomys, to humans it was dreadfully virulent, and it seemed to come from nowhere. but, after a few years, we know that (with one exception from a bat) all arenaviruses are well adapted to particular rodent groups; most rodents are grass eaters, and lots of crops are grasses (wheat, maize, sugarcane, rice, etc.); therefore, the arenaviral fevers are seen primarily in agricultural settings and with stored grain. yet, for the more recently known hantavirus group, or even less with the multiferous arboviruses, we do not have good data on ecological determinents, or even host-reservoir relationships. at the same time, people are modifying environmental conditions rapidly and extensively, and we have little information indicating whether such changes will eliminate potential disease cycles or exacerbate them. this may be even more important for diseases like influenza. if they have obligate or opportunistic vertebrate hosts and these are coincidentily reduced in number or eliminated, what selection pressures are set in action on the virus population to select new hosts? and when it comes to modifying environments, man has no equal. yet we know that this microbial evolution is going on at a rapid pace-just look at how fast drug-resistance develops and spreads! in reading articles and researching references for this document, i was amazed to discover again how many human illnesses have their direct animal (zoonotic) counterparts, or were vectored/hosted by arthropods, rodents, or snails, and how an avian influenza can become a mammalian influenza very quickly, and how a bat or an oppossum can do the same for the chagas disease trypanosome. it is in this context that i feel that we know very little of the natural history or the ecological dynamics of the disease transmission cycles we teach. especially disturbing is to read of a new strain of asian avian influenza and the necessity, around the world, to kill millions of birds. if one was to dream up a model pathogen incubator and dissemination engine, the perfect model would be a modern chicken farm of 500,000 birds, defecating as birds do, and that at a constant temperature and with residues of organic chicken feed all about. and we wonder why new diseases emerge? any farmer worth his/her salt knows that monoculture breeds pests. this is a good place to bring up one other difficult subject-bioterrorism. it is difficult for one dedicated to public health principles to imagine why anyone would even consider using infectious diseases as a weapon, but it is being done, and we need to be able to distinguish between a natural epidemic and one orchestrated by man. again, knowledge of the natural history of the organisms, their natural hosts and reservoirs, will help. already the u.s.a. is stockpiling smallpox and anthrax vaccines in large quantities. one final point; most people concerned with new versus old pestilences work as epidemiologists, infectious disease specialists, hospital officials, and so forth. but public health work is broader than the study and treatment of infectious diseases, and the study the global burden of disease, sponsored by the who, world bank, and harvard university, based on measuring dalys, predicts that fewer infectious disease will be as important in the future as they are at present. for example, "the next two decades will see dramatic changes in the health needs of the world' s populations, and non-communicable diseases such as depression and heart disease . . . are replacing the traditional enemies, such as infectious diseases and malnutrition." maybe toxic smog and non-communicable diseases will replace pestilences, both old and new! the great influenza control of communicable diseases manual exotic viral diseases death by migration plague: an ancient disease in the twentieth century (rev emerging infections plagues and people the global burden of disease the doctor's plague viruses, plagues, and history emerging infectious diseases (vol. 1-10). www.cdc.gov/eid or hard copy from cdc key: cord-022512-939pika7 authors: merck, melinda d. title: clinical management of large-scale cruelty cases date: 2015-12-04 journal: august's consultations in feline internal medicine, volume 7 doi: 10.1016/b978-0-323-22652-3.00069-4 sha: doc_id: 22512 cord_uid: 939pika7 nan large-scale feline cruelty cases typically arise from investigations involving individual hoarders, rescue organizations, sanctuaries, or breeders. these cases involve the crime scene, animal processing at the scene and temporary shelter, animal removal and transport, sheltering, and final animal disposition. the veterinarian plays a role in the planning of the operation, at the crime scene, the temporary shelter, and the hospital receiving cats for more advanced treatment. the clinical management of these cases is different from traditional shelter medicine or feline practice. to act and respond appropriately, the veterinarian must have an understanding of all aspects of the operation. these are legal cases requiring certain types of documentation and procedures. the organization of the entire operation should be conducted similar to disaster response using the incident command systemwhere the medical management on scene and at the temporary shelter is incorporated into this reporting structure with direct communication to the lead investigator. 1 extensive planning is needed, anticipating the number of cats, age ranges, known or likely medical issues, and socialization status. decisions must be made considering practicality, stress on the animals, costs, resources, future animal placement, and any legal impact. the goal is a successful outcome for the individual cat and the criminal case. supplies needed for on-scene and shelter medical operations need to be planned very early in the investigation. all team leaders should be involved in this planning, including those from the medical, forensic evidence, and shelter branches, with a supply list compiled for each branch (table 69-1) . arrangements should be made to obtain supplies during the case response for items that were not anticipated or depleted due to use. the use of color codes is important for large-scale cases to implement a visual system of communication at the scene, from scene to the shelter, within the shelter, and for forensic examinations. the colors are used to designate triage categories, initial cat temperament, and other conditions that determine placement and housing within the shelter. in addition, this system serves to establish the examination sequence. the assignment of color categories may vary with each case based on the availability of the color devices used (table 69-2) . colored duct tape is easily used and readily purchased at home improvement, hardware, office supply, and general merchandise stores. colored flagging tape may also be used but poses a risk of animal ingestion and can be difficult to remove from transport carriers or cages. a piece of the appropriate colored duct tape is placed on the animal identification (id) label located on the transport carrier or cage; some animals may have more than one color assigned (figure 69-1) . the primary use of the color system is at the scene, but some colors may continue to be used within the sheltering system. the color system and their assigned categories should be posted at the scene, within each forensic examination area, and at multiple areas within the shelter. the purpose of the critical triage team, comprised of veterinarians with or without veterinary technicians, is to identify any animals in need of urgent care. the critical triage team assesses animals after the initial scene walk-through is complete and as the physical evidence, animal id, and animal removal teams begin their work. any critically ill or injured animals should have priority for id assignment and removal from the scene. provisions for an on-site critical examination area should be made, such as a mobile veterinary clinic, trailer, tent, or one of the buildings within the scene itself. cats requiring more extensive diagnostics or treatment should be transported to a local veterinary hospital. the forensic examination teams are responsible for examination of all the animals. these teams are comprised of a veterinarian, scribe, and animal handler. it is important that all animals are examined in a timely fashion, within 72 hours, to ensure the examination evidence is representative of the the effect of stress on cats has been well documented. 2 in large-scale cases, stress can come from crowding, prolonged confinement, isolation, lack of environmental enrichment, an unsanitary environment, poor food and water resources, and untreated medical conditions. every consideration should be made to minimize stress during removal, transport, sheltering, and examination of the cats. physical handling of the cats and change of environment are additional but necessary stressors in these cases that carry a risk of exacerbating any pre-existing medical conditions. it is not unusual for these large groups of cats to be subclinical disease carriers that have reached a state of homeostasis that when disrupted results in new outbreaks. in addition, many of these cases involve animals that lack proper socialization and/or have been under severe emotional stress. they may exhibit aggression (due to fear or pain), or they may be completely unable to interact. some cats may exhibit subtle behavioral clues of extreme distress. it is the initial handling at the scene that can set the animal up for success or failure more so than any setbacks that may occur during examination or sheltering. resistance to handling and restraint is almost always the result of fear, anxiety, or pain, which is compounded when force is used. if possible, it is preferable to take several hours to remove a cat from the scene rather than forcefully remove it, which can reinforce the animal's fear and behavioral response. it is important to recognize that these cats may have reactions to novel stimuli different from those raised as pets in private homes. therefore, any negative behavioral designations assigned at the scene should be considered temporary and only for the purpose of cautionary handling. the key to successful handling of animals involves the accurate appraisal of behavior, an adequate number of staff, and the appropriate equipment; proper training in each of these areas being the most important aspect. transportation of cats from the scene to the sheltering site should be done in such a manner to minimize stress on the animals and to maintain compliance with applicable laws and regulations. considerations should be given to the environmental conditions and the time required to load the vehicle (which directly affects the first animals loaded). the spacing of the animal transport carriers within the transport unit should be planned to provide adequate ventilation for the cats inside. individual transport carriers should be of sturdy construction, put together properly to prevent animal escape or injury, have adequate ventilation, and consist of a design that allows stacking of carriers. special areas may need to be animal's condition at the original scene and any health issues are addressed as quickly as possible. the examination and treatment process should be complete and efficient. these teams usually work best when the scribe, who must be familiar with medical terminology, fills out the examination form as the veterinarian dictates the findings. the creation of special laboratory and treatment teams can improve efficiency and free the veterinarian to focus on examination and documentation. it is preferable to have one team designated to examine the more severe medical cases and animals that may require sedation to handle. this team should have ample medical supplies, controlled drugs, and the most secure examination area. there are several assessments that must be made at the crime scene that directly impact handling of the cats throughout the operation and have important legal ramifications. these evaluations may be conducted by veterinarians along with case investigators. it is critical that the veterinary teams, offsite hospitals, and sheltering operations take this into consideration. the assessment should include information about the cats and current housing conditions along with medications and supplies present. the most important consideration, and most overlooked, is the evaluation and continuous monitoring of stressors on the cats. it is important to document any obvious medical, husbandry, or behavioral care needs at the scene. this includes the presence of neonates, nursing queens, chronic medical conditions, infectious diseases, and potential zoonotic diseases. any records present should be assessed for pertinent information, including intake date, medical history, and individual cat names. any medications, supplies, and supplements should be documented, noting any prescriptions for individual cats, the date prescribed, expiration dates, the original amount and quantity remaining, and the prescribing veterinarian's information. the medications and supplies should be assessed for indicators of current or past medical issues. the general housing of the cats should be assessed and documented with photography and mapping. this assessment is an important consideration for housing decisions at the sheltering site. special note should be taken of the number of cats in a room or cage and natural groupings or obvious social bonds. the flow of people and animals within and between housing areas should be documented, with special focus on the potential for fomite transmission of pathogens. the type and brand of food offered should be recorded as well as the number of feeding stations and the number of cats that have access to each one. the availability and potability of water sources should be assessed. the litter boxes should be evaluated, including the type of litter, the amount of urine and feces, the type of litter box, and the number of cats with access to each litter box. samples of diarrhea should be taken for individual cats and representative samples for group population medicine whenever possible, the same persons should care for the same cats to help reduce stress, enhance opportunities for socialization, and decrease risk for injuries. assessments and monitoring may be improved when performed by personnel familiar with individual cats. creating separate sections in housing areas should be carefully considered, because they can create more stress on the cats and complicate management of the shelter. these separations are often based on infectious diseases, zoonotic diseases, critical medical observation, bite quarantine, reproductive status, and special needs (such as those cats that are difficult to handle, pregnant, neonates, or nursing). decisions for separate housing areas must be done with consideration of the crime scene assessment. most of these cases involve cats that have previously been exposed to each other either through housing design or fomite transmission; therefore, separation based on infectious disease is neither necessary nor recommended. in large open shelters, separate areas can be created by hanging tarps or clear plastic sheets that are open at the ceiling for airflow. other considerations for housing of cats include lighting, number of separate rooms, secure areas, and airflow. cats are more stressed when at ground level, so every effort should be made to elevate the housing. 3 in the planning stages, it is important to develop medical protocols, including those for examinations, diagnostic testing, and all treatments. planning should include what tests will be conducted at the shelter and what outside laboratories will be used. because this is a legal case, the use of standard examination forms that are developed specifically for these types of cases is recommended (box 69-1) . these forms should be structured to prompt and guide the veterinarian through a complete examination. photographic id of the cat can be performed by one of the forensic examination team members, or a separate floating photographer may support multiple examination teams. the photographs should begin with the case information, including the case number, date, and animal id written on a card or dry erase board. this first picture with the case and animal information may be taken with or without the animal in the photo, and all photographs following this initial picture should only be of that animal. general photos should be taken showing the entire body of the cat: right and left sides, front (facial), hind (rear), dorsal, and ventral (if possible or appropriate) views. photographs, with and without a photo scale, should be taken of any obvious lesions, abnormal physical findings, and any evidence found on the body. 6 it is important that each veterinarian use the same examination form (see box 69-1). it is the responsibility of the scribe to ensure that the entire form is complete, and it is the veterinarian's responsibility to review it for accuracy. in designated at the offloading area for animals requiring immediate veterinary care or assessment upon arrival to the shelter, especially neonates. planning for animal carrier placement based on infectious disease status is difficult and usually unnecessary as in most cases there has been ongoing exposure among animals due to their living conditions. for large-scale cat cruelty cases, there is rarely an existing animal shelter that can house all of the animals at one location. this requires creating a temporary shelter, which follows the same principles used for animal disaster sheltering, with the primary difference that the animals are evidence in a criminal case. protocols must be in place for monitoring, treatment, daily assessment, and progress documentation, including change of medical or behavioral status. there should be a shelter mapping system to identify and track the location of every animal, including when animals are moved. it is important to have the veterinarian's input on the initial design and any adjustments made based on the crime scene assessment or forensic examinations. the shelter should be designed to be flexible based on changing medical or behavioral conditions of the animals. shelter design should take into account the number of cats, special housing needs, ease of handling, infectious disease, temperature control, airflow, foot traffic, noise levels, animal stress, and environmental enrichment. continued assessment and reassessment of cat behavior and temperament should be conducted, with changes made to address any issues noted. any final behavior or temperament designations should only be assigned after the cats have had time to adjust to their new environment and recover from all stressors, which may take several days to weeks. consideration should be given to the original housing, grouping, and social bonds of the cats at the scene. often these cases involve group housing or free-roaming shelter settings, and the temporary shelter results in isolation of cats into individual cages. although this can be another stressor on the cats, it can be offset (at least for the short term) by the positive change to a sanitary environment and the elimination of competition for resources of space, litter, food, and water. cats should be paired or group-housed whenever possible and practical to reduce stress. shy or feral cats are often less stressed when paired with another cat. wire cages are especially amenable to alteration, creating a "double-wide" space for this purpose. environmental enrichment is important to incorporate into the sheltering design and protocols. this includes soft bedding, boxes, or small carriers to hide in, toys, treats, and catnip. it is critical to offer canned food, especially for cats with upper respiratory or dental disease, to assess and stimulate appetite, and for further enrichment. it is important to minimize changing of bedding and other items within the cages, which can create additional stress. 3 the use of synthetic pheromones (e.g., feliway, ceva animal health) may be effective in stress reduction when sprayed on bedding and through the use of plug-in diffusers. 4 addition, the veterinary teams should use consistent terminology and abbreviations, which may be posted in the exam areas. breed descriptions should be done carefully. unless the breed is known, it is often best to use "breed-type" or predominant "breed-mix." the body condition score (bcs) to be used should be posted in each examination area so that the veterinarians can refer to it. the age or estimated age of a cat should only be documented if supported by examination findings, such as dentition or ocular changes. it is preferable to use designations of neonatal, juvenile, adult, or geriatric with documentation of the basis for the determination. after the examination is complete, a check mark should be placed on the animal's id marker to create a visual indication that the animal has been examined. any color-coded markers should be assessed and removed or changed based on the examination; those that may impact shelter operations should be communicated to the shelter manager. the standard medical protocols enacted depend on the known existing conditions and diseases within the population, the expected length of stay in the temporary shelter, and the expected disposition of the cats; however, a variety of infectious diseases (including respiratory, enteric, and dermatologic pathogens) should be expected. 7 depending on the legal process, the cats may be adopted directly from the shelter, placed at another animal facility within or outside the area, placed in foster homes, or euthanized. these potential scenarios and outcomes may be fluid and will dictate what protocols are most appropriate for the cats at any given time. the medical protocols should be implemented during the forensic examination process. standard vaccination protocols should follow the american association of feline practitioners (aafp) recommendations for shelter-housed cats. 8 vaccinations should be given at the time of intake to the temporary shelter and should include feline herpesvirus type 1, feline calicivirus, feline panleukopenia, and rabies. vaccination for feline leukemia virus (felv) may be considered based on viral test results and group housing. a majority of intensively housed cats will harbor at least one enteropathogen, often of zoonotic significance; the presence of such pathogens is not limited to cats with clinical population medicine euthanasia protocols for euthanasia, necropsy examinations, and the storage of deceased animals should also be determined in advance. because the animals are evidence and also to remain sensitive to the case responders and public sentiment, the criteria and parameters for euthanasia decisions should be discussed with the lead investigator and prosecutor prior to the day of seizure. the decision for euthanasia should first be agreed upon by the examining veterinarian and the case lead veterinarian. the lead veterinarian should obtain permission from the lead investigator to perform euthanasia according to the agreed upon parameters. another factor to address in the planning phase is the use and storage of controlled drugs, including euthanasia solution and sedatives. a controlled drug log must be maintained according to applicable laws. many of the cats may have obvious medical needs. regardless of the presence or absence of clinical signs, it should be expected that most of the cats are likely carrying multiple different pathogens. 13 these may or may not become clinically relevant, depending on the individual cat and the stressors placed on it. the most common medical finding is upper respiratory tract disease (urtd). this may vary from mild to severe with secondary pneumonia, ulcerations of the nasal or oral area, nasopharyngeal polyps, and ocular involvement ( figure 69-2) . mild cases may not need to be treated while being closely monitored for disease progression; most cases will resolve within 7 to 10 days. 14 the more severe cases typically involve secondary bacterial infection, regardless of the primary pathogen, which should be treated aggressively. considerations for choices of medication should include the availability of personnel and their ability to administer signs of illness. 9 therefore, routine deworming for roundworms, hookworms, and tapeworms should be part of the standard medical protocol. fecal testing is not necessary unless there is unresolved diarrhea after deworming, or it is otherwise dictated based on clinical illness. coccidiosis is a common finding in large-scale cat cruelty cases; it is found in up to 23% of cats with diarrhea and 33% of clinically healthy cats. 9 based on observed diarrhea findings at the scene, within the temporary shelter, or diagnostic test results, it may be reasonable to treat all cats for coccidiosis. ponazuril is an effective treatment for coccidiosis and is easily administered to large numbers of cats, given orally once daily at 15 to 30 mg/kg for 1 to 3 days and repeated if necessary in 1 week. 3, 10 the stressors the cats have been exposed to along with dietary changes at the temporary shelter can contribute to diarrhea. in addition, many cats from these cases may harbor other enteropathogens, such as tritrichomonas, giardia, or cryptosporidium species, as well as feline enteric coronavirus, which may or may not be a cause of refractory diarrhea. 9, 11 any additional testing and interpretation should be conducted as discussed later in this chapter. all diagnostic testing decisions for the cat population should be done with consideration for the goals of the operation; the legal impact; the health of the individual cat and the general population; the stress on the cat; the expected time length for housing; the future placement of the cats and the placing agency's responsibilities; the financial and personnel resources; and clinical relevance. for each test considered, both the test itself and the action to be taken based on its result must be appropriate and reasonable. in a large-scale case, tests that may be recommended or conducted in private or shelter medicine practice may not be appropriate or reasonable. the goal is not to cure all animals and eliminate all pathogens prior to the cat leaving the temporary shelter, rather it is to immediately identify and address any important medical issues, improve overall physical and mental health, and address any obvious zoonotic issues. this should be a clear and constant guidepost for decisions made. it cannot and should not be the expectation to test and treat for everything. as with any shelter situation, these cats may be asymptomatic carriers of a variety of pathogens. testing can result in information that is not currently relevant to the health of the animal yet force decisions for unnecessary and inappropriate actions for that individual animal or the general population. for large-scale cruelty cases, it is recommended to test cats for felv and feline immunodeficiency virus (fiv) based on the guidelines established by the aafp. 12 depending on the test results and the original housing at the crime scene (i.e., pre-existing exposure), changes in the housing location within the temporary shelter may or may not be recommended. cats that appear ill without a grossly apparent cause should have routine laboratory work analyzed, including a full biochemistry profile, complete blood count, and urinalysis. such documentation is especially important in legal cases. medication, the possibility of administering medication through canned food or treats, dosing intervals, possibility of multiple pathogens, and risk of side effects. stress reduction plays the single greatest role in the remission and prevention of urtd in cats; 14 therefore, particular attention should be given to providing an enriched environment as a component of the treatment plan. skin and fur conditions are quite common in large-scale cruelty cases. the cats often have urine and/or fecal staining of fur, especially on the feet, usually with associated dermatitis. other issues related to the feet include contact pododermatitis, plasma cell pododermatitis, and overgrown or embedded claws (figure 69-3) . cat bite abscesses are not uncommon. severe matting may be seen on longhaired cats with fecal soiling or impaction in the perineal area with associated dermatitis (figures 69-4 and 69-5). in addition to fleas and associated dermatitis, there may be other external parasites, such as cheyletiella, lice, and demodectic or sarcoptic mange. stress can cause alopecia due to overgrooming or self-mutilation (figure 69-6 ). less commonly, cases of streptococcus canis have been reported in intensively housed cats, often manifesting with skin ulceration and necrotizing fasciitis (figure 69-7) . 15 dermatophytosis may be seen in some cats but rarely is a widespread issue within the population. cats may be mechanical carriers and never develop clinical lesions themselves; such cats are seldom clinically relevant to the case, the individual animals, or the general population. for these reasons, dermatophyte testing is not routinely recommended on intake of cats or for those without dermatologic lesions. rather, the focus should be on diagnosing and treating cats with suspicious lesions and enacting precautions for handling and cleaning. housing placement within the shelter for cats suspected or diagnosed with dermatophytosis should be based on the considerations previously discussed. population medicine in the distal extremities, may be found. other common findings include decubital ulcers, poor wound healing, and secondary bacterial or yeast skin infections. 16 there are numerous other medical findings that may be seen or develop in these cases: a variety of clinical abnormalities of the skin result from nutritional deficiencies, including starvation. a common finding is a sparse, dry, dull, and brittle hair coat with hairs that epilate easily. seborrhea sicca and patchy alopecia may be seen. loss of hair occurs due to anagen and/or telogen defluxion. in the anagen or growth phase of the hair cycle, there can be loss and/or abnormal growth of hair, resulting in broken hair shafts. with telogen defluxion, there is widespread loss of hair in the quiescent phase of the hair cycle due to large numbers of hair follicles being synchronized in this phase. loss of normal hair color and hair keratinization abnormalities can occur. in cats, decreased melanin deposition due to amino acid deficiencies results in a reddish cast to the hairs, sometimes called red coat (figure 69-8 ). erythema or crusting in areas of friction or stretching, such as primary or secondary to other medical conditions, malnutrition, dehydration, stress, and/or an unsanitary environment. often, the investigation findings may provide information on pre-existing conditions through intake paperwork or hospital records. these underlying issues may only be detected with routine blood work and urinalysis; affected cats may require further diagnostic testing, imaging, or hospitalization. the clinical management of large-scale feline cruelty cases starts at the scene and continues to the temporary housing in a shelter or hospital. it is critical that veterinarians understand the importance of the role they play as part of the investigative team and the requirements for documentation in a legal case. the veterinarian may become involved in these cases directly at the scene and shelter or by receiving a patient into the hospital. by the very nature of large-scale cruelty cases, the cats will have a wide range of issues associated with high volume housing and long-term neglect. it is important to be mindful that this will be a fluid situation requiring a process of continuous assessment of the animal's health and behavior to detect early signs of developing issues and initiate treatment. the simple actions of providing appropriate housing, food, routine medical treatments on intake, and overall stress reduction will often result in rapid improvement of cat health. there is a greater chance of recovery-even for the most severely affected cats-when both physical and mental needs are addressed. â�¢ oral lesions, including ulcerations associated with urtd, stomatitis, and dental disease â�¢ urinary tract issues (e.g., urinary tract infection, idiopathic cystitis, nephroliths, uroliths, crystalluria, and vaginitis) are often the reason the cat was presented to the original shelter or sanctuary the clinician should be cognizant of the possibility for these and other underlying medical issues that may be special considerations in animal cruelty cases recognition and management of stress in housed cats feline care in the animal shelter effects of a synthetic facial pheromone on behavior of cats a synthetic fraction of feline facial pheromones calms but does not reduce struggling in cats before venous catheterization crime scene investigation prevalence of infectious diseases in large scale cat hoarding responses aafp feline vaccination advisory panel report prevalence of enteropathogens in four management models for unowned cats in the southeast united states clinical trial to assess the efficacy of ponazuril for the treatment of coccidiosis in dogs and cats enteropathogens identified in cats entering a florida animal shelter with normal feces or diarrhea american association of feline practitioners retrovirus management guidelines prevalence of upper respiratory pathogens in four models for unowned cats in the southeast united states feline infectious respiratory disease fatal streptococcus canis infections in intensively housed shelter cats veterinary forensics: animal cruelty investigations key: cord-004211-58x3nnsc authors: javelle, emilie; lesueur, alexandre; pommier de santi, vincent; de laval, franck; lefebvre, thibault; holweck, guillaume; durand, guillaume andré; leparc-goffart, isabelle; texier, gaëtan; simon, fabrice title: the challenging management of rift valley fever in humans: literature review of the clinical disease and algorithm proposal date: 2020-01-22 journal: ann clin microbiol antimicrob doi: 10.1186/s12941-020-0346-5 sha: doc_id: 4211 cord_uid: 58x3nnsc rift valley fever (rvf) is an emerging zoonotic arbovirus with a complex cycle of transmission that makes difficult the prediction of its expansion. recent outbreaks outside africa have led to rediscover the human disease but it remains poorly known. the wide spectrum of acute and delayed manifestations with potential unfavorable outcome much complicate the management of suspected cases and prediction of morbidity and mortality during an outbreak. we reviewed literature data on bio-clinical characteristics and treatments of rvf human illness. we identified gaps in the field and provided a practical algorithm to assist clinicians in the cases assessment, determination of setting of care and prolonged follow-up. rift valley fever virus (rvfv) is an arbovirus, mainly transmitted by mosquitoes, responsible for a zoonosis disease that affects cattle, sheep, camels and goats. it was first identified in 1931 during an investigation into an epidemic among sheep on a farm in the rift valley of kenya [1] . the virus infects also humans through inoculation after contact with infected animals or through ingestion of unpasteurized or uncooked by-products of infected animals, or also through inhalation of aerosols produced during the slaughter of infected animals. however, human infections occurred also from the bites of infected mosquitoes, mainly aedes and culex but also anopheles or mansonia, and other blood-feeding vectors such as flies and ticks have been identified [2] [3] [4] . to date, no human-to-human transmission of rvfv has been documented. rvfv belongs to the phenuiviridae family (formerly bunyaviridae), member of the phlebovirus genus. the enveloped virion contains a tripartite, predominantly negative-sense, single-stranded rna genome, which codes for structural and non-structural proteins the virus needs to replicate both in mammalian hosts and insect vectors. rvfv attach to cells via the interaction between the viral structural proteins gn and gc and c-type lectins, dc-sign and i-sign [5] . cells become infected with rvfv by receptor-mediated endocytosis, followed by ph-mediated fusion of virus-endosomal membranes to release nucleocapsids into the cell cytoplasm. transcription, translation, and genome replication occur in the cytoplasm. the non-structural protein nss is known to be a major virulence factor allowing the virus to escape host innate immune response. only one serotype is recognized but strains exist of variable virulence. moreover, rvfv is classified as a risk group 3 agent, and biosafety page 2 of 18 javelle et al. ann clin microbiol antimicrob (2020) 19:4 level (bsl)-3 containment requirements are needed to work with the virus in the laboratory [6] . as other arboviral infections including dengue, chikungunya and zika, rvf is emerging worldwide, due to the globalization of arthropod vectors, mainly mosquitoes, which efficiently transmit an increasing number of old, unrecognized and new viruses. arboviruses pose a major threat of introduction to several continents, including europe and north america, with the possibility of cocirculation [7] . the widespread presence of competent vectors, the high viral load in infected animals, trade and global travel, all increase the likelihood of rvfv exportation and establishment outside endemic regions [8] [9] [10] . cases have already been imported to europe and asia [9, 11, 12] and concerns have raised about its potential to extend to other parts of asia, europe [13] and united states [14, 15] . such an introduction would cause significant losses to the livestock industry and substantial human morbidity and mortality [16] . clinicians need to consider rvf in the differential diagnosis for febrile illnesses in a suitable context, however manifestations of rvfv in humans are varied and unspecific including hepatitis, encephalitis, hemorrhagic disease, and retinitis with potential dramatic consequences. the overall case fatality rate is estimated from 0.5 to 2% [8, 17] , but higher mortality rates were recorded, as for example 18% by the saudi health ministry in 2000 [18] , around 22% in east africa, west africa, south africa and madagascar from 2006 to 2010 [16] , and 28% in tanzania in 2007 [19] . in 2019 rvfv emerged in mayotte, a french overseas department and region and gave growth to this work [20] . strategies of rvfv control appeared us challenging because of its complex biological cycle and its multiple routes of transmission to humans [21] . besides, the wide clinical spectrum over a long-period of time made very difficult the establishment of standard definitions of human cases and recommendations for their management. we conducted a literature review on the rvf clinical disease and treatments in humans. we identified the state and frontiers of knowledge. lacking guidelines on the rvf human disease, we proposed an algorithm to assist physicians on the field in the evaluation of cases. this algorithm could help and be improved during next epidemics. we based on preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines to conduct this clinical review and build the flow diagram ( fig. 1) [22] . we did a literature search for english and french language studies published in electronic databases for an unlimited period until december, 2019: pubmed central, embase, medline and scopus. we used the keywords "rift valley fever" and "human". along with this, we conducted targeted search within the online archives of journals of tropical medicine, which published the first clinical reports of rvf in humans since 1930s, i.e. "south african medical journal" and "transaction of the royal society of tropical medicine and hygiene". we contacted editorial office of other journals to get relevant articles published between 1930s and 1990s. if available, we reviewed citations in pubmed of these first articles in literature. besides, we consulted the databases, datasets and official reports of the world health organization (who) and the centre for disease control (cdc) on their official websites and the mentioned references. we removed duplicates and screened titles and abstracts of all these records to include manuscripts reporting clinical descriptions and/or treatments of rvf in humans (case reports and case series). animal models for rvfv pathogenicity studies published during the last 10 years were also considered. serological surveys, works on vaccines, immunology, biology, veterinary science and entomology were excluded. totally, 129 articles resulting from these searches with full-text available were assessed for eligibility. among them, 35 with uncertain rvf diagnosis or without significant content or input were removed. relevant references cited in the eligible articles were reviewed and other records were manually searched and added for specific purposes of our article using the following terms "rift valley fever" and "severity", "severe", "prognosis", "death", "fatal", "risk factors" and "scores". at the end, 107 articles were referenced in the final review ( fig. 1) . we used data on rvf human cases reported by the who in the rubric "disease outbreak news" [23] and the cdc outbreak summaries [24] to build an epidemiological overview and we used the software adobe illustrator 22.1 and macrovector official freepik for figures. human cases have been reported from many african countries following the virus introduction via infected livestock trade [25] . since the end of 1900s, the virus has extended outside the african continent to indian ocean islands: madagascar [26] , comoros, and mayotte [27] [28] [29] [32, 33] , but methods used to calculate this basic reproductive ratio have some limits [34] . infection with rvfv is mostly pauci-symptomatic in humans. general signs may occur in 50 to 95% of infected cases after an incubation period of 2 to 6 days. the typical presentation includes headache, fever, backache and generalized aches in muscles and joints, lasting 4 to 7 days [4, 17, 35, 36] also reported [17, 37] . rvf differs from influenza, dengue and chikungunya as to whether cough, skin involvement (i.e. rash or pruritus) and arthritis are respectively uncommon signs. a slight meningism at the acute stage is not rare, however its prognosis value has never been evaluated [37] . basically, retro-orbital pains and neck stiffness are features hard to classify because both of them were commonly reported in uncomplicated rvf cases [36] , but were also associated with the occurrence of complications [37, 38] . incidences of complications are uncertain because rvfv infection can go unrecognized or be misdiagnosed considering the unspecific symptoms of suspected cases, which overlap with many other co-circulating pathogens [39] . no standard definition of suspected cases exists. rates of complications measured in studies depend on the definitions and methods of recruitment. indeed, the use of clinical or biological criteria specific to rvf complications to define suspected cases could lead to underestimate the mild forms [40] [41] [42] . globally, since the first description of the spectrum of rvf in humans by laughlin et al. during the major outbreak in egypt in 1977, it is considered that less than 5% of symptomatic cases will present complications including ocular, neurologic and hemorrhagic symptoms, while favorable outcome will occur within 1 week for the others [17] . in this historical series, the different known complications occurred in equal proportions (30-35%), but hepatic or renal failures were not identified. during the 2007 kenyan outbreak, kahlon et al. described a clinical syndrome suggestive of severe rvf, characterized by fever, large-joint arthralgia, and gastrointestinal complaints, later followed by jaundice, right upper-quadrant pain, and delirium, often coinciding with hemorrhagic manifestations [43] . complicated forms could have represented up to 20% of symptomatic cases during recent epidemics [36] . morbidity, as well as mortality, varied from one to another outbreak. for example, in south africa in 1975 [44] and in tanzania in 2007 [19] most of rvf severe cases presented with encephalopathy (respectively 71% and 89%), whereas hemorrhagic manifestations predominated in mauritania in 2015 (81%) [45] and madagascar in 2008 (88%) [46] . in saudi arabia in 2000, hepatic insufficiency (75%) and renal failures (41%) were the most frequent complications [47] . moreover, during the epidemic in madagascar, highly fatal associations of two or more complications were highlighted. these occurred in 11/16 (69%) severe cases, of whom 5 (45%) had encephalitis with hemorrhagic symptoms which were lethal in 2/5 (40%), representing half of the deaths (4/16) [46] . variations in the rvfv tropism and virulence are hypothesized according to the lineage involved and the possible accumulation of genetic mutations or genomic reassortments [17, [48] [49] [50] , despite a low overall genomic diversity (∼ 5%) at the nucleotide level [51] . genetic, ethnic or epidemiologic factors in the population exposed to the virus, as well as access to care also play a role [17, 52] . manifestations of rvf in humans are represented in fig. 3 . alternative diagnoses concern a broad array of conditions which may be worldwide distributed or restricted to endemic areas. characteristics and differential diagnoses of rvf manifestations are summed-up in table 2 . determinants for severe rvf outcome are poorly known. a number of retrospective studies suggest that touching, handling, living close to, and consuming animal products are factors associated with increased likelihood of rvf virus infection and possibly more severe outcomes [19, 53] . this is probably linked with a significant exposure to the virus that results in higher inoculation rate. indeed viremic loads have been reported correlated with severe rvf diseases [54] . single nucleotide polymorphisms (tlr3, tlr7, tlr8, myd88, trif, mavs, and rig-i) were also associated with severe symptomatology [55] . acute malaria co-occurrence was observed in severe forms and hiv-positive status was associated with a 75% case fatality rate in tanzania in 2007 [19] . schistosomal liver co-involvement and bacterial or fungal co-infections were also documented in fatal cases [37] . liver is the primary site of rvfv replication, so that it is frequently early involved during rvfv acute infection [56, 57] . a severe acute hepatotropic disease may occur with liver failure and jaundice within the first 3 weeks of the disease [43] . tenderness, palpable enlargement and more than threefold elevation in transaminases are criteria of severity [42, 58] . jaundice was proved to be independently associated with a high mortality rate [40] . acute hepatitis may complicate with prolonged blood coagulation times and may occur together with or precede fatal hemorrhages or neurologic complications. autopsy studies and pathogenesis characterization in mouse model found evidence of liver necrosis with rvf viral antigens identified within hepatocytes and küpffer cells, arguing for a direct virus-induced cellular necrosis [19, 37, 44, 57, 59, 60] . a rvf case with a co-existing condition of cirrhosis after hepatitis b infection died as a result of gastrointestinal bleeding and hepatic encephalitis in mayotte [28] , and 4/31 (13%) severe cases described during the epidemic in mauritania in 2015 had chronic hepatitis b [61] , suggesting that patients with chronic hepatic acute or delayed encephalitis day vision disorders day [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] disorders-mainly hepatitis b chronic infection-could be at higher risk of unfavorable outcome. soon after the onset of flu-like illness or acute hepatitis, patients may present bleeding from the nose or gums (gingivorrhagia being a key early warning sign) [62] , hematemesis or melaena, petechial/purpuric rash or ecchymoses, menorrhagia, hematuria, or bleeding from venipuncture sites [46, 63] . yellow fever-like expression were also reported with a first improvement at day 3 followed by a rebound of fever [62] . epistaxis is not considered a reliable sign of how serious the illness is [64, 65] . thrombocytopenia is invariably present. hepato-renal failure with jaundice, disseminated intravascular coagulation and encephalitis can be associated [44, 66] . overall prevalence is estimated 1%, but prevalence was rather 10% in hospital cohorts [40, 47] . a population-based survey during the 2007 outbreak in kenya even reported 26% of hemorrhagic rvf disease with a mortality of 23% in this group of cases [67] . indeed, the mortality rate associated with bleeding manifestations is the highest, up to 65% [40, 68] . viral load could play an important role in the hemorrhagic expression. in humans studies, it exhibited positive correlation with markers of inflammation (ip-10, crp, eotaxin, mcp-2 and granzyme b), markers of fibrinolysis (tpa and d-dimer), and markers of endothelial function (sicam-1), but a negative correlation with p-selectin, adamts13, and fibrinogen, which are associated with coagulation pathways occurring on the endothelial surface [69] . the onset of meningoencephalitis usually occurs 1 to 4 weeks after the first symptoms (which may be very mild or subclinical), and in some cases neurological complications can manifest beyond 60 days after the initial symptoms of rvf. clinical features may include intense headache, neurological deficit, rigor, neck rigidity, hyperreflexia, hypersalivation, choreiform movements, loss of memory, hallucinations, confusion, disorientation, vertigo, convulsions, ataxia, lethargy, decerebrate posturing, locked-in syndrome and coma [17, 35, 44, [70] [71] [72] [73] . in a human outbreak in mauritania in 1989, up to 5% of observed infections had encephalitis [71] . two types of pure encephalitis were described: acute febrile forms with short duration and possibility of death, and subacute forms with a longer duration, a lower fatality rate but frequent sequelae [71] . pulmonary complications may occur [71, 74] , and malaria can worsen the severity of neurologic symptoms [19] . lethality may be as high as 50% in this form [40] . from mauritania in 1989, clear a cellular csf were documented in all cases with encephalitis [71] . in a 18-year old woman treated for chronic myeloid leukemia with acute rvfv encephalitis acquired in saudi arabia, csf was documented predominantly with polynuclear leukocytes. magnetic resonance imaging (mri) showed high signal intensity on t2-weighted images in frontoparietal and thalamic regions, with multiple bilateral asymmetrical cortical hyperintense areas consistent with inflammation or ischemia in axial diffusion, while changes in ct-scan of her brain appeared much later [70] . in delayed meningoencephalitis normal glucose and protein concentrations will lymphocytic pleocytosis were found in csf [17] . in a kidney transplant recipient with cured hepatitis b, presenting acute hepatitis followed by delayed pachymeningitis, specific rvf-igm were detected in lymphocytic csf at day 58, while igg were positive in blood at the first screening at day 44 [74] , which was consistent with the first neurologic description in literature [72] . in a mouse model of rvf infection, survivors to primarily hepatitis cleared the virus from liver and blood, but exhibited neuro-invasion and fatal encephalitis [57] . active viral replication in brain leading to necrotizing encephalitis was documented in several animal models [75, 76] . the route of transmission and prompt robust immune response could be a determining factor of the rvf neurologic disease course [77] . indeed, whatever the routes of inoculation, rvfv rna was detected in the brain of infected rats confirming the virus neurotropism [78] , but aerosol exposure to rvfv caused earlier and more severe neuropathology in the murine model and fatal encephalitis in primates [75, 76] . in aerosol-infected rats with lethal encephalitis, neutrophils and macrophages were the major cell types infiltrating the cns, and this was concomitant with microglia activation and extensive cytokine inflammation [78] . differences in the peripheral blood biomarkers during the course of the neurological disease in african green monkeys were measured with defect in early t-cells, proinflammatory and antiviral responses in lethal encephalitis [79] . other immune disorders and alteration in vascular permeability in the brain could be more involved in delayed forms [80] . during rvfv infection, elevated urea and creatinine levels may be secondary to hypovolemia, multipleorgan dysfunction, or hepatorenal syndrome [39, 81] . acute hepatonephritis, possibly related to direct rvfv injury, characterized by proteinuria and oliguria were also reported with a bad prognosis [62] . in mauritania in 2015, creatininemia was meanly more than fourfold upper the reference range in severe cases [61] . in saudi arabia in 2000, renal impairment concerned up to 60% of rvf inpatients and dialysis was needed in 90% of them [81] . the mortality rate was 31% in patients with acute renal failure, 25% in those with hepatorenal syndrome, and 31% in patients with primary hepatic involvement and mild renal impairment [81] . progression to chronic renal failure was not seen [40, 47, 81] . in 2008 in mayotte, an acute pericarditis with symptoms of right-sided heart failure, relapsing at 1 month, was documented in a 53-year old farmer diagnosed with rvfv infection [28] . in historical post-mortem examinations, fragmentation in myocardial muscle was found in two cases and rvfv was isolated from one pericardial fluid [37, 44] . macular exudates with potential permanent loss of central visual acuity were firstly described in 7 among 20,000 estimated cases (< 0.05%) during the 1950-51 outbreak in south africa [82, 83] . the prevalence of ocular manifestations has been estimated to 1% during epidemic outbreaks in egypt in 1977, and up to 15% both in patients with mild and severe rvf disease during the 2000 outbreak in southwest saudi arabia [84] . unilateral or bilateral symptoms generally occur 5 to 14 days after the rvfv infection, but can be more delayed, and may include decreased visual acuity, scotoma, acute hemorrhagic conjunctivitis and retro-orbital pain [85] . the most frequent and most specific ocular lesion is a macular or paramacular retinitis [86, 87] . the funduscopy by indirect ophthalmoscopy usually shows a single well demarcated necrotic lesion with ill-defined creamy-white patchy lesions of macular retinitis with hemorrhages [84] . the other retinal signs include arterial occlusions, vasculitis (mostly phlebitis and sometimes arteritis) [84] , sheathing of the vessels, which are best explored using fluorescein angiography. in series, vitreous reaction with vitreal haze or vitritis occurred in less than one third of patients, optic-nerve head edema or palor were described in 15% of cases with retinal involvement, and no infectious optic neuropathy was reported [84, 85] . anterior uveitis was associated with a posterior uveitis, defining a panuveitis with aqueous flare and fine non-granulomatous keratic precipitates [84, 85] . fluorescein angiography performed during the active phase of the disease may show early hypofluorescence with delayed filling of the arterioles and venules, associated with late staining of the lesions [84] . it also helps for the diagnosis of vasculitis, showing vessels sheathing and staining, and vascular occlusions when present. follow-up fluorescein angiography performed several months after rvf diagnosis have revealed window defect in the area of the retinitis, vascular occlusions and obliterated macular vessels [84] . however, ophthalmoscopic and angiographic features of the rvfv associated retinitis are not specific and can be encountered in several viral or bacterial infections (table 3 ) [85, [87] [88] [89] . ocular active lesions resolve spontaneously in 10 to 12 weeks. macular or paramacular scarring, vascular occlusions and post-infectious optic atrophy associated to the central scarring lead to poor visual acuity outcomes. retinal complications may cause 40-50% of permanent vision loss, and up to 71% of the affected eyes reached the criteria for legal blindness [38, 84, 85] . no chronic anterior uveitis, posterior synechiae, iris nodules, uveitic glaucoma nor cataract were described [38, 84, 85] . it is not known if the ocular manifestations of the rvf result from direct toxicity of the virus or from an immune response to the infection. post-mortem examination suggested the presence of focal areas of retinal necrosis and retinal pigment epithelium (rpe) degeneration with round cell inflammatory infiltration and perivascular cuffing but the presence of the virus in the ocular tissues has not been proved. most of animal models for rvf do not show any ocular disease [35, 57] . in a sheep model quantitative rt-pcr (qrt-pcr) was positive on eye tissues after the viremic phase [90] , but retinal complications of rvf could also be caused by antibodyrelated auto-immune reactions [86] . in a seroprevalence study, mothers experiencing fetal death or miscarriage had the same rvfv antibody prevalence as those with normal deliveries [91] . a retrospective study in egypt in 1980 found no increase in the risk of abortion in humans [92] . however a recent cross sectional study has demonstrated an association between infection with rvfv and miscarriage in sudanese pregnant women (54% versus 12% of risk in non-infected pregnant women with p < 0.0001 and or 7.4 with 95% ci [2.7-20.1] in multiple logistic regression analysis) [93] . the teratogenic potential of the rvfv is unknown. occasional vertical transmission has been reported, sometimes with fatal outcome in the newborn [94, 95] . few symptomatic infections were described in pregnant women [91] and children under the age of 10 years old [96, 97] . the question is remaining whether it is the result of a lack of exposure to infected mosquitoes and infected animals, or if there are differences in susceptibility between animals and humans [8] . importantly, hemorrhagic complications require high cautious infection control measures, following the cdc guidance on infection control precautions for hemorrhagic viral fevers (hvfs), while waiting for the exclusion of other hvfs such as ebola virus disease or crimean-congo hemorrhagic fever [39, 98] . standard precautions with personal protective equipment (ppe) were reported sufficient to prevent from nosocomial transmission of rvfv during the outbreak in arabian peninsula [99] , and must be implemented according the who checklist [100] , to care any suspected case regarding the theoretic risk of rvfv transmission through contact with infected blood, tissues, or other body fluids, secretions and excretions. considering rvf is also mosquito-borne disease [56] , we recommend for all-day preventive measures against vectors using physical (long clothing and bed nets), chemical (topical repellents and insecticide impregnations) barriers in the environment of viremic patients. considering the mean length of the viremia, these measures could be reasonably stopped 1 week after the illness onset, but there is no evidence-based cut-off time to allow donation of blood and removal of tissue or organs for transplantation from a rvfv-infected patient. samples of suspected cases must be collected with ppe and safe handled adhering to bsl-3 precautions. specimens must be labelled, packaged in accordance with the guidelines for the transport of dangerous biological goods (triple packaging), stored at 4 °c and addressed to a reference center. if necessary, whole-blood specimens can be dried on blotting paper, stored 30-60 days and transported without refrigeration for retrospective diagnosis confirmation [9] . according to who a confirmed rvf infection relies on (i) detection of rvfv rna by reverse transcriptasepolymerase chain reaction (rt-pcr) on sera or plasma; (ii) igm and igg detection by enzyme-linked immunosorbent assay (elisa). viral isolation is also an assay for laboratory confirmation of rvfv infection but this assay needs to be done on bsl3 and is less sensitive than detection of viral rna by rt-pcr. interestingly, rt-pcr for rvfv was reported positive for a prolonged period in urines, semen [74] and whole blood [9] . the rvfv rna load in blood usually decreases between days 1 to 4 and may be detectable until day 8 after the onset of symptoms [9] . prolonged and intense viremias were reported during acute encephalitis and hemorrhagic fever. thus, testing of serial patient specimens collected 24 to 48 h apart may have prognostic value in determining patient outcome. the decrease in viral loads coincides with a rise in rvfv specific igm and igg antibodies that may be testing using elisa. the presence of igm antibodies appears as an early transient response (day 4 to 60) and protective igg antibodies persist for several years [36] . a second convalescent blood sample collected 7-14 days after the first is necessary to confirm the seroconversion making a definitive diagnosis of a recent rvf infection. in case of delayed-onset of encephalitis or ocular complications, imputation to rvfv may be difficult if only igg are identified in blood at this stage. in human cases with encephalitis, specific igm and igg can be detected in csf [72, 74] . no positive rvfv cultures or rt-pcr have been reported in csf, or on aqueous or vitreous samples and should be further tested. management of rvf human cases comprises ipc measure implementation and general supportive therapy. no specific treatment is currently available. iatrogenic use of medications such as hepatotoxic analgesics (acetaminophen), aspirin or non-steroid anti-inflammatory drugs, which enhance the risk of hemorrhagic complications, must be avoided in the early stage. co-infections or alternative diagnosis with parasitic, bacterial, fungal or viral pathogens must be considered and treated as early as possible to improve the outcome. severe patients should be treated empirically with broad spectrum antibacterial drugs and antimalarial molecules according to the local epidemiology. in 2000, the saudi arabian ministry of health evaluated the feasibility of a randomized, placebo-controlled trial using intravenous ribavirin in patients with suspected severe rvf, but no official result was published. in a who report of the emerging and dangerous pathogens laboratory network in 2016, it was briefly mentioned that ribavirin was used without efficacy in saudi arabia [101] . evidence suggests ribavirin efficacy in animal models [102, 103] , but it failed to prevent from neuropathology in mice infected by rvfv by aerosol exposure [75] . ribavirin is recommended for the treatment and the prophylaxis of hemorrhagic fever due to arenaviruses and bunyaviruses [104] and was successfully used to cure and prevent from lassa fever [105] . to date, its use is not indicated when rvf diagnosis is confirmed [39, 56] . antiviral drugs are under development including favipiravir t-705, 2′-fluoro-2′-deoxycytidine (2′-fdc), and benzavir-2 [106] [107] [108] [109] . molecules targeting viral components, host cellular components or pathways, such as the ubiquitin proteasome system, autophagy system, kinases and oxidative stress responses, have demonstrated in vitro efficacy against rvfv [110] . the use of polyclonal immunoglobulins or serum of recovered patients has not been reported. specific monoclonal neutralizing antibodies could be developed in the coming years [111] . to date, liver transplantation has never been attempted in rvfv fulminant hepatitis. in case reports of encephalitis, the use of amantadine, rifampicin, and dexamethasone [72] , doubled prednisone doses with a stop in immunosuppressive drugs [74] , and phenytoin [70] were reported but not evaluated precisely. early renal substitution therapy in patients with severe acute renal failure improve the prognosis and survival [39, 56, 81] . for eye involvement, artificial tear preparations may maintain corneal lubrication and provide temporary comfort for ocular irritation. topical ophthalmic steroids were used in the rvfv anterior segment manifestations [84] . aciclovir was used in eyes lesions of other mosquito-transmitted viral disease notably chikungunya, dengue and west nile (table 2 ) [112] . other antiviral drugs (e.g. ganciclovir, foscarnet) could be administrated through intravitreal routes. in case of elevated intraocular pressure, antiglaucoma medications could be useful. ocular surgery including cataract removal, retinal hole and detachment repair, vitrectomy, and laser ablation for neovascularization could be additional therapeutics to be evaluated. based on the clinical and biological scoring system for the prognosis of rvf established by adam et al. [64] , the cdc definitions of suspected severe rvf cases during the major epidemic in saudi arabia in 2000 [42] , the updated guidelines for health workers [39] , and clinical series in literature [19, 40, 43, 45, 47, 61, 63] , we propose an algorithm to help clinicians at the bedside in the classification and referral of patients during a rvf outbreak (fig. 4) . using the dengue model for case management, we identified clinical and biological warning signs defining complicated cases at risk of severe illness and requiring hospitalization for medical supervision, as we already proposed for chikungunya [113] . severe illness included hemorrhagic fever, neurological disorders or hepatic/ renal failures requiring intensive cares. ocular signs were classified as complications. ophthalmologic examination should be prospectively performed in all confirmed cases to detect early asymptomatic rvfv ocular signs and evaluate their potential ability to predict the occurrence of neuro-ophthalmologic complications. this should at least include visual acuity determination, intra-ocular pressure measurement, slit-lamp biomicroscopy and funduscopy by indirect ophthalmoscopy. in case of rvf signs, a fluorescein angiography should be performed as well as fundus photography if available. indocyanine green angiography has not been evaluated yet in fvr ocular manifestation but could possibly bring arguments for a choroidal involvement since a delayed peripapillary choroidal filling in the arteriovenous phase of fluorescein angiography has been described [38, 85] . optic coherence tomography (oct) is a recent technique that is yet to be evaluated in the retinal complications of rvf. oct could be helpful to describe the retinal lesions and their evolution through time, and could help elucidate the nature of the macular exudate-like lesions described before [114] . we recommend to follow-up rvf patients for at least 1 month after the onset of symptoms to monitor for possible delayed neurological and/or ocular complications. in the lack of adequate medical support, considering the diversity and time course of rvf complications, medical evacuation of confirmed cases may be considered, except if hemorrhages because this presentation is a highly contagious vital emergency. rift valley fever has emerged and extended outside africa in the 2000s, leading to the re-description of the human disease. there is a global concern about the risk of rvfv exportation in areas where competent vectors are present including europe and north america. economic losses, human morbidity and mortality may be significant during epidemics. infection with rvf has a wide clinical spectrum and may result in delayed complications. there are no commercially licensed vaccines nor antiviral treatment for humans. human cases are often detected when the virus has already spread among livestock and people, hence outbreak control is challenging. thus, human case surveillance systems for early detection and correct management are essential to reduce global morbidity and mortality. we proposed a tool for physician guidance on the field. this algorithm should be evaluated during ongoing and coming outbreaks, and could help neighboring places in the detection of cases. we identified frontiers of knowledge and remaining uncertainties concerning rvf which deserves more interest. in particular, therapeutic trials on specific supportive care, antiviral molecules or immunotherapies, should be anticipated to be implemented at the start of future epidemics. enzootic hepatitis or rift valley fever. an undescribed virus disease of sheep cattle and man from east africa a review of mosquitoes associated with rift valley fever virus in madagascar rift valley fever in namibia rift valley fever rift valley fever: biology and epidemiology biosafety in microbiological and biomedical laboratories (bmbl), 5th edn. cdc laboratory portal mosquitoes of north-western europe as potential 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rift valley fever virus evolved with increasing severity in human populations in east africa? rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the m rna complete genome analysis of 33 ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry rift valley fever virus: unanswered questions factors associated with severe human rift valley fever in sangailu using a field quantitative real-time pcr test to rapidly identify highly viremic rift valley fever cases association of symptoms and severity of rift valley fever with genetic polymorphisms in human innate immune pathways rift valley fever the pathogenesis of rift valley fever virus in the mouse model liver involvement in patients with moderately severe rift valley fever ultrastructural pathology of human liver in rift valley fever pathologic studies on 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description of a case clinical studies on rift valley fever. part 2: ophthalmologic and central nervous system complications rift valley fever in kidney transplant recipient returning from mali with viral rna detected in semen up to four months from symptom onset, france, autumn 2015. eurosurveillance aerosol exposure to rift valley fever virus causes earlier and more severe neuropathology in the murine model, which has important implications for therapeutic development aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets emerging infections of cns: avian influenza a virus, rift valley fever virus and human parechovirus neutrophil and macrophage influx into the central nervous system are inflammatory components of lethal rift valley fever encephalitis in rats peripheral blood biomarkers of disease outcome in a monkey model of rift valley fever encephalitis vascular permeability in the brain is a late pathogenic event during rift 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study case report: rift valley fever with vertical transmission in a pregnant sudanese woman vertical transmission of fatal rift valley fever in a newborn an outbreak of rift valley fever in northeastern kenya, 1997-98 rift valley fever among children and adolescents in southwestern saudi arabia isolation precautions. guidelines library. infection control the risk of nosocomial transmission of rift valley fever standard precautions in health care fièvre de la vallée du rift en afrique: aspects cliniques chez l'homme enhanced efficacy of liposome-encapsulated ribavirin against rift valley fever virus infection in mice prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug bichat guidelines for the clinical management of haemorrhagic fever viruses and bioterrorism-related haemorrhagic fever viruses review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever anti-rift valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound t-705) protects against peracute rift valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment 2′-fluoro-2′-deoxycytidine is a broad-spectrum inhibitor of bunyaviruses in vitro and in phleboviral disease mouse models lethal mutagenesis of rift valley fever virus induced by favipiravir recent advances in the development of antiviral therapeutics for rift valley fever virus infection a protective monoclonal antibody targets a site 19:4 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from: of vulnerability on the surface of rift valley fever virus ocular manifestations of mosquitotransmitted diseases french guidelines for the management of chikungunya (acute and persistent presentations) the role of optical coherence tomography angiography in the management of uveitis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are very thankful to julien ripamonti for his precious help in figures.marseille, france. 3 authors' contributions ej made the literature review and wrote the first draft. al made literature review and wrote the draft on ocular diseases and built table 2 . gh reviewed and corrected the ophthalmological part of the draft. vps, fl, gt are the epidemiologists involved in the response to rvf cases in soldiers in mayotte, they provided epidemiological data, asked for questions to clinicians and reviewed the draft. tl was the french military physicians deployed in mayotte who was at the origin of this work. gd and ilg from the french national reference center for arboviruses, confirmed cases in mayotte and helped in the discussion and the draft for the biological part. fs is national referent in infectious diseases for the french armed forces; he managed the working group, helped in the selection of references and the algorithm development. he reviewed and corrected the draft. all authors read and approved the final manuscript. authors did not receive any funding for this work. not applicable. not applicable. not applicable. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential competing interests. key: cord-006818-2lclcf1x authors: tibary, a.; rodriguez, j.; sandoval, s. title: reproductive emergencies in camelids date: 2008-06-02 journal: theriogenology doi: 10.1016/j.theriogenology.2008.04.024 sha: doc_id: 6818 cord_uid: 2lclcf1x emergencies in theriogenology practice go beyond just saving the life of the patient, but also preserving its reproductive abilities. camelid emergency medicine is a relatively new field. this paper discusses the most common reproductive emergencies, their diagnosis, treatment, and prognosis in male and female camelids. the conclusions drawn are based primarily on clinical observations by the authors over the last 25 years. special consideration is given to peculiarities of the species, particularly in the choice of obstetrical manipulations and therapies. in theriogenology practice, emergencies are defined not only in terms of concerns for the welfare of the patient, but also for its future reproductive life. the challenge often faced with reproductive emergencies is how to preserve the life of the patient and maximize the chance to maintain reproductive ability. in camelids, this is even more important, as assisted reproductive technologies have either not yet been perfected (semen preservation, in vitro embryo production, nuclear transfer) or are not allowed (embryo transfer) by some breed registries. in the case of pregnant females, which constitute the majority of reproductive emergencies, the life and welfare of the neonate must also be considered. the objective of the present paper is to review the most common reproductive emergencies in male, female, and neonatal camelids. this review draws primarily on the clinical experience of the authors, as there are very limited controlled studies regarding clinical reproduction and emergency care in camelids. although many disease processes that present as emergencies may have some severe repercussions on the reproductive process in the male, our review will be limited to diseases and accidents with sudden onset that are directly linked to the urogenital system. reproductive emergencies in the male camelid are primarily due to sudden onset of visible abnormalities in the external genitalia. these abnormalities can be summarized as acute scrotal or preputial swelling, preputial prolapse or paraphymosis, and post-surgical emergencies [1] [2] [3] . in camels, preputial swelling is also a primary clinical sign of acute trypanosomiasis, a disease with high morbidity and mortality in many countries where camel breeding is important [3] . www.theriojournal.com 2.1. general approach to examination of the male for reproductive emergency as with any other emergency, accurate body weight, body condition score, physical examination, degree of dehydration, baseline complete blood count (cbc), blood biochemistry, and urinalysis should be part of the initial evaluation. immediate placement of an intravenous catheter is indicated in severely compromised or recumbent animals. ultrasonography of the urogenital organs should also be conducted. testicular thermoregulation is very important for normal spermatogenesis in camelids. therefore, compromised testicular thermoregulation in these animals should considered serious, as the effect on spermatogenesis can be long lasting or permanent. acute scrotal swelling is generally due to heat stress, trauma, or a local or systemic infectious process. testicular torsion and scrotal hernia are commonly considered as differential diagnoses in other large animal species, but have never been encountered in camelids in our practice [2] . scrotal and preputial edema and development of severe hydrocele are features of heat stress in the male llama and alpaca [2] . this syndrome is relatively common in the usa. factors predisposing to heat stress include prolonged high ambient temperature and humidity, inadequate shade, long fleece, dark coat color, and obesity. the risk for heat stress is exacerbated by stresses such as transportation, exercise, fighting, and breeding [4, 5] . hyperthermia results from impaired evaporative cooling, particularly under hot and humid conditions [5] . scrotal edema may be the first clinical sign in the male. the exact pathophysiology of the scrotal and ventral abdominal edema is not well understood. contributing factors may include inability of the pampiniform plexus and testicular artery to cope with the fluid turnover, or vascular thermal injury resulting in impaired wall permeability and extravasation of intravascular proteins, electrolytes, and fluid into the interstitium. many cases may resolve spontaneously, but leave the male infertile for various intervals, usually lasting from 2 months to years [2] . spermatogenesis (sperm production and semen quality) was severely impaired in llamas housed at an ambient temperature of 29 8c for 4 weeks [6] . these temperatures, relatively common in summer months in many countries outside the native range of south american camelids, can result in infertility due to decreased sperm numbers, decreased motility, and increased abnormalities. the heat index (ambient temperature â humidity) would cause even more severe changes in hot and humid summers [6] . in advanced cases, other clinical signs appear and include hyperthermia, increased salivation, anorexia, depression, ataxia, muscular weakness, dehydration, ketosis/hepatic lipidosis, and dyspnea/hyperpnea [4, 5] . these animals generally display an inflammatory or stress leukogram. anemia may be secondary to hemolysis. serum biochemical abnormalities may include hypophosphatemia, hypocalcaemia, hypomagnesaemia, hyponatremia, hypochloridemia, hypo-or hyperkalemia, hyperglycemia, and elevated serum ast and cpk concentrations. serum glucose concentration >300 mg/dl has been associated with a poor prognosis. severe electrolyte imbalances and damage to the thermoregulatory center in the hypothalamus will be the end point of the disease progress in nontreated animals, leading to multi-organ damage or failure and increased mortality [4, 5] . stabilization of the heat-stressed animal should include urgent cooling of the core body temperature to the normal range (shearing, spraying the ventral abdomen with cold water, fan), and fluid therapy to rehydrate the animal and correct metabolic abnormalities. intravenous isotonic sodium bicarbonate solution may be required to treat metabolic acidosis. maintenance fluid rates are 30-40 and 80-120 ml/kg/day in adults and crias, respectively. pulmonary edema is a serious risk if fluids are administered too fast (>20 ml/ kg/h). palliative therapies against other complications should include nasal oxygen insufflation in hypoxemic patients, nonsteroidal anti-inflammatory drugs (nsaids; e.g. flunixin meglumine), antioxidants (vitamin e and selenium), and broad-spectrum antibiotics. steroids such as dexamethasone may be indicated in advanced cases, but should not be used in females in the second half of pregnancy. therapeutic diuresis with furosemide is indicated in animals with respiratory distress due to pulmonary edema. heat stress is best prevented by timely shearing, adequate hydration (clean, cool water) and providing shade and cooling mechanisms such as sprinklers, a pond, or wading pool. prevention of obesity and reduction of stresses of long transportation, handling and breeding during the hottest part of the day also reduce the risk for heat stress. the primary indicator of heat stress risk is not only the ambient temperature, but also the humidity. the heat stress index (hsi), expressed as the ambient temperature (8f) + humidity (%), is considered too high when it reaches or surpasses 160 (e.g. combination of 100 8f and 60% humidity). traumatic injuries to the scrotal area are relatively common in the male camelid and are usually inflicted by other males; they occur when new, mature males are added to a paddock, particularly when competing for breeding. severe traumatic fighting injuries are more common in camels during the rutting season [7, 8] . scrotal traumatic injuries are relatively rare in wild camelids, probably because of their strict social organization. traumatic injuries are often due to bites and can range from a superficial scrotal laceration to severe testicular rupture and hemorrhage. testicular hemorrhage may occur without external lacerations, but requires ultrasonographic evaluation of scrotal contents [7] . treatment protocols should focus on reducing local swelling, preventing infectious complications, and providing a tetanus toxoid booster. unilateral castration is the treatment method of choice for severe unilateral testicular trauma involving the tunica vaginalis and testis [8] . testicular and epididymal inflammation may present as an emergency in the male camelid. the most common complaint is a sudden onset of lameness or reluctance to breed and visible swelling of the scrotum. various infectious agents have been reported in cases of orchitis that are spread by hematogenous routes, such as brucella abortus, brucella meletensis and streptococcus equi zooepidemicus, or the agent may ascend from scrotal wounds [9, 10] . treatment with systemic antimicrobials is often unrewarding. therefore, for unilateral orchitis, unilateral orchidectomy is the best option for the welfare of the male and salvage of reproductive ability [8, 9] . 2.6. acute penile/preputial swelling acute penile or preputial swelling may be due to complications from urolithiasis or traumatic injuries. the etiology of urinary calculi in the camelid is not well understood, but is suggested to be similar to that in other domestic ruminants [11] [12] [13] [14] [15] [16] [17] . early clinical signs of urethral obstruction often go undetected. some males may show increased straining to defecate, odontoprisis, inappetence and ileus, followed by anorexia, frequent unsuccessful attempts at micturition or dribbling blood tinged urine, and signs of abdominal discomfort [13] [14] [15] [16] . complications of urethral obstruction include urethral or urinary bladder rupture. this may happen within 2 days of the first clinical signs. in emergency cases, the animal presents with anorexia, inability to pass urine, and signs of depression. physical examination often reveals tachycardia, tachypnea, and elevated rectal temperature. complete blood count may reveal an elevated white cell count and neutrophilia with a left shift, increases in fibrinogen, increased creatinine kinase and aspartate aminotransferase activity, hyperglycemia, hypercreatininemia and increased urea nitrogen. serum electrolyte abnormalities included hyponatraemia, hypochloraemia, and hyperkalaemia. fluid obtained by abdominocentesis or from the preputial swelling has increased creatinine concentration [12] [13] [14] [15] [16] . increased serum urea nitrogen and creatinine concentrations suggest uroperitoneum [16] . transcutaneous ultrasonography of the ventral abdomen may enable visualization of subcutaneous free fluid and tissue edema in the case of urethral rupture and a large volume of free fluid in the abdominal cavity in the case of urinary bladder rupture. with the latter, it may not be possible to visualize the urinary bladder. transrectal ultrasonography may reveal dilation of the pelvic urethra if the bladder is intact. the prognosis is grave if there is hydroureter and hydronephrosis [16] . uroliths are often located in the distal penile urethra, approximately 7-12 cm from the penile orifice, but are occasionally immediately proximal to the sigmoid flexure. camelids, like domestic ruminants, have a urethral recess at the ischial arch, making catheterization of the urinary bladder exceedingly difficult. management techniques for obstructive urolithiais is similar to those reported in ruminants and include repair of the ruptured urinary bladder and relief of the obstruction via retrograde flushing and urethrotomy. however, these techniques do not salvage the reproductive career of the animal. flushing, followed by tube cystotomy, may be the only option to try to salvage the reproductive life of the animal [16] . postsurgical management should include multiple therapies, including antimicrobial (procaine penicillin, 30,000 iu/kg im, twice daily; and gentamicin sulphate, 6.6 mg/kg iv, once daily), anti-inflammatory (flunixin meglumine, 1 mg/kg iv, twice daily) and intravenous fluids. prognosis for life is fair, but prognosis for return to breeding is usually guarded [16] . preputial lacerations are relatively common in breeding males. they are usually a consequence of masturbation (breeding the ground or objects) or complications from foreign objects within the prepuce. hair-ring lacerations of the penis are common in llamas and suri alpacas. males may present because the owner has observed an abnormal protrusion of the prepuce, or discomfort during urination or mating. however, bloody or purulent discharge may be the only clinical sign. preputial and penile lacerations can quickly become complicated and jeopardize the reproductive life of the male due to development of severe inflammation and adhesions. injured males may continue to attempt breeding, further exacerbating the lesions [7] . evaluation of penile and preputial injuries is best performed under heavy sedation or general anesthesia. the penis should be completely exteriorized and inspected for lesions. early management of preputial and penile injuries should center on providing adequate protection of the traumatized tissue and prevention of infection and complication with urine scalding. the initial treatment is to replace viable prolapsed preputial mucosa and maintain it in place with a purse string suture. daily cleaning of the sheath with saline, and application of local anti-inflammatory and antimicrobial ointment (petercillin) for 3-5 days will reduce the chance of further complications. sutures may be removed after 7-10 days. excessive preputial prolapse with slight necrosis requires circumferential resection and anastomosis of the prepuce. prognosis for return to normal breeding activity is poor if adhesions or abscesses develop at the base of the prepuce [2, 18] . the most common post-surgical complication in the male camelid is post-castration hemorrhage, often secondary to inadequate time to insure hemostasis of the testicular cord. management of these conditions is not different from other species and includes placing the male in a calm environment and packing the bursa for 6-24 h. several commercial hemostatic agents are available and may be helpful [19] . exteriorization of the soft palate (dulla) is a characteristic rutting behavior in the dromedary camel [7] . furthermore, permanent exteriorization of the soft palate during the rut season is a common in the dromedary. this usually starts with an impaction of the diverticulum with food or a foreign body [20, 21] . part of the impacted soft palate becomes trapped under the molars and is traumatized during mastication. traumatic lesions of the soft palate range in severity from superficial cuts and bruises to severe lacerations accompanied by hemorrhage; these lesions are rapidly complicated by infection and development of severe inflammation and edema. formation of large abscesses is not uncommon. in most cases, the inflamed organ is permanently hanging from the side of the mouth and becomes progressively necrotic [22] [23] [24] . in a few cases, the soft palate is swollen, but not exteriorized, and blocks air exchange, which may lead to asphyxiation. if the condition is not treated, the animals become emaciated due to dysphagia and impairment of mastication and deglutition. management of these cases requires surgical ablation of the soft palate [20] . surgical excision of the soft palate can be performed under heavy sedation and a local block. large vessels are ligated with resorbable suture material. laser ablation is the best approach. postsurgical management includes administration of nsaids, antimicrobials, and tetanus prophylaxis. animals should be on soft feed for at least 3-4 days after surgery. urethral rupture and subcutaneous infiltration of urine is relatively common in draught camels and is due to a tight strap. advanced stages are managed surgically by complete urethrostomy. animals present with varying degrees of ventral swelling and prolapse of mucocutaneous junction of the penis and prepuce. tissue necrosis is common, and may include the penis due to pressure ischemia. surgical debridement [25] , phalectomy, or both, may be required [26] . reproductive emergencies in female camelidae can be divided into emergencies occurring in the nonpregnant female, severe pregnancy complications, obstetrical emergencies, and postpartum emergencies. emergencies requiring intervention during parturition and the immediate postpartum phase must concurrently take into account emergencies pertaining to the neonate. the most common reproductive emergencies in nonpregnant females are traumatic injuries during breeding or iatrogenic injuries during reproductive examinations. although rare, breeding trauma may occur during an unsupervised paddock mating. in camels, traumatic injuries are not always restricted to the reproductive tract, and include bite wounds and fractures of the pelvis and/or dislocations. these traumatic injuries are seen in multiple-sire breeding systems. in south american camelids, breeding trauma may occur by heavy llama males trying to breed alpacas. discussion of these types of traumatic injuries are beyond the scope of this paper, but should be considered in downer syndrome in females with a history of recent (<24 h) mating. iatrogenic traumatic injuries are by far the most common emergency in camelid practice; they include perforation of the rectum, colon, vagina, or uterus. anal sphincter bleeding due to excessive stretching and rectal prolapse can occur secondary to transrectal palpation, particularly when there is already a predisposing factor for excessive straining (e.g. pelvic mass, urinary bladder disease). however, these are not life threatening and can be managed successfully with sedation, a caudal epidural, and protection of the prolapsed tissue. rectal and colonic injuries have been reported in llamas and alpacas, and are a common reason for malpractice suits. rectal or colonic injuries may happen during breeding, but they more commonly are due to excessive manipulation during transrectal palpation or ultrasonography [27, 28] . the examiner will usually recognize that an injury has occurred when palpating llamas and camels. however, in alpacas, when the ultrasound transducer is mounted on an extension for reproductive examination, the practitioner may not detect evidence of perforation until it is too late. the amount of blood retrieved with the palpating hand is variable; it is the sensation of rupture or tear that is most indicative of the seriousness of the injury. since the distance between the anus and the peritoneal reflection is very short (2-3 cm in alpacas, 4 cm in llamas, and 6-10 cm in camel), complete rectal tears in camelids are rapidly complicated by peritonitis. often the only clinical sign is reluctance to stand, lethargy and progressive dehydration a few hours after a reproductive examination. severe toxic shock and death follows within 8-24 h if no medical action is taken. all suspected rectal or colonic injuries should be immediately referred to a surgical facility. the animal should be sedated and started on intravenous antimicrobial and anti-inflammatory therapy for transport. further evaluation at the referral facility includes cbc, blood chemistry, transabdominal ultrasonography, and abdominocentesis. animals with evidence of peritonitis should be immediately prepared for surgical correction by celiotomy or celiotomy and pubic symphysiotomy to allow peritoneal lavage. stable patients without alarming changes in their blood and peritoneal fluid characteristics may be further evaluated under epidural anesthesia to decide if a transanal repair is possible. evaluation of the injury can be performed under general anesthesia. the anal sphincter is dilated using stay sutures on the mucocutaneous junction. gentle evacuation of the rectal cavity may be attempted by low-power vacuum aspiration until the lesions can be visualized. use of a flexible videoendoscope can facilitate this evaluation. in llamas and camels, lacerations due to transrectal palpation are usually located in the ventral aspect of the rectum, 5-20 cm anterior to the anus. however, in alpacas, particularly when the perforation has occurred with a transducer mounted on an extension, the lesion can be dorsal. also, in these cases, the presence of more than one perforation is possible, perhaps due to faulty alignment between the extremity of the transducer and the extension rod. transanal repair is successful if the laceration is not deep [28] . celiotomy with pubic symphysiotomy is the only option for caudal injuries and in particular for alpacas. successful repair of rectal and colonic injuries by celiotomy or celiotomy/pubic symphysiotomy has been reported in a few llamas [28] . preventive measures for colonic rectal injuries include use of caution when choosing the candidate for transrectal palpation, ample lubrication, and cautious use of an extension rod, particularly in maiden or agitated females. sedation of the female or relaxation of the rectum and rectal sphincter may be obtained by epidural anesthesia or instillation of 2% lidocaine into the rectal cavity before examination. most cases of uterine perforations seen in our practice are iatrogenic, due to aggressive placement of foley catheters, infusion pipettes, and biopsy forceps. these are more common in alpacas than in llamas and camels. they become an emergency if a major blood vessel is damaged, or if an irritating substance (e.g. iodine) is infused into the abdominal cavity. females with these injuries may present with colicky signs consistent with peritonitis or hemoperitoneum. anemia is a feature if there is sufficient blood loss; for example, one animal had a pcv of 10% following an endometrial biopsy. suspicion is based on a history of recent gynecological examination and the feel of a ''pop'' during manipulation. the patient should be worked up as for any case of colic of abdominal origin [12] . supportive therapy includes nsaid's and antimicrobials. blood transfusion and surgical intervention may be indicated if the pcv is <8%. vaginal perforation with severe bleeding may be controlled by vaginal compression packs. any clinical syndrome occurring during pregnancy may have a serious effect on the fetus. therefore, monitoring fetal well-being should be part of any protocol for medical management of the pregnant female and particularly in the case of emergencies. camelids rely exclusively on the cl for progesterone secretion and maintenance of pregnancy. therefore, severe illness associated with an inflammatory or extreme stress response may rapidly lead to luteolysis and abortion (with all its complications). pregnant females may present with a variety of emergency clinical syndromes, ranging from severe colic, downer (lateral or sternal continuous recumbency), anorexia, diarrhea, depression, neurologic conditions, excessive straining, vaginal discharge, premature lactation, vulvar dilation, or vaginal prolapse. some of these presentations may have a genital origin. the cardinal rules in handling emergencies in the pregnant females are a thorough physical evaluation of the dam, evaluation of the fetus, and ruling in or out the genital origin of the presenting complaint after stabilization of the dam. the main emergencies of genital origin in the pregnant female are uterine torsion, vaginal prolapse, impending abortion, and uterine rupture. the main complication of any emergency in late pregnant females is hepatic lipidosis. pregnancy can also exacerbate clinical diseases. for example, in a recent outbreak of respiratory diseases in alpacas and llamas in north america, morbidity and mortality was highest in females in their last trimester of pregnancy. an important principle in our practice is that any suspicion by an owner that ''something is wrong'' with a pregnant female is taken seriously. behavioral assessment may be conducted while taking history, unless the female is obviously depressed or painful. a detailed history should be obtained and include breeding dates, time and methods used for pregnancy diagnosis, history of previous illness of reproductive disorders, onset and duration of the clinical problem, and recent treatments. if the female is obviously in severe distress, blood samples should be taken immediately and the female stabilized before further examination. oxygen therapy may be indicated for severely compromised females. a jugular catheter should be placed immediately to allow fluid therapy and emergency anesthesia if needed. sedation may be needed for some females in order to complete evaluation. choices of drugs and dosage for sedation should take into account their effect on the fetus. butorphanol tartrate (0.05-0.1 mg/kg) provides good sedation and has minimal effect on the cardiovascular system. however, there is a mild decrease in systemic vascular resistance that can be relevant if uterine blood flow is already compromised [29] . transabdominal ultrasonography should be used to determine fetal well-being, and the integrity of the uterus and placenta. in addition to the reproductive organs, abdominal viscera and the peritoneal cavity should be assessed [30] . in advanced pregnancy, imaging of abdominal viscera becomes very difficult in the absence of severe displacement. for complete imaging of abdominal contents, the lower abdomen should be clipped and cleaned with alcohol from the xyphoid region to the base of the mammary gland. the area to be examined may need to be extended dorsally to the flank in order to visualize the dorsal aspect of the abdomen and the kidneys. cranially, the projection area of the liver may also need to be prepared for examination. for transabdominal ultrasonography, a 5 mhz linear-array transducer may be sufficient for mid-pregnancy and in small patients, whereas in the last trimester, the use of a 2.5-3.5 mhz sector transducer provides better penetration and imaging of the abdomen. transabdomimal ultrasonography may also be used to locate distinct pockets of free peritoneal fluid and to perform abdominocentesis. other imaging techniques such as radiography, mri or ct scanning may be indicated in the case of downer females, but they are not routine procedures and are only a possibility in referral centers. following transabdominal ultrasonography, transrectal palpation and ultrasonography should be performed, albeit, cautiously, as this may cause additional stress. administration of an epidural and infusion of a mixture of lidocaine and lubricant in the rectal cavity may reduce straining, provide some relaxation, and facilitate the examination in llamas and alpacas. the primary objective of transrectal palpation is to determine the location and direction of the broad ligaments and evaluate the caudal abdomen for any masses or abnormalities of the pelvic area, kidneys, and urinary bladder. transrectal palpation in the female sitting in a sternal position may offer some challenges for the inexperienced practitioner. the quantity and quality of fecal material in the rectal cavity should be evaluated. severely stressed camelids often have profuse diarrhea, whereas an absence of fecal material and/or the presence of scant mucoid feces may be due to intestinal transit disorders or tenesmus. vaginal examination should be performed with a speculum after thoroughly cleaning the perineal area. the speculum should be advanced slowly, while concurrently examining the vagina for any abnormalities. the cervix is evaluated for the degree of relaxation and opening. the cervix of the llama and alpaca is often difficult to visualize during late pregnancy, but it should be obvious if it is patent. manual examination of the vagina and cervix may be indicated in some cases, but this procedure is often limited by the size of the examiner's hands. assessment of fetal well-being is an important component in the evaluation of medical crises. unfortunately, there is a paucity of information regarding fetal biophysical characteristics in camelids. however, based on clinical experience in the authors' laboratory, the two main indicators for fetal distress are fetal heart rate and rhythm. normal fetal heart rate in mid-to late-pregnancy range from 1.6 to 1.8 times that of the dam. in that regard, fetal heart rate is usually 80-115 bpm in the last trimester of pregnancy, but decreases to 80 bpm a few days before parturition. fetal heart rates that are consistently >130 or <50 bpm suggest fetal distress. the fetal heart rhythm should be regular and respond to phase of activity by a 10-20% increase in rate. fetal activity is maximal in the first half of pregnancy, but substantially reduced in the last 2 months. the entire fetus should be examined to determine fetal position and number. normal fetal positioning for parturition appears to occur a few hours before parturition. it is not uncommon to image the fetus low in the abdomen with the dorsum against the diaphragm and all limbs pointing to the pelvic area. transverse position of the fetus in the abdomen does not mean a transverse position inside the horn, but rather reflects the position of the entire pregnant horn. that the fetus is entirely in the left horn and the special arrangement of the pregnant horn vis-à-vis the abdominal viscera may contribute to signs of discomfort in some females in late pregnancy. late in pregnancy, the presence of twins is best confirmed by abdominal radiography [31] . fetal biometrics may provide data regarding fetal growth and stage of pregnancy, but in our experience, most measurements are not very accurate and cannot be used for physical bioprofiling [32, 33] . fetal fluids are difficult to assess, due to the low volume of amniotic and allantoic fluid in camelids. uteroplacental thickness should be evaluated in the horn containing the fetus (left horn) only, as the placenta may appear thicker in the nonpregnant horn. the combined uteroplacental thickness should be <8 mm in the last trimester. excessive edema of the uterine horn or premature placental detachment are relatively easy to detect and require immediate intervention if the female is at term. a minimum baseline evaluation of a severely depressed or colicky pregnant female should include complete blood count (cbc), blood chemistry, and fibrinogen. evaluation of peritoneal fluid (abdominocentesis), fecal evaluation, and urinalysis should be considered in select cases. although a stress leukogram is often present in many females, neutrophil count, immature neutrophil count, neutrophil morphology, packed cell volume, and fibrinogen concentration are very valuable in evaluating inflammatory and toxic states. anemia may be due to blood loss, or the onset of other problems such as mycoplasma hemolamae. blood chemistry will determine electrolyte imbalances and risk for hepatic lipidosis, a major concern in anorectic, stressed pregnant females. hypoprotenemia is often present in old pregnant females and may predispose to metabolic complications. in some cases, the serum may be grossly hyperlipemic (white). however, lipemia and ketonemia are not always present in hepatic lipidosis. elevated concentrations of nonesterified fatty acid (nefa; >400 mmol/l) and b-hydroxybutyrate (bhb) are important indicators of stress and liver compromise. liver compromise is also indicated by elevated bile acids, gamma-glutamyl transferase, aspartate transaminase, and sorbitol dehydrogenase [17, 34] . furthermore, arginal calcium and magnesium concentrations or hypocalcemia may be present in late-pregnant females and require correction and monitoring. progesterone is the major hormone evaluated routinely during pregnancy [35, 36] . determining baseline progesterone concentration is a good practice if an assay is readily available. the cl is the primary source of progesterone throughout pregnancy in camelids; pregnancy cannot be maintained if blood progesterone concentrations are <1 ng/ml [37] . progesterone concentrations may be substantially altered by level of hydration and weight and body condition score of the female. progesterone supplementation is still a subject of debate. estrone sulfate concentrations in plasma increase after 80 days of pregnancy, reaching a peak immediately before parturition. determination of relaxin concentration may be helpful in the evaluation of placental function, but this assay is not widely used [35] . there are no studies on the effect of a compromised liver (typically due to hepatic lipidosis) on steroid metabolism and blood steroid concentrations. supportive therapy in pregnant females depends on the symptoms and degree of compromise. it may include oxygen therapy, fluid therapy, antimicrobials, and nsaid's. compromised pregnant females should be placed immediately on broad spectrum systemic antimicrobials. our primary choices of antimicrobials have been ceftiofur in alpacas and llamas and longacting tetracycline in camels. uterine torsion remains the main genital cause of colic or depression in pregnant new world camelids. there are no detailed studies regarding the epidemiology of this disorder. it is noteworthy that uterine torsion is not common in camels (a. tibary, unpublished observations), nor is it common in llamas and alpacas in south america (j. sumar, personal communication). perhaps this apparent difference is due to nutrition or body size. in our experience, there are two common stages of pregnancy at presentation: 8-10 months and at parturition. clinical signs of uterine torsion are quite variable, ranging from mild discomfort to severe colic, diarrhea, and anorexia. we have had cases present simply as ''quieter than usual'' and ''decreased appetite'' or ''just a little off her normal routine'' [31] . the female may display signs of pain, circling, kicking at the belly, lateral recumbency, and excessive vocalization. tachypnea and tachycardia are very common. the cbc and blood chemistry are consistent with a stress leukogram, with various metabolic changes (hepatic lipidosis) depending on the duration and severity of the problem [38] . diagnosis is based on transrectal palpation of the broad ligaments, as described in other large animal species [31, 38] . clockwise torsion is indicated if the left broad ligament is stretched across midline to the right and over the uterus, whereas the right ligament is shorter and pulled ventrally and medially under the uterus. palpation of the broad ligament may elicit a severe painful reaction. difficulties encountered in transrectal evaluation for uterine torsion include physical limitations, particularly in alpacas (tight anal sphincter, narrow pelvis and size of the examiner's hand and arm), as well as a lack of experience palpating late-pregnant camelids in a sternal position. although diagnosis by vaginal palpation has been reported by practitioners, in our experience, it is not reliable unless the torsion includes the cervix. with a severe colic, a definitive diagnosis may not be possible until exploratory laparotomy. alternatively, the female could be palpated under general anesthesia, which provides greater relaxation of the anal sphincter and perineal area [39] . transrectal ultrasonography may sometimes reveal increased dilation of the blood vessels. although it was reported that the majority (>90%) of camelid uterine torsions are clockwise [38] , this has not been our experience; therefore, direction of the torsion needs to be ascertained before attempting nonsurgical correction. correction of uterine torsion can be accomplished nonsurgically by rolling or surgically after coeliotomy. both techniques are very efficient. rolling should be considered only if the uterus and its vasculature are not compromised. rolling may be performed done under general anesthesia, sedation, or without sedation. the female is placed on lateral recumbency on the side of the direction of the torsion and rolled while the fetus is maintained in position with a small plank or with the fists [31, 38] . the pain usually disappears immediately after correction of the torsion and females may return to normal activity immediately. however, if they have been anorexic, correction of metabolic disorders should included in post-surgical management. surgical correction may be performed following flank or midline laparotomy. midline laparotomy is the preferred method in late pregnancy [39] [40] [41] [42] . the success rate of both rolling and surgical correction is very high, as is survival of the fetus. no special management is needed if the torsion has been diagnosed and corrected early. however, anorexia and pain may cause hepatic lipidosis, in which case the patient should be placed on broad spectrum antimicrobial therapy [39] . monitoring blood progesterone is useful, particularly if an assay is readily available. the need for progesterone supplementation after correction of a torsion remains controversial. complications of uterine torsion include abortion, uterine rupture/hemorrhage, endotoxemia, and death of the dam [38, 40] . splenic torsion concurrent with uterine torsion has been described in one case, with persistent pain following correction of the uterine torsion [39] . uterine rupture is often secondary to severe or inadequate clinical management of a uterine torsion. females usually present in an advanced stage of shock, in lateral recumbency. abdominocentesis may reveal large amount of serosanguinous or bloody fluid. severe pain with presence of serosanguinous peritoneal fluid may also be due to splenic torsion [39] . the only option is surgical intervention to remove the fetus and salvage the uterus. complete hysterectomy should be considered if the uterus is severely compromised. vaginal prolapse has been described during the first half of pregnancy, but the condition is more common during the last 2 months of pregnancy [31, [43] [44] [45] . it is likely due to softening of tissues due to increased estrogen concentration during the last part of pregnancy. predisposing factors include age (older females), parity, and body condition (obese and very thin females) [31, 45] . the prolapse tissue may be limited to 3-5 cm, and visible only in the recumbent female. however, with increased inflammation and edema of the tissues, the degree of prolapse increases and becomes permanently exteriorized. prolapse of the entire vagina and exteriorisation of the cervix is rare, but possible. prolonged periods of prolapse increase inflammation and can cause severe necrosis of the vaginal mucosa, potentially resulting in ascending infectious placentitis. increased tenesmus with risk of abortion and/or rectal prolapse occurs in chronic cases. furthermore, rectal and vaginal prolapse may be the only indications of dystocia or abortion [45] . the prognosis for the life of the fetus and dam is relatively good if the condition is treated early. in camels, the vaginal tissue is maintained in place with a bühner suture around the vulva. in the alpaca and llama, a shoelace suture pattern is sufficient. more advanced cases of prolapsed vagina with increased tenesmus may require epidural anesthesia [44] . the animal should be monitored regularly and the suture removed if signs of impending parturition are observed [41] . other complications of pregnancy in camelids include ventral abdomen herniation, prepartum downer syndrome, metabolic diseases, and premature lactation/ placentitis. hydrops of fetal fluid is extremely rare in camelids. ventral herniation during pregnancy is often a complication of previous abdominal surgeries, including cesarean section. in addition to determining the primary cause of these disorders and assessing the chances for survival of the female, determination of fetal well-being and the possibility of induction of abortion or parturition should be contemplated. abortion can be induced with the prostaglandin f 2a analogue, cloprostenol (250 mg in llamas and alpacas, and 500 mg in camels). the same dose is sufficient for induction of parturition, with good neonatal survival at >330 days of pregnancy and sufficient mammary gland development and colostrum production. abortion or parturition occurs approximately 18-22 h after prostaglandin treatment. in a few situations, a second treatment with a prostaglandin f 2a analogue is necessary [45] [46] [47] . giving llamas or alpacas >5 mg of pgf 2a (dinoprost thrometamine) has been associated with severe respiratory distress. most neonatal deaths occur during birth or shortly thereafter. adequate obstetrical management and monitoring for early signs of distress are closely linked with the chances of survival of the cria and the reproductive future of the dam. proper procedures, immediate neonatal care, and close of observation of the newborn, are the best means of reducing neonatal losses. normal parturition and proper obstetrical techniques have been reviewed in detail elsewhere and are not very different from the approach used in other large animal species (especially horses) [31] . it is estimated that approximately 5% of all camelid births will require some assistance and $2% will require advanced obstetrical expertise. obstetrical problems are an emergency in camelids, due to the relatively explosive and short duration of stages of parturition (similar to the mare). all normal births are in an anterior longitudinal presentation. dystocia of maternal origin include uterine inertia, uterine rupture, and failure of appropriate dilation of the cervix or vestibulum [31, 45, 48] . uterine torsion and failure of cervical dilation require delivery by cesarean section. however, it is important to confirm that the dam is at term and to first rule-out uterine torsion [31, 49] . dystocia of fetal origin occur most commonly as a result of malpositioning or malposture, and to a lesser degree, presence of malformations, twins, and large fetuses. the most common fetal causes of dystocia are carpal or shoulder flexure or head deviations (lateral and ventral). breech and transverse presentations are possible and are common reasons for cesarean section [31, 50] . fetal abnormalities causing dystocia include schistosoma reflexus, contracted tendons, and ankylosis of the hind limbs or neck [7] . other anomalies that may complicate delivery include fetal anasarca and an emphysematous fetus resulting from fetal death and gas production during decomposition [31] . although twining is rare in camelids, a few twin births have been reported. delivery of twins may be complicated by both fetuses in the birth canal at the same time. in our experience, all dystocias due to twins required a cesarean section to preserve the integrity of the female reproductive tract [31] . regarding obstetrical procedures, there are three major differences between camelids and ruminants: (1) the pelvic inlet is narrower; (2) the cervix and vaginal are more prone to laceration and severe inflammation (often leading to adhesions); (3) risks for neonatal hypoxia and death are increased by the forceful uterine and abdominal contractions and the rapid detachment of the microcotyledonary placenta. consequently, (1) early recognition of dystocia is paramount, (2) obstetrical decisions and manipulations should be rapid, and (3) supportive care should be provided to the dam and fetus (if alive) before and during manipulation. dystocia is recognized by prolongation of the first or second stage of labor. assessment of the health of the female and viability of the fetus is the first step in managing obstetrical cases. providing analgesia (epidural and administration of butorphanol) may facilitate examination of the parturient alpaca. prolongation of the first stage of parturition is primarily due to failure of cervical relaxation and uterine torsion [50] . examination of the parturient female is continued by vaginal palpation to judge cervical dilation, determine the presentation, posture and position of the fetus and its viability, and to formulate a course of action based on the findings. abdominal radiography may be helpful in determining position, posture and number of fetuses in alpacas [31, 50] . fetal manipulations are similar to other species, but need to be restricted to a maximum of 15-20 min. a different approach should be attempted if fetal position, presentation, and posture suggest that manipulation is not possible, or if manipulations are not fruitful after 15 min. we consider that fetotomy is not an option in alpacas and most llamas and camels. surgical relief of dystocia (cesarean section) remains the best approach if controlled vaginal delivery cannot be achieved in <20 min. techniques for cesarean delivery in camelids are well described [41, 51] . we recommend a flank approach in camels and any severely compromised dam. this technique does not require deep general anesthesia and can be performed under sedation and a regional block, which is a good choice under field conditions. a midline celiotomy approach is ideal if the uterus is compromised or needs to be completely exteriorized [41, 51] . regardless of the type of obstetrical intervention, adequate oxygen delivery to the uterus is essential for a healthy neonate. reducing uterine blood flow or oxygen-carrying capacity of the blood is liable to harm the fetus and may increase fetal or neonatal mortality. in most species, uterine blood flow is reduced when the dam is exposed to pain or stressful conditions. sedatives, analgesics, and anesthetics may all supress cardiac output and therefore decrease blood flow to the fetus. in addition, certain drugs or drug combinations may further decrease uteroplacental perfusion, due to their tonic effect on the myometrium. unfortunately, there are no studies on the effects of anesthetics on the uterus and fetus in camelid. xylazine, a drug of choice for sedation of camelids in the field, markedly reduced blood flow (by as much as 59%) and availability of oxygen to the uterus. furthermore, 5 min after xylazine treatment, uterine artery resistance increased by 165%. xylazine has also been associated with increased myometrial contraction in ruminants and could cause increase fetal morbidity and mortality, at least in these species [52, 53] . this effect was not significant in mares. there are no studies on the effect of xylazine on uterine perfusion in camelids. in sheep, the fetus responds to hypoxia, hypotension and hypovolumia with increased concentrations of acth, vasopressin and cortisol, via activation of the hypothalamic-pituitary axis, mediated by changes in afferent neural activity of arterial baroreceptors and chemoreceptors; it has been suggested that the fetal response is primarily mediated through chemoreceptors [54] . ketamine, a dissociative anesthetic and known noncompetitive inhibitor of glutamatergic n-methyl-daspartate (nmda) receptors, blocks the fetal reflex bradycardic response to maternal ventilatory hypoxia and may not be a good choice for anesthesia. this corroborates our observations in camelids where use of ketamine as a preanesthesic has been associated with severely depressed neonates. propofol (2,6-di-isopylphenol compound) is a small molecule that is rapidly metabolized; its advantages are rapid onset and offset of action and redistribution from the central nervous system. even with continuous propofol anesthesia, maternal and fetal heart rate and blood pressure were not affected in pregnant ewes [55] . this makes the drug ideal for induction of anesthesia for cesarean section or for surgical management of uterine torsion. propofol decreased myometrial activity in the gravid ovine uterus in vivo [56] and in uterine muscle from gravid humans in vitro [57] . in vivo, there is no effect on placental perfusion. it can induce a transient tachycardia and decrease in po2 and ph in the dam, but these effects have minimal repercussions on fetal heart rate and blood pressure. because propofol is primarily metabolized by the liver, it should be used with caution in females with hepatic lipidosis. maintenance of general anesthesia with isoflurane or sevoflurane are ideal, because these inhalation anesthetics are rapidly eliminated [56] . the combination propofol/isoflurane has been used successfully by our group in emergency cesarean section in camelids; a similar combination was also very good for cesarean section in the bitch [60, 61] . it is noteworthy that the effects of these anesthetics may be exacerbated by pre-existing conditions in the fetus (e.g. hypoxia) [56, 58, 59] . postpartum emergencies are often due to complications of obstetrical situations. however, females may present for emergency critical care with a history of what appears to have been an uncomplicated parturition. in addition to the primary genital problems that may alarm the owner (i.e. traumatic injuries, bleeding, uterine prolapse, and retained placenta) some of these cases present with ataxia, prolonged recumbency, and varying degrees of anorexia or depression as primary complaint. evaluation of the postpartum female should include a complete history and a detailed account of the obstetrical situation, including delivery of the placenta. the female should be assessed by complete physical examination, cbc, blood chemistry, transabdominal and transrectal ultrasonography, and vaginal examination. excessive fluid in the abdomen would warrant abdominocentesis. due to their small perineal body and powerful expulsive efforts, rectal-vaginal tear is common following overt obstetrical manipulations in camelids. a common cause of these tears is rapid vaginal delivery of the fetus without sufficient preparation of the vulva and vestibular area. episiotomy should be considered in females with insufficient dilation of the vulva, particularly maidens. cases seen in our practice are often a complication of fetotomy. rectal-vaginal tears may be repaired immediately, or a few weeks later, after second-intention healing [41] . postpartum uterine tears are not as dramatic as in the mare, unless there is involvement of a large vessel or severe contamination of the uterus and peritonitis. uterine bruising is often seen following excessive obstetrical manipulation (particularly fetotomy). uterine involution is very rapid in the camelid and small, dorsal uterine tears may heal spontaneously; the only sequela may be infertility due to peri-uterine adhesions. complications from uterine tears are often due to severe contamination, either during obstetrical manipulation or following partial or total retention of the placenta [62] . these females may initially appear comfortable, then slowly develop a fulminating peritonitis. clinical signs of toxemia may appear within the first 24 h, but it may take as long as 3-4 days for the clinical picture to become recognizable. it is important that these cases be stabilized, with antimicrobial and anti-inflammatory therapy initiated at the first sign of compromise. uterine lavage should be considered only after verification of the integrity of the uterine wall and should be monitored by transabdominal ultrasonography to visualize remnants of the placenta. a case of complete passage of the placenta into the abdominal cavity was described in a llama with progressive deterioration of health, which eventually succumbed to peritonitis 11 days after dystocia [62] . it is not clear how uterine tears occur in camelids; although most are associated with obstetrical manipulation, we have seen cases following spontaneous and apparently uneventful parturition. therefore, every female should be monitored to ensure delivery of the placenta, followed by inspection of the placenta to ensure that it is complete. the camelid placenta is epitheliochorial, mircocotytledonary and is rarely retained more than 36 h, even after dystocia. if a uterine tear is detected in the early postpartum period by direct vaginal palpation, an attempt could be made to induce uterine prolapse after treatment with epinephrine and epidural anesthesia. alternately, the uterine tear can be repaired after celiotomy. if the placenta is still present, it should be pealed from the endometrium around the tear before suturing. in cases of unexplained fever, abdominal pain or anorexia in the postpartum female, exploratory celiotomy or laparoscopy should be considered. adjunctive therapy for peritonitis is indicated and should include abdominal lavage and systemic broad-spectrum antimicrobial and anti-inflammatory therapy, along with intravenous fluid therapy for cardiovascular support. postpartum hemorrhage from the uterine arteries is less common in camelids than mares. most of the postpartum hemorrhage cases diagnosed by our group consist of rupture or laceration of the vaginal uterine artery. this artery is easily recognized by palpation per vaginum during obstetrical manipulation and is peculiarly large in camelids. excessive manipulation, and in particular fetotomy, may cause erosion of the mucosa and laceration of the artery. unfortunately, many of these hemorrhages are missed, as no outward signs are apparent until it is too late. typically, blood accumulates within the uterus for a few hours, followed by cardiovascular collapse. in one case, the female was found dead in her stall 2 h after delivery. ruptured vaginal arteries may be sutured and blood transfusion should be considered in females with a pcv <10%. packing of the vaginal with compresses, i.e. a device similar to the ''umbrella pack'' used in humans, may be helpful. partial or total uterine prolapse occur secondary to dystocia, manual removal of a retained placenta, and excessive use of oxytocin (dose and frequency). uterine prolapse is far more common in camels than in llamas and alpacas, and is often associated with hypocalcemia, selenium deficiencies, and retained placenta [49, 50] . dairy camels seem to be more prone to uterine prolapse [63] [64] [65] [66] [67] [68] [69] . uterine prolapse occurs generally immediately (first 30 min) after parturition or abortion [50] . techniques for replacement are similar to those reported in cattle and small ruminants, and are usually done under sedation and epidural analgesia. the placenta is often easily peeled off and should be removed if possible before replacement of the uterus. the female is positioned in sternal recumbency, with the hind quarters slightly elevated. the uterus should be inspected for any lacerations or hemorrhage. the area of major risk for hemorrhage is located near the cervix where the uterine artery may be exposed. the uterus is cleaned with warm dilute povidone iodine solution before replacement. a bühner suture is used in camels and a shoelace pattern can be used around the vulvar lips in alpacas and llamas. uterine prolapse tends to reoccur if the uterine horns are not fully extended. hysterectomy should be considered if the uterine tissue has sustained severe damage [8, 31, 70] . rectal prolapse has been reported in llamas and camels. pregnant females with tenesmus and diarrhea are predisposed. rectal prolapse can be intermittent. in a case of a dromedary female near term, rectal prolapse was noticed intermittently, without vaginal prolapse. treatment of the underlying cause and surgical repair have been successful [3, 71] . emergency postpartum complications in camelids include a vast array of conditions which often manifest themselves as lethargy, depression and progress towards a downer female syndrome. the approach to diagnosis of the causes of downer syndrome is similar to that used in cattle [72] . predisposing factors include septic metritis, necrotic vaginitis, retained placenta, hypocalcemia, dystocia, pelvic injuries, hemorrhage, and presence of compressive lesions. a milk fever syndrome (hypocalcemia), similar to the condition in dairy cattle, is also observed in dairy camels. toxic mastitis has been described in dairy camels, but not in south american camelids [73] . in addition to physical evaluation, cbc and blood biochemistry, the evaluation of the downer postpartum camelid should include transrectal and transabdominal ultrasonography and potentially collection and evaluation of cerebrospinal fluid. more advanced imaging techniques may be required in some cases in order to detect neoplastic masses. although, retained placenta is not usually an emergency in camelids, failure of delivery of the placenta following a cesarean section may lead to severe complications. severe swelling of the vulva and vagina are painful conditions associated with overt obstetrical manipulation. females experiencing these complications may have persistent straining and abandon their neonate. untreated vaginal and cervical inflammation may lead to adhesions and development of pyometra. females with severe inflammation of the birth canal should be treated with systemic and local anti-inflammatory drugs. daily application of cold compresses and treatment with ointments with anti-inflammatory and antimicrobial properties may reduce inflammation and adhesions. in an epidemiological study in the united kingdom, 4-11% of deaths amongst llamas and 17-33% of deaths in alpacas occur during the first 6 months of life. a high proportion of these deaths occur within the first week of life [31, 74] . in camels, neonatal mortality can reach 50% of the calf crop in the first 10 days of life [45] . newborn morbidity and mortality is very high in the immediate neonatal (<1 week old) period following obstetrical manipulations, cesarean section, prematurity, or dysmaturity [75] . these losses are often due to complications from hypoxia, failure of passive transfer, and intrapartum infection. the clinical signs are often nonspecific and vague, resulting in an individual that is slow to adapt to extrauterine life, or that dies suddenly within the first few days of life. infections may be acquired in utero or intrapartum, and should be suspected if the newborn has elevated plasma fibrinogen concentrations in the first 12-24 h of life, the placenta appears abnormal, or the dam exhibited uterine discharge peripartum [75] . therefore, immediate identification and care of the newborn camelid at high risk for sepsis is an important part of reproductive emergencies. the newborn should be evaluated within the first hours of life to detect any abnormalities of development or maladjustment to extra-uterine life. physical and behavioral parameters of the normal newborn are shown ( table 1 ). assessment of the newborn cria should include evaluation of the epidermal membrane and placenta, respiration, cardiac function, and the presence of obvious congenital abnormalities. the epidermal membrane, which is normally translucent, may become yellow or brownish due to meconium staining in case of fetal stress due to dystocia. many congenital abnormalities have been described in camelids, some of which can be lethal. amongst the most important are: cleft palate, choanal atresia, atresia ani, and heart defects. the initial examination of the cria should establish if any of these abnormalities are present (table 2) , so they can be corrected early or a decision made to humanely euthanize the cria. neonatal cases are presented with a wide variety of nonspecific complaints based on deviations from the normal appearance and behavior presented above. the minimum database used to evaluate the cria include: evaluation of maternal transfer of immunogobulins, cbc (including differential count and determination of plasma fibrinogen concentration), arterial blood gas analysis, serum chemistry, and aerobic and anaerobic blood cultures. contrast radiographs of the nasopharyngeal area may be indicated if choanal atresia is suspected as a cause of dyspnea [76] . any cria delivered before day 315 of pregnancy should be considered premature. premature birth may be a consequence of a stressful illness during pregnancy or due to a decision to induce parturition because of severe compromise to the dam. recently, the authors have seen a high rate of premature births following an outbreak of respiratory diseases. premature birth may also be secondary to uterine pathology (i.e. placentitis or placental insufficiency) [77] . premature crias display specific phenotypic characteristics, including a birth weight significantly (>20%) lower that that the average for the farm, and a thick epidermal membrane firmly attached to the foot pads and the mucocutaenous junctions. a ''floppy'' syndrome, often seen in premature camelids, includes inability to rise, to hold the head up, or to maintain sternal recumbency and floppy ears (new world camelids), due to immaturity of the cartilage. the coat appears silky and the limbs are overextended at the carpus and fetlock, due to laxity of the tendon and poor muscle tone. the incisors are not erupted and the suckling reflex is absent or weak. premature neonates adapt to extrauterine life very slowly. due to the normal elevated fetal cortisol concentrations, they may appear healthy initially, but become comprised a few hours later due to developing metabolic problems. these problems are often due to hypoxemia, acidosis, hypoglycemia, and limited body reserves or poor thermogenic ability. premature neonates are exposed to a wide range of respiratory and intestinal compromise due to immaturity of these systems. respiratory distress may be notice by labored or even open-mouth breathing. this syndrome is likely due to lack of surfactants required for normal air sac expansions and inefficient oxygen absorption. mortality rate is very high is these crias if they do not receive immediate attention [31, 50] . intestinal immaturity in premature crias predisposes them to failure of passive transfer, even if colostrum is ingested orally in the first hours of life (failure of absorption). they also tend to be more at risk for bloating and meconium retention due to poor gut motility. dysmature or hypoxic neonates are often the result of induction of parturition, severe illness during pregnancy, or prolonged gestation. they usually present with similar biophysical characteristics as the premature neonates, except that they may have normal body development. mature compromised crias are usually the result of lengthy obstetrical manipulation or delivery via cesarean section. the degree of compromise depends on several factors. there is a complete lack of evidence-based medicine in emergency critical care of newborn camelids; most of the available information is anecdotal and based on clinical experience with other species. premature or stressed neonates require intensive care in the first few hours of life. they should be placed immediately in a warm environment. baseline cbc and blood biochemistry are indicated to determination status of hydration and electrolytes, blood glucose concentration, total protein and igg at 24-36 h. at-risk patients should receive an intravenous plasma transfusion. if the suckling reflex is absent, tube feeding is necessary and should be restricted to small volumes every 2-3 h, to reach 10-15% of body weight by 24 h of life. oxygen supplementation may be required if respiratory distress is pronounced. lung function should be monitored by blood gas analysis. aminophylline, an adenosine a (2a)-receptor antagonist like caffeine, has been given for 3 days to stimulate the central nervous system and regulate breathing and to stimulate the type ii pneumocytes to produce components for the surfactant production [78, 79] . intraoperational administration of aminophylline to the dam may be advantageous if a cesarean section is planned [79, 80] . doxapram is routinely used to stimulate the central nervous system and relieve neonatal apnea following dystocia or cesarean section [81, 82] . we general administer a small dose sublingual (5 mg in llama and alpaca crias and 50 mg in camels) initially after a cesarean section or dystocia. in neonates with severely depressed respiration, this dose, or up to twice this dose, should be given iv or iv. the neonate should be monitored closely for convulsions or hyperventilation. sepsis is a major concern in all compromised neonates. in one study, the median age at presentation of [75, 76, 83] . both gram + and gram à organisms have been isolated from neonates with septicemia. based on common isolates, the antibiotics of choice for camelids at high risk of sepsis include the following combinations (enrofloxacin and ppg, enrofloxacin and ceftiofur, ceftiofur and gentamicin) [75, 76, 83] . gentamicin should be used with care, as it can be extremely nephrotoxic to severely dehydrated newborn camelids, or if there is already evidence of renal dysfunction. blood cultures may be submitted, but broad-spectrum antimicrobial treatment should be started without delay. supportive treatment should include nsaids (ketoprofen 4 mg/kg sid) to control pain and toxemia and antiulcer medication (omeprazol, given orally, 2 mg/kg daily) to offset the effect of stress and nsaid. intravenous fluid therapy is indicated in all dehydrated, hypoglycemic newborns, however caution should be exercised regarding the rate of fluid replacement, as camelids are prone to pulmonary edema. severely dehydrated crias require fluid therapy. the type of fluid should be determined based on glucose, electrolyte and blood gas evaluation. generally, a balanced isotonic solution with 2% dextrose and bicarbonate to correct metabolic acidosis are sufficient. dextrose concentration may be increased to 5% in hypoglycemic crias. rate of administration should aim to correct half of the deficit over the first hour, and the other half over the next 2 h. total or partial parenteral nutrition should be considered in severely depressed crias that are unable to nurse [84] . prognosis for life and normal growth depends primarily on the interval between birth and providing emergency care. diseases in the first 24 h of life are usually associated with congenital abnormalities, digestive (meconium retention), urinary problems (urine retention), exposure or malnutrition. the most common lethal congenital abnormalities that affect the camelid neonate are: choanal atresia, atresia ani or coli, and heart defects. most commonly, affected animals will suffer from severe respiratory, circulatory or metabolic complications. heart defects can be very severe and lead to death of the cria within a few hours, but most will survive for a few days to months, with the only abnormality being failure to thrive. syncope or fainting were observed in crias with severe heart defects. choanal atresia is the lack of opening of the nasal air passages, resulting from the presence at the level of the choanae of a membranous or osseous separation between the nasal and pharyngeal cavities [85, 86] . diagnosis can be confirmed by mouth to nose artificial breathing or by contrast radiographs of the head after injection of a radio-opaque substance in the nasal cavity. maxillofacial agenesis or dysgenesis ''wry face'' is a head deformity characterized by varying degrees of deviation of the maxilla. this abnormality may be associated with choanal atresia. there is no treatment for this condition and the cria should be euthanized. respiratory distress associated with congenital goiter has been described in camels [45] . atresia ani and atresia coli are, respectively, the lack of opening of the anal sphincter and lack of connection between the colon and the rectal cavity. these abnormalities results in the blockage of the intestinal transit and accumulation of fluid in the gastrointestinal tract. the cria becomes progressively bloated and depressed. ultrasonographic and radiologic examination of the abdominal cavity allows confirmation of the diagnosis. atresia coli may be mistaken for meconium retention. in the female cria, these abnormalities may involve the genital tract. surgical correction of the atresia ani has described [87] . congenital blindness associated with different ocular defects has also been reported and will impact neonate behavior and wellness [88] [89] [90] . it is important that the practitioner established the diagnosis of congenital abnormalities with certainty, because some of these may be hereditary [91, 92] . meconium is the amniotic fluid ingested by the fetus during pregnancy. meconium is usually passed within 18-24 h after birth as dark pasty or stringy feces. clinical signs of meconium retention include straining, squatting, tail wagging, anorexia, and signs of abdominal discomfort. initial treatment consists of one or two warm soapy water enemas (20-40 ml). if after two enemas, the meconium has not passed, intravenous fluids may be indicated, as multiple soapy water enemas may irritate the rectal mucosa, resulting in severe straining and rectal prolapse [93] . crias that have retained meconium may have other abnormalities and should be examined closely. routine administration of enemas to every newborn cria should be discouraged. urine retention may be associated with congenital abnormalities of the urinary and genital tracts [94] . in males, urethral blockage (aplasia) results in bladder rupture. in females, vulvar agenesis or atresia vulvi present with an obvious bulging of the perineum and often symptoms of pain, due to the large quantity of urine in the uterus and abdominal distension [31, 71] . accidents to the umbilical stump are not uncommon. the simplest form is persistent bleeding, which can be treated with hemostasis provided by a hemostat or sutures. persistent urachus is not as common as in other species. umblical hernia and rupture of the abdominal wall with eventration has been seen by the author following dystocia due to uterine torsion and may be due to wrapping of the cord around the fetus. these are easily replaced surgically. failure of passive transfer is a major cause of neonatal mortality in camelids [95] . assessment of igg concentrations can be performed 18 h after birth [96] [97] [98] . serum total protein concentrations <5 mg/ dl are also very indicative of failure of passive transfer. in these cases, hyperimmune plasma should be given iv or ip (15-25 ml/kg). commercial products are now available (triple j farm, kent laboratories, 777 jorgensen place, bellingham, wa 98226, usa). this product is collected from llamas regularly immunized with clostridium perfringens type c, escherichia coli bacterin-toxoid, clostridium chauvoeisepticum, clostridium haemolyticum, clostridium novyi, clostridium tetani and clostridium perfringens types c and d bacterin-toxoid, killed equine herpes virus-1, bovine rota-coronavirus modified live virus, j-5 e. coli bacterin, imrad 3 killed rabies vaccine, and inactivated cultures of leptospira canicola, leptospira grippotyphosa, leptospira hardjo, leptospira ichterohaemorrhagiae and leptospira pomona. reproductive emergencies involve not only saving the health but also the reproductive future of the patient. emergencies in the pregnant female present an additional challenge, in that the fetus has to be considered regarding response to treatment and viability. at times, it is important to make a decision as to which of the two (dam or fetus) has more economic or sentimental value, or chances to survive. one of the main challenges in emergency care in camelids is the lack of evidence-based scientific data on treatment and outcome assessment. although extrapolation from other species has been possible, it is important to remember species peculiarities, especially with regard to fluid therapy. handling of obstetrical situations is particularly important, as many female camelids loose their ability to reproduce due to iatrogenic vaginal adhesions and cervical trauma from prolonged manipulation. in the male, hyperthermia (environmental or pathologic) is the leading cause of reproductive loss and client education regarding its prevention and early recognition is paramount for successful preservation of fertility. veterinarians involved in camelid practice, of which reproductive services (including reproductive emergencies and neonatology) represents over 70% of the complaints, should have a very good understanding regarding anatomical, physiological and medical peculiarities of camelids, and utilize their experience in other species. this makes an excellent point for the importance of comparative approach to training theriogenologists and large animal veterinarians. emergency drugs and protocols (table 3) should be in place to ensure timely delivery of critical care and improved outcomes. uro-genital defects, renal agenesis, atresia vulvi preputial prolapse in an alpaca reproductive physiology and infertility in male south american camelids: a review and clinical observations pathology and surgery of the reproductive tract and associated organs in the male camelidae heat stress in a llama (lama glama): a case report and review of the syndrome hyperthermia in llamas and alpacas changes in testicular histology and sperm quality in llamas (lama glama), following exposure to high ambient temperature theriogenology in camelidae: anatomy, physiology, bse, pathology and artificial breeding. actes ed., institut agronomique et veterinaire hassan ii reproductive disorders in the male camelid infectious causes of reproductive loss in camelids septic orchitis in an alpaca common surgical procedures in camelids gastrointestinal causes of colic in new world camelids surgical management of a ruptured bladder secondary to a urethral obstruction in 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cardiopulmonary function in isoflurane-anesthetized alpacas transabdominal ultrasonographic appearance of the gastrointestinal viscera of healthy llamas and alpacas obstetrics and neonatology prediction of gestational age by ultrasonic fetometry in llamas (lama glama) and alpacas (lama pacos) real-time ultrasonic biparietal diameter measurement for the prediction of gestational-age in llamas hepatic lipidosis in llamas and alpacas hormonal indicators of pregnancy in llamas and alpacas plasma concentrations of 15-ketodihydro-pgf(2 alpha), progesterone, oestrone sulphate, oestradiol-17 beta and cortisol during late gestation, parturition and the early postpartum period in llamas and alpacas reproduction in female south american camelids: a review and clinical observations surgical and nonsurgical correction of uterine torsion in new world camelids: 20 cases (1990-1996) splenic torsion in an alpaca surgical treatment of uterine torsion in a llama (lama glama) surgery of the reproductive tract in camelids surgical correction of an acquired vaginal stricture in a llama, using a carbon-dioxide laser vaginal prolapse in a camel simple management of vaginal prolapse in the camel (camelus dromedarius) institut agronomique et veterinaire hassan ii use of cloprostenol as an abortifacient in the llama (lama glama) induction of parturition in alpacas and subsequent survival of neonates obstetrics, neonatal care, and congenital conditions ventral midline caesarean section for dystocia secondary to failure to dilate the cervix in three alpacas recent advances in camelid reproduction. ithaca: international vet information service recent advances in camelid reproduction. ithaca: international vet information service cardiopulmonary effects of xylazine and acepromazine in pregnant cows in late gestation the effects of xylazine on intrauterine pressure, uterine blood flow, maternal and fetal cardiovascular and pulmonary function in pregnant goats ketamine inhibits fetal acth responses to cerebral hypoperfusion a comparison of the haemodynamic effects of propofol and isofluronae in pregnant ewes effects of propofol-sevoflurane anesthesia on the maternal and fetal hemodynamics blood gases, and uterine activity in pregnant goats the effect of propofol on isolated human pregnant uterine muscle maternal and fetal effects of propofol anaesthesia in the pregnant ewe transplacental transfer of propofol in pregnant ewes use of propofol-isoflurane as an anesthetic regimen for cesarean section in dogs periparturient and neonatal anesthesia peritonitis associated with passage of the placenta into the adbominal cavity in a llama diseases and causes of mortality in a camel (camelus dromedarius) dairy farm in saudi arabia an outbreak of nutritional muscular dystrophy in dromedary camels uterine prolapse in a camel (camelus dromedarius) rectal prolapse caused by a fibroma in a she camel-a case report uterine prolapse in a camel uterine prolapse in the dromedary camel uterine prolapse in a camel (camelus dromedarius) reproductive disorders of the female camelidae rectal prolapse, surgery and radiology of the dromedary camel al ahsa. saudi arabia: ramadan, r.o. king faisal university a review of the causes, prevention, and welfare of nonambulatory cattle lactation and udder diseases south american camelids in the united kingdom: population statistics, mortality rates and causes of death gram-negative bacterial-infection in neonatal new-world camelids-6 cases (1985-1991) neonatal care of camelids: a review and case reports body condition and blood metabolite characterization of alpaca (lama pacos) three months prepartum and offspring three months postpartum adenosine a(2a)-receptor blockade abolishes the roll-off respiratory response to hypoxia in awake lambs the combined maternal administration of magnesium sulfate and aminophylline reduces intraventricular hemorrhage in very preterm neonates surfactant administration to the human fetus in utero: a new approach to prevention of neonatal respiratory distress syndrome (rds) a new look at the respiratory stimulant doxapram oral pharmacokinetics of doxapram in preterm infants culturepositive sepsis in neonatal camelids: 21 cases practical fluid therapy in llamas and alpacas complete choanal atresia in a llama what is your diagnosis? [complete bilateral choanal atresia in a llama surgical correction of anorectal atresia and rectovaginal fistula in an alpaca cria congenital glaucoma in a llama (lama glama) congenital cataracts and persistent hyaloid vasculature in a llama (lama glama) congenital coloboma in a llama congenital abnormalties an overview of camelid congenital/genetic conditions perinatal and neonatal care of south-american camelids urinary obstruction in a hermaphroditic llama failure of passive immunoglobulin transfer: a major determinant of mortality in newborn alpacas (lama pacos) a note on colostral immunoglobulin g concentrations versus subsequent serum concentrations in naturally suckled llama (lama glama) and alpaca (lama pacos) crias evaluation of assays for determination of passive transfer status in neonatal llamas and alpacas passive transfer of colostral immunoglobulin g in neonatal llamas and alpacas congenital defects in the llama medicine and surgery of south american camelids: llama, alpaca, vicuna, guanaco renal agenesis in an alpaca cria anderson kl. bilateral renal agenesis in an alpaca cria atresia vulvi in camels (case reports) a case of atresia ani with rectovestibular fistulae in an alpaca (l. pacos) surgical repair of a cleft soft palate in an alpaca surgical repair of a bilateral choanal atresia in a llama surgical treatment of a congenital flexural deformity of the tarsal joint in a llama management of bilateral flexural deformity of the metacarpophalangeal joints in three alpaca crias surgical correction of carpal valgus deformity in three alpacas multiple non-lethal congenital anomalies in a llama diaphragmatic hernia in a llama key: cord-253638-5f9ofdsc authors: alsaied, tarek; aboulhosn, jamil a.; cotts, timothy b.; daniels, curt j.; etheridge, susan p.; feltes, timothy f.; gurvitz, michelle z.; lewin, mark b.; oster, matthew e.; saidi, arwa title: coronavirus disease 2019 (covid‐19) pandemic implications in pediatric and adult congenital heart disease date: 2020-06-10 journal: j am heart assoc doi: 10.1161/jaha.120.017224 sha: doc_id: 253638 cord_uid: 5f9ofdsc the corona virus disease ‐2019 (covid‐19) is a recently described infectious disease caused by the severe acute respiratory syndrome corona virus 2 with significant cardiovascular implications. given the increased risk for severe covid‐19 observed in adults with underlying cardiac involvement, there is concern that patients with pediatric and congenital heart disease (chd) may likewise be at increased risk for severe infection. the cardiac manifestations of covid‐19 include myocarditis, arrhythmia and myocardial infarction. importantly, the pandemic has stretched health care systems and many care team members are at risk for contracting and possibly transmitting the disease which may further impact the care of patients with cardiovascular disease. in this review, we describe the effects of covid‐19 in the pediatric and young adult population and review the cardiovascular involvement in covid‐19 focusing on implications for patients with congenital heart disease in particular. corona viruses are important pathogens that can infect humans and animals. at the end of 2019, a novel corona virus was isolated in wuhan, a city in china, as a cause of a cluster of severe cases of pneumonia and acute respiratory distress syndrome 1 . the virus rapidly spread around china and the world. on march 11 th 2020, covid-19 was declared as a pandemic by the who and the virus has infected hundreds of thousands of patients and thousands of health care providers 2, 3 . while children in general seem to have milder or even asymptomatic forms of the illness, there is little to no data in children with chronic medical conditions, specifically heart and lung disease 4, 5 . the knowledge and experience in caring for patients with covid-19 is rapidly evolving and thus it is crucial for all health care providers to be aware of the potential impact of covid-19 on patients with congenital heart disease (chd). covid-19 is particularly important for cardiac team members for the following reasons: 1-covid-19 has higher morbidity and mortality in adult patients with acquired cardiac disease. 2-covid-19 may affect and impact the cardiovascular system. 3-covid-19 may cause symptoms that mimic symptoms of cardiovascular disease like cyanosis and shortness of breath commonly seen in forms of worsening cardiac disease. 4-cardiac care team members are at risk for acquiring covid-19 and may play a role in spreading the disease between patients and in the society at large. 5-patients with chd are known to have higher risk for complications with viral illnesses 6, 7 . 6-covid-19 may stretch our health care system and our resources which may impact the care of patients with cardiovascular disease. based on the covid-19 pandemic trends it is expected that many of the cardiovascular care team members will be exposed to patients with covid-19 in the coming weeks and months. in anticipation of the spread of covid-19 many hospitals and cardiology practices have this article is protected by copyright. all rights reserved changed their care models and policies to accommodate the care of patients impacted by covid-19 and prevent the spread of the disease. the objective of this review is to explore the current knowledge about covid-19 potential effects on the cardiovascular system in the pediatric cardiology and adult chd population. in addition, we will review the prevention practices and the impact on the health care team wellness. due to the emergent and evolving nature of covid-19 we reviewed the published literature in medline through pubmed and critically assessed early reports using a search for covid19 and sars-cov-2 in medrxiv and biorxiv from january 1 st 2020 until march 23 rd 2020. we had personal communications with multiple organizations around the world caring for children and adults with chd and we reviewed social media reports from leaders in the field of congenital cardiology. in addition, the websites of the health organizations including who and cdc were reviewed to provide up to date numbers and infection control recommendations. a congenital cardiology email list serve was initiated on the 15 th march to facilitate discussion and share experiences and ideas on topics such as: strategies to manage outpatient clinic, inpatient service, procedure prioritization, screening, telemedicine and dealing with staff illness and school closures and we share some thoughts and information from physicians on the frontline of care of patients with covid-19. sars-cov2 belongs to the coronaviridae family. as a group this family has a single strand rna genome 8 . the viral rna closely resembles the rna of two bat corona viruses (88% identical) 9 . the hypothesis is that the bat is likely the primary source of the virus although the this article is protected by copyright. all rights reserved mechanism of transfer from bat to human is still unclear (i.e. whether it was through direct transmission or whether there was an intermediate host) 8, 9 . the virus is also closely related (79% identical) to the sars corona virus which was responsible for an outbreak in china in [2002] [2003] and 50% identical to the middle east respiratory syndrome corona virus that caused an outbreak in the arabian peninsula in 2012 [8] [9] [10] . importantly the virus enters the cells using the angiotensin converting enzyme 2 (ace-2) 11, 12 . sars-cov2 spike protein binds to the ace-2 and the virus enters the cells through endocytosis 13, 14 . ace-2 are expressed on the surface of epithelial cells within the alveoli and small intestine which explain the respiratory and gastrointestinal symptoms associated with the disease 14, 15 . ace2 is a membrane bound protein that is also expressed in vascular endothelium, renal tissue and cardiovascular tissue 16 . ace converts angiotensin i to angiotensin 2 while ace-2 deactivates angiotensin 2 17 . angiotensin 2 stimulates lung injury and ace-2 serves a role in lung protection as it deactivates angiotensin 2. viral binding to ace-2 deactivates the protective role of ace-2 and that may contribute to severe lung injury seen with covid-19 13, 15 . more than 2.5 million cases of covid-19 have been confirmed worldwide with more than 175,000 deaths attributed to covid-19 according to the who website as of 04/23/2020 2, 18 . of these cases 84,000 cases were reported from china where the disease peaked between mid-january and early february 2, 19, 20 . the initial patients had worked or visited a seafood "wet" market in wuhan that sold live animals and was closed subsequently for infection control 21, 22 . human to human spread subsequently became the main source of transmission 2, 19 . the disease then spread around the world initially through patients travelling from china followed by local transmission 5 . as of april 23rd 2020, there were 800,926 confirmed cases in the united states and over 40,000 deaths due to covid-19 according to the centers for disease this article is protected by copyright. all rights reserved control and prevention (cdc) website 5 . transmission is thought to occur mainly via respiratory droplets produced during talking, coughing or sneezing similar to other corona viruses and similar to the influenza virus 23 . transmission from asymptomatic individuals or patients in the incubation period has been documented 24, 25 . the virus can also stay viable in aerosols up to 2.6 hours and can stay for hours to days on surfaces, especially plastic and stainless steel 23 . in adult patients the incubation period is up to two weeks with a median of 4-5 days [25] [26] [27] . more than 80% of infected individuals will remain asymptomatic or develop mild pneumonia 11 . about 15% of patients will develop severe pneumonia and another 5% of patients will develop critical disease with respiratory and multi-organ failure and shock 11 . fever is the most common symptom reported in most adult patients followed by fatigue and dry cough 1, 28 . other symptoms include myalgia, anorexia, dyspnea, loss of sense of taste and smell (anosmia) and less commonly rhinorrhea, headache and sore throat [28] [29] [30] . gastrointestinal symptoms including diarrhea and nausea have also been reported in up to 51% of patients 31, 32 . the median time to hospital admission is 7 days from the start of symptoms 31 . according to the who, recovery happens in two weeks in mild cases and in up to 6 weeks in severe cases 3, 33 . the median time from symptoms to death in severe cases is estimated to be between 13-17 days 34 . estimating case fatality rate is challenging and is evolving depending on the testing policies in each country and the age and risk profile of the infected patients. the rates reported range from 0.4 % up to 5.8 % 11, 35-37 . this article is protected by copyright. all rights reserved while the severity and mortality due to covid-19 is higher in older patients, the virus can also infect children and young adults 38, 39 . overall only 2% of the reported cases from china were in individuals younger than 20 years and less than 1% were younger than 10 years 11, 40 . fortunately, the disease in children is reported to be milder than adults although severe cases with multi-organ involvement have been reported 41, 42 . a nationwide case series from china included 2143 pediatric patients with covid-19 43 . of those, 731 had positive testing while the rest had highly suspicious clinical picture 43 . severe disease with hypoxia was reported in 5% of the cases while a total of 13 patients had critical disease with acute respiratory distress syndrome or multi-organ failure and one death was reported in a 14 year-old 43 . compared to older children, infants had a high risk for severe disease 43 . another study looked at all the cases tested and treated at the wuhan children's hospital which is the only hospital to test and treat children in wuhan for covid-19 11 . of 1391 patients tested, 171 (12.3%) were confirmed to have sars-cov2 infection at a median age of 6.7 years. of these 16% were asymptomatic and only 42% were febrile at some point during the illness. during the course of the admission only 3 of 171 patients required intensive care unit and mechanical ventilation and all 3 had preexisting conditions (intussusception, leukemia on maintenance chemotherapy and hydronephrosis). one 10 month old patient died but the patient also had intussusception with multi-organ failure 11 . the data on cardiovascular involvement with covid-19 is evolving 44 . covid-19 may result in cardiac injury through multiple potential mechanisms, including: 1. cardiomyocyte injury due to an acute and severe inflammatory response as seen during a cytokine storm this article is protected by copyright. all rights reserved a recent meta-analysis evaluated the prevalence and effect of preexisting cardiovascular disease in patients with covid-19 45 . the study evaluated 1527 patients and reported the prevalence of hypertension, cardiac and cerebrovascular disease, and diabetes to be 17%, 16% and 10% respectively 46 . studies from china and italy consistently show that preexisting cardiovascular disease increase the risk of mortality in patients with covid-19 11, 47 . the mechanism for increased risk of mortality is likely multifactorial including older age in patients with cardiac morbidities and altered immunologic response due to age and diabetes 48, 49 . a population with potentially increased risk is patients with heart transplantation due to immunosuppression and two case reports describe covid-19 in heart transplant patients [50] [51] [52] . one patient had mild disease while the other had severe disease. both patients were successfully treated with steroids, intravenous immunoglobulins and antibiotics and both survived without rejection 50, 51 . multiple cardiovascular sequelae are reported in patients with covid-19 which may lead to or exacerbate cardiac disease 44 . myocardial injury evidenced by elevated troponin is common in covid-19 especially in patients with severe disease 44, 53 . arrhythmia is also commonly seen in covid-19 patients and while nonspecific, palpitations are one of the presenting symptoms in 7% of the patients 31, 44, 54 . arrhythmia was noted in 16% of a group of hospitalized adult patients in china and was more common in the icu setting 31 . there remains uncertainty about the specifics of the type of arrhythmia and there are no data in children. arrhythmia could be related to stress, inflammation and metabolic abnormalities but the development of new malignant arrhythmias should raise the suspicion for an underlying myocarditis 44, 54 . myocarditis is a significant contributor to mortality in up to one third of cases with severe covid-19 and was the primary cause of death in 7% of the patients 55 . autopsy found evidence this article is protected by copyright. all rights reserved of fulminant myocarditis with inflammatory mononuclear infiltrates in the myocardial tissue in some cases although the prevalence and risk factors for myocarditis are yet to be described 44 . heart failure was reported in up to 23% of patients with covid-19 with a high prevalence of mortality 56 . whether this is due to unmasking of pre-existing left ventricular dysfunction or new onset of cardiomyopathy due to myocarditis remains unclear 56 . finally some severe cases may progress to cardiogenic shock and extracorporeal membranous oxygenation support may be considered in these cases although the prognosis is poor with 83% mortality in one report 44, 57 . population chd affects up to 1% of the general population and remains the leading cause of infant mortality due to congenital malformations 58 this article is protected by copyright. all rights reserved summarized in table 2 . in addition to the cardiac morbidity, many patients with chd also have other organ involvement including chronic lung disease, cirrhosis and renal disease which may also increase the risk of covid-19 64 . we want to emphasize that there is very limited evidence to make firm recommendations and that these are based on the experience with previous viral diseases 6, 7 . of note cardiovascular morbidities like diabetes and hypertension may also increase the risk of covid-19 in pediatric patients similar to that seen in adult patients. additionally, diabetes, hypertension, and acquired cardiovascular disease may coexist in adults with chd, potentially further increasing their risks. it is also important to note that many symptoms of covid-19 may mimic symptoms of worsening cardiac conditions in the chd population. symptoms such as shortness of breath, palpitations and fever are commonly seen in endocarditis which is a major concern in patients with chd 65 . the same symptoms can also be seen in heart failure decompensation due to a viral illnesses and these considerations should be kept in mind during the covid-19 pandemic. aspirin is commonly used in the pediatric and adult population with chd for its antiplatelet effects at a low dose of 3-5 mg/kg/day 66 . there is an association between the use of aspirin and the development of reye syndrome, a rare form of hepatic encephalopathy, in young children taking high dose aspirin for kawasaki disease who have concurrent infection with influenza or varicella 67, 68 . there are no reports of reye syndrome in patients on low dose aspirin typically used with chd or with covid-19 60 . due to that we currently do not stop aspirin in children or adults with chd and covid-19. this article is protected by copyright. all rights reserved there is no convincing evidence that nsaids are harmful in covid-19 although some clinicians suggested a possible negative impact if used early in the course of the disease for fever 69 . the who and the european medicines agency currently approve the use of ibuprofen and nsaids in covid-19 70, 71 . the british congenital cardiac association suggested avoiding nsaids to treat fever in covid-19 63 . the ace-2 is used by the virus for cell entry 44 . at this point there is insufficient data to suggest that ace-i/arb could affect the course of covid-19 11, 44 . ace-i/arbs suppress angiotensin ii or blocks its effect on its receptor. this is a positive effect to blunt the pulmonary inflammatory response and may result in upregulation of ace-2 which can be a positive effect for the same reason but at the same time carries the potential risk of increasing sars-cov-2 entrance to the alveolar cells 56 . in the chinese data the rate of hypertension was significantly higher in patients who died post covid-19. whether this is because of the older age and multiple comorbidities in the patients with hypertension or if it has any relationship to the ace-i/arbs use is unclear 56, 72 . it is our practice currently not to stop ace-i/arb in patients who have covid-19 as stopping these medications could have harmful effects. the acc, american heart association and the heart failure society of america issued a statement recommending not adding or removing these medications for patients who are taking these medications if diagnosed with covid-19 and to follow an individualized approach 73 . this article is protected by copyright. all rights reserved coagulation cascade abnormalities and disseminated intravascular coagulation (dic) are reported in patients hospitalized with severe covid-19 disease 44, 56, 74 . we suggest continuing anticoagulation in mild cases with monitoring from the prescribing physician. adjusting anticoagulation can be done as needed in cases of severe covid-19. there is no data to suggest any interaction of covid-19 with these medications and we suggest continuing these medications and adjusting as needed by the prescribing physician. the interaction between covid-19 treatments and the cardiovascular system are previously reviewed in jacc 44 . several medications are under active investigation to treat covid-19 75 . antiviral medications being trialed include ritonavir/lopinavir which can cause qt prolongation 44 . ribavirin, another antiviral being investigated, and ritonavir/lopinavir can also interact with anticoagulants 44, 76 . remdesivir, an investigational antiviral medication used to treat ebola virus, is being trialed and although cardiovascular toxicities have not been reported, among 175 patients one developed hypotension and cardiac arrest after the loading dose 44, 77 . chloroquine and hydroxychloroquine, antimalarial agents, have been shown in vitro to have inhibitory activity on sars-cov2 due to increasing cellular endosomal ph and thus preventing the viral endocytosis into the cells 75, [78] [79] [80] . this medication has been trialed in vitro and has been shown to be effective. however, there is limited human data to support effectiveness in humans 80, 81 . one small study treated 20 patients who were asymptomatic or had mild covid-19 with hydroxychloroquine in combination with azithromycin and showed a significant decrease in viral load in treated patients 81 . the results should be taken with this article is protected by copyright. all rights reserved caution as the medication was previously tried in cases of influenza, hiv and zika virus and showed effectiveness in vitro without any positive effect in humans 80 . of note these agents have qt prolonging effects which should be taken into consideration especially when combined with hypokalemia, hypomagnesemia, myocarditis or other qt prolonging medications. this includes azithromycin, another commonly used antibiotic in respiratory infections with qt prolonging effects that is being trialed in covid-19 infections 44, 75 . this would be of particular concern in patients in an intensive care unit setting where a variety of qt affecting medications and physiologic conditions might put patients at risk of myocardial hypersensitivity and arrhythmia risk. a list of these medications can be found at https://www.crediblemeds.org/. screening all patients entering a health care facility for covid-19 symptoms including fever and respiratory symptoms, travel and exposure to covid-19 cases is essential for early identification of potential covid-19 patients and to allow care team members to wear appropriate personal protective equipment (ppe) (figure 1) 82 . screening may be through the phone before the patient arrives and should employ the use of interpretation services when needed. non-urgent cardiac visits should be rescheduled for a later date, particularly if the patient has viral symptoms without concern for possible worsening cardiovascular disease. urgent cardiac visits with symptomatic patients (or if asymptomatic but with a high-risk exposure) should include the use of a facemask by the patient and ideally a separate waiting rooms 82 . any caregiver entering the room for a confirmed or suspected covid-19 case should wear a gown, gloves, eye protection and a respirator (e.g. n95). when the supply of respirators is limited a facemask is an acceptable alternative for routine interaction but a respirator this article is protected by copyright. all rights reserved (preferably a powered or controlled air purifying respirator) should be worn for any aerosol generating procedures 82 . these procedures include intubation, cardiopulmonary resuscitation, and manual ventilation before intubation 44, 82 . transesophageal echocardiography (tee) has been included on this list; but the data is currently unavailable as to whether the risk of aerosolization during tee is similar in both the setting of the paralyzed and intubated patient as opposed to the sedated patient without cuffed intubation who might be expected to gag or cough during the procedure. in light of this data gap, a conservative approach should be employed. during this pandemic, patient to provider, provider to patient and provider to provider transmission represent major challenges. it is important to know that 3.8% of the cases reported from china were of healthcare team members suggesting that health care providers are at a significantly increased risk of contracting covid-19 11, 83 . this emphasizes the need for selfprotection by all providers. this is especially a challenge in cases where ppe are limited and in emergent interventions such as cardiopulmonary resuscitation; providers should prioritize following ppe guidelines and should limit exposure. similarly, elective and nonessential visits and procedures should be postponed. in the chd settings, these include non-urgent cardiac surgeries, cardiac catheterization, tee and electrophysiological procedures. office visits can be postponed or scheduled through phone calls or telemedicine. telemedicine technologies limit the patient and provider exposure while still allowing for patient care 44 . teams must go through the exercise of considering unintended consequence to delayed access to care for the patient and put into place safeguards in their strategy. the duration of the corona virus pandemic is uncertain and therefore requires frequent reassessment of care plans. specific definitions of urgent versus non-urgent should be created so that schedulers and care providers this article is protected by copyright. all rights reserved are in alignment. the definition of non-urgent should include the concept that "delay of a certain duration unlikely to result in patient harm". other strategies may include limiting the number of team members with exposure to suspected cases by allowing phone and chart review consults in patients admitted to the hospital. most congenital cardiac centers hold weekly multidisciplinary conferences to discuss the cases and all team members including cardiologist, surgeons and anesthesiologists attend these conferences. due to the requirement of social distancing to limit viral transmission management, and teaching conferences should be limited and virtual meetings should replace in person meetings (figure 2) 84 . in addition, many hospitals are implementing staff screening strategies for symptoms and taking temperature for all employees and is assigning those staff non-essential for patient care to work remotely during these times. as age is a risk factor for severe disease, decisions must be made as to whether to restrict older physicians and care team members to work in settings with lower potential for exposure. in addition to the physical risks this is a stressful time for the care team members and it is important to keep provider wellness in mind. resources can be found on the american college of cardiology website and other professional societal websites 85 . during this time of "physical" distancing it is important to maintain "social connectivity" by supporting each other through our professional societies, our teams and our personal support networks. this has become increasingly via available video communication enterprises 85, 86 . new information is emerging every day on covid-19. it is important to continue to follow the guidelines from the who, the cdc, your local health department, hospital and practice. it is also crucial to make this information easily available and accessible in real time and utilize data visualization tools to allow smooth transformation of the data to the public. in addition, social distancing has limited trainee education. online education is now accessible and this article is protected by copyright. all rights reserved resources from the acc 2020 virtual national meeting will be available free of charge. efforts to make other recorded material available for all providers are ongoing. finally technology allows virtual meetings with video communications and conferences to allow for fellows teaching and continue to make multidisciplinary discussions accessible to all care team members. while data are emerging on covid-19 effect on the cardiovascular system, there are several areas where further research is needed. identifying high risk patients among children and adults with chd is a priority. furthermore, studies to understand the mechanisms of cardiovascular involvement in covid-19 will facilitate the efforts to discover treatment options. in addition, several trials are ongoing searching for identification of the significance of the infection in patients with chd, treatments and post exposure prophylaxis. an example is a collaboration effort between the adult congenital heart association and the international society for adults with congenital heart disease in order to better understand the impact and burden of covid-19 on adults with chd in the united states and globally. during these trying times of covid-19 pandemic, cardiovascular team members will play a central role in controlling the disease. patients with chd are likely to become increasingly affected with covid-19 and may have more grave outcomes than other populations during the pandemic due to their perturbed physiologic state and co-morbidities. infection control processes are crucial. continuing research and education about the disease may provide us with insights to alleviate morbidity and mortality in the near future. includes simple native lesions (isolated small asd or vsd, mild isolated pulmonary stenosis) or repaired lesions (ligated or occluded pda), repaired asd or vsd without residual shunting or chamber enlargement. repaired or unrepaired lesions including avsd, aortic or mitral valve disease and coarctation of the aorta, anomalous pulmonary venous return, alcapa, aaoca, ebstein, tof, ps (moderate or greater), vsd with moderate or greater shunt. cyanotic congenital heart defect (unrepaired or palliated, all forms), double-outlet ventricle, fontan procedure, iaa, single chd: congenital heart disease. this article is protected by copyright. all rights reserved theoretical concern for reye syndrome although the only reports were at high doses used in kawasaki disease in the setting of influenza or varicella. no reports with covid-19. suggest continuing aspirin. theoretical concern due to potential upregulation of ace-2. no data to support an interaction with the disease. suggest continuing the medication. covid-19 may cause dic and coagulation cascade abnormalities in severe cases. suggest continuing the medication in mild cases and adjust as needed in severe cases. some clinician suggested worsening course of the disease if given early. there is no data to show any association of nsaids with the course of the disease. the who does not recommend avoiding nsaids. there is no information to suggest any harmful effect in covid-19 patients thus it is reasonable to continue these medications. this article is protected by copyright. all rights reserved clinical features of patients infected with 2019 novel coronavirus in wuhan, china world health organization declares global emergency: a review 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briefing on covid-19 -24 incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response 2019-novel coronavirus (2019-ncov): estimating the case fatality rate -a word of caution updates on covid-19 in korea clinical features in pediatric covid-19 a review of coronavirus disease-2019 (covid-19). indian journal of pediatrics sars-cov-2 infection in children a 55-day-old female infant infected with covid 19: presenting with pneumonia, liver injury, and heart damage county teen's death may be coronavirus related. here is what we know epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china cardiovascular considerations for patients, health care workers, and health systems during the coronavirus disease 2019 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is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-011245-nkr0998x authors: yokomichi, hiroshi; tanaka-taya, keiko; koshida, rie; nakano, takashi; yasui, yoshinori; mori, masaaki; ando, yuka; morino, saeko; okuno, hideo; satoh, hiroshi; arai, satoru; mochizuki, mie; yamagata, zentaro title: immune thrombocytopenic purpura risk by live, inactivated and simultaneous vaccinations among japanese adults, children and infants: a matched case–control study date: 2020-04-06 journal: int j hematol doi: 10.1007/s12185-020-02866-1 sha: doc_id: 11245 cord_uid: nkr0998x this case–control study investigated immune thrombocytopenic purpura (itp) risk following live, inactivated, and simultaneous vaccination, with a focus on infants aged < 2 years. we matched case patients with itp to one or two control patients with other diseases by institution, hospital visit timing, sex, and age. we calculated mcnemar’s pairwise odds ratios (ors [95% confidence interval]) with 114 case–control pairs. the case group had 27 (44%) males and 22 (35%) infants, and the control group included 49 (43%) males and 42 (37%) infants. for all age groups, the mcnemar’s or for itp occurrence was 1.80 (0.54–6.84, p = 0.64) for all vaccines. among infants, these were 1.50 (0.17–18.0, p = 0.50) for all vaccines, 2.00 (0.29–22.1, p = 0.67) for live vaccines, and 1.00 (0.01–78.5, p = 0.50) for inactivated vaccines. sex-adjusted common ors for simultaneous vaccination were 1.52 (0.45–5.21, p = 0.71) for all vaccines, 1.83 (0.44–7.59, p = 0.40) for inactivated vaccines only, and 1.36 (0.29–6.30, p = 0.69) for mixed live and inactivated vaccines. in infants, these were 1.95 (0.44–8.72, p = 0.38), 1.41 (0.29–6.94, p = 0.67) and 2.85 (0.43–18.9, p = 0.28), respectively. these limited data suggest no significant itp risk following vaccinations or simultaneous vaccination in any age group, including infants. electronic supplementary material: the online version of this article (10.1007/s12185-020-02866-1) contains supplementary material, which is available to authorized users. immune thrombocytopenic purpura (itp) is a haemorrhagic disorder characterised by thrombocytopenia, a purpuric rash, normal bone marrow and the absence of signs of other identifiable causes of thrombocytopenia [1] [2] [3] [4] [5] . previous studies have shown that itp risk increases after measles, rubella, chickenpox and influenza infection [2, [6] [7] [8] . recently, live measles-mumps-rubella (mmr) [8] [9] [10] [11] and varicella [12] vaccines have been reported to potentially increase the risk for itp. studies have also suggested that inactivated hepatitis b [13] and diphtheria-tetanus-acellular pertussis (dtap) [12] vaccines may increase itp risk. evidence suggests that itp risk after vaccination increases through the same mechanism as that by which microbial infections induce antiplatelet autoantibodies [14] . because vaccines are designed to induce protective immunity by mimicking infections in the human body, both live and inactivated vaccinations can theoretically trigger the development of itp [15] . researchers have investigated associations between multiple vaccinations and itp risk, but findings regarding inactivated vaccinations were inconsistent [16] [17] [18] [19] [20] . however, because many vaccines are administered to children in infancy, an epidemiological study for vaccine safety conducted some time after a single vaccine dose is difficult to design. therefore, more studies are needed to clarify the itp risk associated with live and inactivated vaccines. a combined vaccine is defined as a single product in which equivalent component vaccines are administered as a single vaccine to prevent more than one disease or to protect against multiple strains of infectious microbes [21] . for example, mmr, diphtheria-pertussis-tetanus and multivalent pneumococcal conjugate vaccines have been licensed for large-scale supply. the nature of combined vaccines means it is difficult to separately measure the adverse reaction rate of each component. simultaneous vaccination, which is commonly conducted for children in europe [22] , north america [23, 24] and asia [25] , is defined as administering more than one vaccine at different anatomic sites during the same clinic visit, without combining these vaccines in the same syringe [21] . early research reported that parents were concerned about the efficacy of simultaneous immunisation [26] . however, experimental and clinical epidemiological studies provided a scientific basis for the efficacy of this practice [27] . recently, the focus of interest in simultaneous vaccination has moved from questions of efficacy to questions of safety [28] [29] [30] . if a vaccination has adverse reaction risks, these risks may accumulate in simultaneous vaccination [31] . however, no research has compared the risks when administering vaccines simultaneously versus separately in the same population. studies conducted in the 1990s indicated rates of adverse reactions were similar between simultaneous and separate vaccine administration [32] [33] [34] . correspondingly, the centers for disease control and prevention issued a guideline recommending that parents use simultaneous vaccination for children younger than 2 years to avoid missing the appropriate vaccination timing [21] . however, some studies suggested that rates of several adverse reactions may increase with simultaneous vaccination [27, 35] . therefore, the risk associated with simultaneous vaccine administration needs to be investigated more comprehensively. in this case-control study, we aimed to determine the itp risk after live, inactivated and simultaneous vaccination in japan. we also examined the risk associated with simultaneous vaccination among subjects aged < 2 years, referred to in this study as infants [36] , because this age group are frequently immunised using this method. we requested physicians from paediatrics and internal medicine departments at seven university hospitals and 18 regional centre hospitals in japan to recruit inpatients and outpatients for this study. recruitment occurred from 1 october 2015 to 27 march 2017. during this period, participating physicians enrolled in all new cases with itp that attended their hospital and met the inclusion criteria for this study. these physicians filled in the study questionnaire with the requested data for the case and control patients. we defined exposure as vaccination within 28 days before the onset of itp. to measure this exposure, participating physicians who treated case (itp) and control (other diseases) patients completed questionnaires covering retrospective information on vaccination history and other characteristics. in japan, vaccination history for infants and preschool children is recorded in the maternal and child health handbook, which is provided by the municipality. vaccination history for other children and adults was captured in a medical interview conducted by physicians or obtained from medical records. additional information was obtained from hospital medical records. in japan, the immunization law defines itp as an adverse event following immunisation that occurs within 28 days after vaccine administration [37] . based on this law, we considered ≤ 28 days after administration as the duration in which vaccinated people could potentially develop itp as a result of vaccination. the live vaccines investigated were the bacillus calmette-guérin (bcg), rotavirus, varicella, mumps and measles-rubella (mr) vaccines (in japan, mr and mumps vaccines are administered separately). inactivated vaccines were the influenza, diphtheria-pertussis-tetanus-polio (dpt-ipv), hepatitis b, 13 following japanese and american guidelines [38] [39] [40] [41] , cases with itp were identified when patients met all of the following conditions. (1) peripheral blood platelet count ≤ 100,000/µl [41] [42] [43] . (2) without anaemia unless the patient was bleeding as a result of itp. (3) without deformation of red or white blood cells. (4) without aplastic anaemia, myelodysplastic syndrome, leukaemia, malignant lymphoma, paroxysmal nocturnal haemoglobinuria, systemic lupus erythematosus, disseminated intravascular coagulation, bone marrow metastasis, myelofibrosis, thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome, liver cirrhosis, hypersplenism and megaloblastic anaemia. (5) without bernard-soulier, wiskott-aldrich, may-hegglin and kasabach-merrit syndromes. (6) condition was not pseudo-thrombocytopenia due to ethylenediaminetetraacetic acid. (7) condition was not thrombocytopenia caused by pharmacological agents, radiation or measles. because this study included patients with new onset itp, all itp cases were considered to be as acute [42] . each patient with itp (case) was matched with one or two control patients [29, 30] . participating physicians matched controls with case patients by the institution, timing of hospital visit (within a 1-month difference), sex and age. in the matched study design, we permitted overlapping use of case patient datum to be paired with two different control patient data; within an institution, one control datum was paired with two different case patient data. we requested that participating physicians reduced the difference in timing of hospital visit between case and control patients to within 2 weeks where possible. age was matched as age in months for case patients aged under 1 year, and within 2 years for case patients aged 1-17 years. for case patients aged ≥ 18 years, age was matched within 2 years where possible, with a maximum difference of 5 years. all participating patients were of asian ethnicity. sex, age at hospital visit and consulted department were measured; height and body weight were not measured. to exploit the matched study design, we calculated mcnemar's odds ratios (ors) and their 95% confidence intervals (cis) [44] for the primary outcome. mcnemar's or is suitable for matched case-control design. briefly, in this calculation method, vaccination histories for case-control pairs were grouped into four profiles: (1) vaccination ( +) for a patient with immune thrombocytopenic purpura (case) and vaccination ( +) for a patient with other diseases (control); (2) vaccination ( +) for case and vaccination ( −) for control; (3) vaccination ( −) for case and vaccination ( +) for control; and (4) vaccination ( −) for case and vaccination ( −) for control. in calculating mcnemar's ors, only two of these profiles (2 and 3) were used based on an assumption of the binary distribution. for example, if the number of the case-control vaccination profile 2 equalled that of profile 3, mcnemar's or = 1 (null hypothesis) [45] . if the former number of profile 2 doubled the latter number of profile 3, mcnemar's or = 2. this calculation method originated when mcnemar's or was developed to evaluate the results of a matched case-control study. subsequently, conditional logistic regression was developed to further adjust for covariates. we also calculated mcnemar's ors stratified by age group. because vaccination for measles, rubella and varicella are known risk factors for itp onset [8] [9] [10] [11] [12] , we calculated those ors as the reference values. tests for p values were based on a binary distribution with the null hypothesis being the same pair of numbers between the two vaccination history profiles, which is usually used in calculating ncnemar's or [44] . we also calculated common ors to exploit all available data for unmatched case and control patients. data for those patients whose paired partner was excluded from the matched data analysis were included in the unmatched analysis. common ors were the secondary outcome. we aimed to measure confounders of history of infection with helicobacter pylori [42, 46] , other viruses and bacteria, but only obtained these data for a subset of patients. therefore, in estimating common ors, we only adjusted for sex [10, 47, 48] , and calculated ors stratified by age group. the age groups were < 2, 2-5, 6-17 and ≥ 18 years, based on the vaccination schedule for children, adults and older adults recommended by the japan pediatric society and the infectious disease surveillance center (english versions) [25, 49] . because the risk associated with simultaneous vaccination could not be assessed in the matched analysis because of the small sample size, this risk was only assessed in the unmatched analysis. we calculated sex-adjusted common ors for itp occurrence in the simultaneous administration of two or more vaccines. we performed all statistical analyses using sas version 9.4 (sas institute, cary, nc, usa). all reported p values were two-sided, and p < 0.05 was considered significant. in total, 114 pairs were included in the matched analyses, and 175 patients in the unmatched analyses. most patients with itp were enrolled from paediatrics departments. in the itp case and control groups, varied infectious diseases were reported 1 month before the hospital visit. there were no underlying diseases involving an immunocompromised status, but there was an epilepsy case in the itp case group and an asthma case in the control group. table 1 shows the characteristics of the matched case and control patients. in total, 64 participants (36.5%) were under 2 years of age, 52 (29.7%) were aged 2-5 years and 76 (43.4%) were male. for the 16 cases on whom data was provided by their physicians, the mean (standard deviation) of duration from vaccination to itp onset was 17.4 (11.1) days. among case patients, there were 18 cases of purpuras, three of bleeding in the oral cavity and difficulty stopping bleeding, three of hypermenorrhoea and one of haematochezia. diagnoses recorded for hospital visits among control patients included 16 respiratory tract infections, nine digestive tract infections, three urinary tract infections, nine allergic diseases and four malignant neoplasms. there were 16 positive results for any helicobacter pylori test in the case group, but no positive results in the control group. figure 1 presents a histogram of the ages of case patients. most case patients with itp were aged < 1-14 years. for simultaneous vaccination, we investigated children aged 2 months to 11 years: six children had two vaccines, four had three vaccines, two had four vaccines and one had five vaccines. table 4 shows the sex-adjusted common ors for itp occurrence in the simultaneous administration of two or more vaccines. the mcnemar's and common ors for itp occurrence for all, live and inactivated vaccines ranged from almost null to more than 1 among people of all ages, children aged < 18 years and children aged < 2 years, but these results were not statistically significant. the common ors for simultaneous administration of two or more vaccines were greater than 1 for all, live and inactivated vaccines; these results were not statistically significant. previous research suggests that people may develop itp as an immune reaction to vaccination in combination with a genetic predisposition [4, [50] [51] [52] [53] [54] . because vaccines are designed to mimic real infections and trigger immune reactions, clinicians have been concerned that vaccinationparticularly the administration of live vaccines-may cause autoimmune disease [50] . mmr-associated acute itp has been frequently investigated in studies on this topic [9, [51] [52] [53] . in 1994, the united states institute of medicine acknowledged that evidence had established a causal relationship between mmr vaccination and thrombocytopenia [55] . in our study, the common or for mr vaccination was 3.55, although this result was not statistically significant (supplementary table 15 ). in summary, the present study did not find many significant effects, but the mcnemar's and common ors estimated for live vaccination ranged from null to high across age groups. itp risk associated with inactivated vaccination has not been sufficiently investigated in previous studies. a postlicensure retrospective study in the united states did not find any itp cases among 2-month-old infants vaccinated with the dtap-ipv/hib or other dtap-containing vaccine from 1 october 2008 to 31 july 2010 [17] . a canadian surveillance study (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) identified eight itp cases after dtp or dtap ± ipv ± hib vaccinations, three after hepatitis b vaccination and two after influenza vaccination in 12 paediatric hospitals [18] . an italian post-licensure prospective study involving a large number of older adults conducted from 2006 to 2009 reported 13 cases of itp after seasonal influenza vaccinations [19] . a study using a taiwanese national database found an incidence rate ratio for itp of 1.09 (95% ci 0.65-1.85) following pandemic influenza (h1n1) 2009 monovalent vaccination without adjuvant versus non-vaccination among people aged 0-42 years, and of 1.05 (95% ci 0.41-2.85) among children [20] . the united states vaccine adverse event reporting system database reported that from 1990 to 2008, influenza vaccination had a proportional reporting ratio (an index of identifying unexpectedly frequent reports) for thrombocytopenia occurrence of 2.10 (17 cases, lower bound of 90% ci of the empirical bayesian geometric mean < 2.0) [16] . however, there is a lack of consistent evidence about the itp risk associated with inactivated vaccines. in our study, the estimated mcnemar's ors for inactivated vaccines varied from under 1 to over 1, and were not statistically significant in the examined age groups. calculation of mcnemar's ors used discordant case-control pairs in terms of vaccination history; therefore, calculating mcnemar's ors required a much larger sample size than would have been required to calculate common ors [56]. because of insufficient power, we were unable to clarify whether itp risk accompanied inactivated vaccination. care should be taken in the interpretation of the ors reported for inactivated vaccines in this study. in the context of japan, many inactivated vaccinations are scheduled for children aged < 1 year [25] , meaning that infants of this age have a high chance of being immunised with inactivated vaccines. previous research suggests that among japanese children, the peak age of itp onset is < 2 years [48] . the ors for inactivated vaccination could, therefore, be overestimated in this study because most data used for their estimation included data for infants aged < 1 year, who frequently receive inactivated vaccines and have a relatively high incidence of itp. therefore, we interpreted the mcnemar's or of 1.00 (table 2 ) and common or of 0.99 (table 3 ) among children aged < 2 years as indicating that this study did not detect itp risk in inactivated vaccination. vaccination is frequently criticised in the media [29, 30] . however, vaccination has three types of benefits: direct effects (immunity of the vaccinated person), indirect effects (herd protection of non-vaccinated people) and reduction in the risk for developing itp caused by infection with viruses or bacteria. the third type of benefit could be rephrased as the observation that the incidence of itp following vaccination is lower than the incidence of itp after infection with wild viruses [50] . the incidence of itp development in children immunised with mmr is reportedly around one in 30,000, whereas it is approximately one in 3000 following natural rubella infection and approximately one in 6000 following measles infection. another report suggested that the incidence of itp caused by mmr vaccination is 10-20 times lower than the incidence of itp following natural infection [6] . as is inherent with a single outcome (in this case, the development of itp), the present findings of ors greater than 1 do not consider the third type of vaccination benefit. in other words, it is difficult to investigate the long-term effect of vaccination on preventing itp development from the perspective of the third type of benefit-if it exists-in cohort or case-control studies. the present study is notable for investigating itp risk following simultaneous vaccination. although we did not find statistically significant results, the sex-adjusted ors were relatively high (or 1.52 among children aged < 12 years and 1.95 among children aged < 2 years; table 3 ). the point estimate of the or was larger than that of single vaccination (1.38 among children aged < 12 years and 1.21 among children aged < 2 years; table 3 ) in children of each age group. few studies have investigated the risks associated with simultaneous vaccination. a randomised controlled trial involving people receiving simultaneous influenza vaccination reported increased haemagglutinin inhibition titre, seroconversion and seropositivity rates, suggesting that the immune response was boosted [57] . because vaccine efficacy and immune-related adverse reactions have the same origin [15] , the rate of itp development may increase depending on the administration method. table 4 suggests that there is potential for a relatively higher risk for itp with simultaneous vaccination compared with separate vaccination. however, further analyses using large databases are needed to confirm this finding. there are several limitations inherent to this study. first, the sample size meant the study was underpowered because we adopted a matched design to adjust for several confounders between cases and controls. this sample size also made it difficult to estimate ors separately by age group. to compensate for this limitation and present additional results, we calculated common ors using all available data, although adjustment for confounders might have been insufficient for this analysis. second, we could not adjust for infection history as a confounder. although we aimed to measure infection history in the questionnaire, we did not obtain sufficient data. third, because the investigation depended on the voluntary replies of physicians by post, reporting bias might have occurred. physicians who were interested in potential adverse events might have been more likely to participate in this study, which could have led to an overestimation of the ors. a national surveillance system of adverse events following vaccinations in japan is needed in an era when people are concerned about drug-induced diseases. fourth, each case was matched to one or two controls. this method might have resulted in an imbalance in the weight of samples for the primary outcome. because several matching factors may restrict selecting potential control patients, we chose a design to counter this limitation. in terms of study strengths, the detection of itp cases by physicians was a main advantage of the present study. the use of physician-registered cases enhanced the internal validity of this investigation. this validity would not be possible in a large database study. second, the data were gathered from hospitals across japan. the reported itp risks, therefore, reflect a broad area, suggesting relatively high external validity. third, the analyses included detailed results stratified by age group and type of vaccine. this information will be informative for researchers and clinicians seeking to assess the potential risks for adverse events for their patients. based on the limited data available in this study, high itp risk was not found following inactivated or simultaneous vaccination. future investigations of itp risk should include analyses using large databases. idiopathic thrombocytopenic purpura recurrent immune thrombocytopenia after influenza vaccination: a case report prediction of response to first-line therapy with itp by flow cytometric analysis of bone marrow lymphocyte phenotypes diagnostic and treatment guidelines for thrombotic thrombocytopenic purpura (ttp) 2017 in japan glucocorticoids promote response to thrombopoietin-receptor agonists in refractory itp: a case series the itp syndrome: pathogenic and clinical diversity virus-associated immune thrombocytopenic purpura in childhood association between drug and vaccine use and acute immune thrombocytopenia in childhood vaccine administration and the development of immune thrombocytopenic purpura in children risk of immune thrombocytopenic purpura after measles-mumps-rubella immunization in children mmr vaccine and idiopathic thrombocytopaenic purpura immune thrombocytopaenic purpura: an autoimmune cross-link between infections and vaccines thrombocytopenia reported in association with hepatitis b and a vaccines consequence or coincidence?: the occurrence, pathogenesis and significance of autoimmune manifestations after viral vaccines immune thrombocytopenic purpura (itp) associated with vaccinations: a review of reported cases thrombocytopenia after vaccination: case reports to the us vaccine adverse event reporting system safety of dtap-ipv/hib vaccine administered routinely to infants and toddlers thrombocytopenia after immunization of canadian children safety of mf59-adjuvanted influenza vaccination in the elderly: results of a comparative study of mf59-adjuvanted vaccine versus nonadjuvanted influenza vaccine in northern italy safety of pandemic (h1n1) 2009 monovalent vaccines in taiwan: a self-controlled case series study best practices guidance of the advisory committee on immunization practices (acip) vaccination schedules for individual european countries and specific age groups advisory committee on immunization practices recommended immunization schedule for children and adolescents aged 18 years or younger-united states canada's provincial and territorial routine (and catch-up) vaccination routine schedule programs for infants and children. 2020. public health agency of canada vaccination schedule recommended by the japan pediatric society addressing parents' concerns: do multiple vaccines overwhelm or weaken the infant's immune system? simultaneous administration of childhood vaccines: an important public health policy that is safe and efficacious children who have received no vaccines: who are they and where do they live? the pandemic influenza a (h1n1) 2009 vaccine does not increase the mortality rate of idiopathic interstitial pneumonia: a matched case-control study safety of the influenza a (h1n1)2009 vaccine in chronic obstructive pulmonary disease: a matched case-control study additive, multiplicative, and other models for disease risks simultaneous administration of measles-mumpsrubella vaccine with booster doses of diphtheria-tetanus-pertussis and poliovirus vaccines safety, tolerability, and immunogenicity of concurrent administration of haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) with either measles-mumps-rubella vaccine or diphtheriatetanus-pertussis and oral poliovirus vaccines in 14-to 23-monthold infants immune response to simultaneous administration of a recombinant dna hepatitis b vaccine and multiple compulsory vaccines in infancy reactions following administration of influenza vaccine alone or with pneumococcal vaccine to the elderly centers for disease control and prevention ministry of health, labour and welfare consensus guideline for diagnosis and treatment of childhood idiopathic thrombocytopenic purpura epidemiology of primary immune thrombocytopenia in children and adults in japan: a population-based study and literature review standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group chinese guidelines for treatment of adult primary immune thrombocytopenia reference guide for management of adult immune thrombocytopenia in japan: 2019 revision thromboembolism in patients with immune thrombocytopenia (itp): a meta-analysis of observational studies 1: the analysis of case-control studies the mcnemar test for binary matched-pairs data: mid-p and asymptotic are better than exact conditional high-dose dexamethasone therapy as the initial treatment for idiopathic thrombocytopenic purpura risk of immune thrombocytopenic purpura after measles-mumps-rubella immunization in children. child care health dev a nationwide survey of newly diagnosed childhood idiopathic thrombocytopenic purpura in japan routine/voluntary immunization schedule in japan vaccination and autoimmune disease: what is the evidence? idiopathic thrombocytopenic purpura and mmr vaccine recurrent thrombocytopenic purpura after repeated measles-mumps-rubella vaccination effect of live measles vaccine on the platelet count persistent changes in circulating white blood cell populations after splenectomy adverse events associated with childhood vaccines other than pertussis and rubella: summary of a report from the institute of medicine on the sample size for studies based upon mcnemar's test safety and immunogenicity of a 2009 pandemic influenza a h1n1 vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the 2009-10 influenza season: a multicentre, randomised controlled trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to express our deep gratitude to the participating patients. for their cooperation as physicians in this study, we would also like to thank dr. katsuko maeda, yamagata city hospital saiseikan; dr. yuya sato, dokkyo medical university hospital; dr. noritaka furuya, saitama citizens medical centre; dr. yoshinori kobayashi, national hospital organization yokohama medical centre; conflict of interest tn received honoraria from daiichi sankyo co., sanofi k.k. and mitsubishi tanabe pharma corporation. mmochizuki received honoraria from pfizer inc. mmori's department received unrestricted research grants from abbvie gk; ayumi pharmaceutical corporation; chugai pharmaceutical co., ltd.; csl behring k.k.; japan blood products organization; nippon kayaku co., ltd.; ucb japan co., ltd.; and asahikasei pharmaceutical corporation. the other authors have no conflicts of interest to declare. the study protocol was reviewed and approved by the ethics committee of the national institute of infectious diseases (the principal institution of this project) in accordance with the ethical guidelines and regulations of the declaration of helsinki (approval number: h27-561).informed consent physicians in hospitals explained the study to their patients and obtained written informed consent from the parent(s) or legal guardian(s) of each child and/or assent by the child (when applicable) before initiating medical interview and record review. hiroshi yokomichi 1 · keiko tanaka-taya 2 · rie koshida 3 · takashi nakano 4 · yoshinori yasui 5 · masaaki mori 6 · yuka ando 7 · saeko morino 2 · hideo okuno 2 · hiroshi satoh 2 · satoru arai 2 · mie mochizuki 8 key: cord-007331-wccmeaep authors: orcutt, connie j. title: emergency and critical care of ferrets date: 2017-04-20 journal: vet clin north am exot anim pract doi: 10.1016/s1094-9194(17)30157-3 sha: doc_id: 7331 cord_uid: wccmeaep ferrets are becoming increasingly popular as pets in the united states. emergency situations involving ferrets are most often caused by gastrointestinal disease, neoplasia, cardiac disease, or endocrinopathy. hospitalization and supportive care of the critically ill ferret, emergency treatment techniques, and diagnostic procedures are discussed. diseases most commonly involved in critical presentations are reviewed along with treatment protocols. localities mandate euthanasia of the animal for rabies testing? if the ferret is active yet tractable, support the body vertically under the forelimbs for examination. with more energetic ferrets or those prone to biting, scruffing and holding the patient vertically elicits relaxation and a yawning reflex. distraction is afforded by offering nutri-cal (evsco pharmaceuticals, buena, nj) or ferretone (8-in-l pet products, hauppauge, ny), but avoid sugary treats in ferrets suspected of insulinoma. fractious ferrets can be scruffed in lateral or sternal recumbency with one hand while placing the other hand cranial to the pelvis and stretching the body slightly? awareness of specific anatomic, physiologic, and behavioral characteristics of the ferret is important for accurate clinical assessment. modify the extent of the physical examination based on the ferret's status. dyspneic animals may tolerate only brief periods of handling without oxygen supplementation. bruxism, ptyalism, or pawing at the mouth most often indicates gastrointestinal discomfort or nausea resulting from hypoglycemia or other causes. hypoglycemic ferrets may also appear dazed. posterior paresis in the ferret can be a manifestation of hypoglycemia, neurologic disease, or weakness of any cause. the ferret's normal body temperature is 100° to 103° f. 42 testing the hydration status of a ferret by tenting the skin can be inaccurate, and evaluating mucus membrane capillary refill time is generally a more reliable method. the normal heart rate of 180 to 250 beats / min often varies greatly due to the ferret's normal respiratory sinus arrhythmiay the thorax is long relative to the total body length of a ferret, and the entire area must be ausculted for murmurs or abnormal arrhythmias. splenomegaly is a common finding in many ferrets, but pronounced splenomegaly or abnormal splenic texture may indicate pathology. most pet ferrets in the united states originate from large breeding facilities in which kits are neutered at 5 to 6 weeks of age, and a tattoo is placed inside the right pinna. although this makes disease involving the reproductive tract less likely, it does not preclude the presence of a reproductive remnant. a swollen vulva in a female ferret may indicate adrenal disease (most commonly, especially in middle-to older-aged animals),42 an intact female in estrus, or the presence of an ovarian or uterine remnant. male ferrets have an os penis, and the prepuce is located on the ventral abdomen. the critically ill ferret is optimally hospitalized in a quiet area separate from dogs and cats. isolation areas must be available for pa-tients suspected of having infectious disease (for example, canine distemper virus or epizootic catarrhal enteritis). hospital personnel with influenza should avoid contact with ferrets. the ferret requiring supplemental oxygen or heat can be hospitalized in the same type of oxygen cage or incubator used for a dog or cat. alternatively, ferrets can be placed in acrylic intensive care cages designed for birds. 42 monitor ferrets in incubators carefully to prevent hyperthermia. the normothermic, eupneic ferret hospitalized in a wellventilated cage must also be monitored closely to prevent hypothermia. placing a towel or disposable diaper in the cage discourages the ferret from expending energy while attempting to burrow under cage paper. anorectic ferrets are at risk of developing hepatic lipidosis or hypoglycemia. enteral feeding is recommended whenever possible. debilitated ferrets refusing their regular food often accept syringe feeding or soft foods offered on a tongue depressor. aid diet (hills pet products, topeka, ks) provides an easily digestible diet accepted by most ferrets. syringe feeding is dosed at 2-5 ml three to four times daily. 42 supplements such as nutri-cal or deliver 2.0 (mead johnson, evansville, il) provide additional calories when added to syringe-fed foods, but these sources are not nutritionally complete for ferrets and should not be fed as their sole diet for extended periods. 42 nutri-cal and other sugarcontaining formulations can cause rebound hypoglycemia in ferrets that have insulinoma and should be avoided in those animals. raw meat or eggs that may contain bacterial pathogens are also not recommended. pharyngostomy tube placement in ferrets has been described? the technique is identical to that described for cats and utilizes an 8 to 10 fr pediatric feeding tube. in cases of protracted diarrhea or vomiting, gastrointestinal ulceration, or resistance to syringe feeding, a total nutrient admixture (tna) has been used successfully to provide parenteral nutrition (pn) to more than 10 ferrets in the author's clinical practice. if a veterinary practice does not have the capability to compound parenteral solutions, some human hospitals mix pn solutions in their own pharmacies and will formulate a bag of tna for a veterinarian when provided with a prescription (table 1) . a mixture of lipid and dextrose provides the ferret's resting energy requirement (rebecca remillard, phd, dvm, acvn, angell memorial animal hospital, boston, ma, personal communication, november 1997). this mixture is supplemented with amino acids, electrolytes, water-soluble vitamins, and minerals, and fluid is added so the total solution meets daily fluid volume requirements. such all-in-one solutions can be refrigerated for at least 7 days. a silicone elastomer or polyurethane jugular catheter (cook veterinary products, bloomington, il) is recommended for delivery of the solution by infusion pump. hospitalized ferrets eating voluntarily and without special dietary requirements are optimally provided with their regular diet. otherwise, offer a premium-quality dry cat, kitten, or ferret food . ferrets have high fat and animal protein requirements and metabolize fat more efficiently for energy than carbohydrates. 7 if a ferret requires fasting before surgery for the evaluation of blood glucose or for radiographic examination of the gastrointestinal tract, do not withhold food for more than 6 hours. 6 ferrets with insulinoma are especially at risk for developing profound hypoglycemia during extended periods of fasting. the critically ill ferret should be stabilized before pursuing extensive diagnostics. however, even severely compromised ferrets usually toler-ate the sampling of a small volume of blood from a peripheral vein for estimated blood glucose (bg), total protein (tp), packed cell volume (pcv), and blood urea nitrogen (bun) measurements. use an insulin syringe with a 28-gauge needle for collecting small volumes of blood «0.5 ml) from either the lateral saphenous or cephalic vein. visualization of peripheral veins is facilitated by using a 1/ 4-in. penrose drain as a tourniquet. for larger volumes of blood, the jugular vein or anterior vena cava are preferred sites of venipuncture. a healthy ferret's blood volume is approximately 5% to 6% of its body weight? as much as 10% of the blood volume can be safely withdrawn in a healthy ferret?42 determine the ferret's initial pcv and level of hydration before withdrawing substantial volumes of blood. for jugular venipuncture, use a 25-gauge needle on a tuberculin or 3-ml syringe. hold the ferret at a table's edge or in lateral recumbency as for a cat, or alternatively, wrap the ferret in a towel and restrain in dorsal recumbency? the ferret's jugular vein lies more lateral than that in a dog or cat?-42 venipuncture of the anterior vena cava for ferrets has been described. 42 this procedure can be performed without sedation in a debilitated ferret. however, if the patient resists restraint or the practitioner is inexperienced with this technique, anesthesia may be required. restrain the ferret in dorsal recumbency while one assistant extends the forelimbs caudally and a second assistant stretches the body with gentle traction applied cranial to the pelvis (fig. 1) . palpate a slight depression at the thoracic inlet lateral to either side of the manubrium and cranial to the first rib. approach the site at a 45° angle to the skin while aiming toward the opposite hip. insert a 25-gauge needle attached to a 3-ml syringe up to the needle hub. apply negative pressure, and slowly withdraw the needle until blood begins to fill the syringe. withdraw the needle if the ferret struggles. it is not necessary to apply pressure to the venipuncture site after the sample is collected. a method for obtaining volumes of as much as 1.0 to 1.5 ml of blood from the ventral tail artery of ferrets has been described? this procedure is painful to perform in the unanesthetized patient. restrain the ferret in dorsal recumbency. insert a 22-gauge needle attached to a syringe along the ventral midline of the tail, 2 to 5 cm distal to the tail base. direct the needle at a shallow angle toward the body while gently aspirating. peripheral catheterization can be performed in the awake debilitated ferret, but more active ferrets often require anesthesia. use a 22-or 24gauge peripheral catheter for placement in the lateral saphenous or cephalic vein. to facilitate intravenous (iv) catheter placement, first puncture the skin overlying or adjacent to the vein with a 20-or 22gauge needle while being careful to avoid the vein. secure the catheter with sterile tissue glue or a tape butterfly sutured to the skin before bandaging the leg. for jugular catheterization, the author most often uses a 22-gauge, 8-in. through-the-needle catheter on a 19-9auge needle (intracath, becton dickinson vascular access, sandy, ut). a cutdown procedure may be necessary to access the vein. ferrets often act depressed with a jugular catheter and neck bandaging in place. access to the vascular system for more than several days can be achieved with a subcutaneously placed vascular access system (vascular-access-port, access technologies, skokie, il). 44 an intraosseous (10) catheter can be placed in the humerus, femur, or tibia, with the femur being the site least likely to impede the ferret's movement,4,7 use a 20-or 22-gauge, 1.5-in. spinal needle or a 20-or 22gauge hypodermic needle with a sterile surgical wire inserted into the lumen as a stylet to prevent occlusion of the needle with a plug of bone. 7 , 42 placement of the catheter is done with the ferret under anesthesia unless the animal is very debilitated. alternatively, the soft tissues and the periosteum can be blocked with local anesthetic. 4 the technique for catheter placement is the same as that described for a cat. 10 catheters can be left in place for several days during which time prophylactic use of parenteral antibiotics is recommended? maintenance fluid requirement for the ferret is estimated at 70 ml/kg / 24 h ? calculate additional fluid requirements for correction of dehydration and compensation for ongoing losses following protocols used for small animals. iv or 10 fluid delivery is recommended in severely debilitated ferrets requiring dextrose supplementation. continuous delivery of fluids using an infusion pump is recommended. alternatively, a buretrol device (baxter healthcare, deerfield, il) can be used to deliver small fluid volumes. 42 monitor the ferret carefully for overhydration, which may first be evident by auscultation of harsh lung sounds or a heart murmur. ferrets with cardiac disease are especially at risk of overhydration. debilitated, anorectic animals may require fluids supplemented with dextrose, vitamin b, and potassium following the protocols used for small animals. 42 in cases of protracted anorexia, a tna delivered iv can be substituted for this fluid mix. use only small volumes of heparinized saline when flushing any catheter in the ferret to prevent heparin overdosage. antibiotics and most other medications are administered at dosages used with cats on a per kilogram basis.42 when an indwelling catheter is not in place, use a 25-gauge butterfly catheter for iv medications. most medications given intravenously, with the exception of some chemotherapeutic drugs, can alternatively be administered via an 10 catheter? limit the use of intramuscular (1m) medications in ferrets owing to their reduced muscle mass. 42 this author prefers the quadriceps for 1m injections because of its increased mass and the reduced risk of iatrogenic nerve damage at this site compared with the caudal thigh. administer subcutaneous injections as for a dog or cat. oral medications are best administered in liquid form, because pill administration is difficult in ferrets.42 most tablets can be crushed and compounded into a suspension with a vehicle such as ora-plus (paddock laboratories, inc., minneapolis, mn), flavoring, and water in a 1:1:2 ratio. 51 refrigerate the medication and shake well before using. there is no guarantee of stability.51 transfusion in the ferret has been recommended when the pcv drops below 12% to 15%, depending on whether the anemia is acute or has developed gradually.42 blood groups have not been demonstrated in ferrets, and there is little risk of a transfusion reaction even without crossmatching and after using a variety of donors?' 33, 42 estimation of the blood volume required by the recipient can be calculated as for a cat. 3d . . rcv desired -fcv of recipient anticoagulated blood volume (ml) = body welght (kg) x 70 x fcv f d . . i o onor ill anhcoagu ant place a 22-gauge jugular catheter or a 20-gauge 10 catheter for transfusion delivery. the author treats the recipient 15 minutes before transfusion with a slow iv bolus of a short-acting corticosteroid (prednisolone sodium succinate at 22 mg/kg or dexamethasone sodium phosphate at 6-to 8-mg/kg)y choose a clinically normal ferret (pc v at least 44%) as a donor.42 the author collects a maximum blood volume (milliliter) equivalent to 0.6% of the donor's body weight. use a 21-gauge butterfly catheter on a 6-to 12-ml syringe for blood collection. an acid-citrate-dextrose (acd) solution is used as an anticoagulant. flush the needle, tubing, and syringe with acd before collection, and use 1.0 ml acd per 6.0 ml whole blood collected from the anesthetized donor ferret. 42 whole blood can be transfused immediately with a slow bolus or with a syringe pump?' 33, 42 sedation is recommended for cystocentesis in all but very debilitated ferrets, because the bladder wall is thin and can be easily lacerated if the patient struggles? use a 25-or 28-gauge needle for cystocentesis. urethral catheterization requires anesthesia even with depressed patients. most cases of urethral obstruction involve male ferrets and are the result of cystic calculi or hyperplastic prostatic-like tissue at the neck of the bladder? catheterization of the male is complicated by the jshaped os penis as well as the very small diameter of the penile urethra. place the male in dorsal recumbency, and aseptically prepare the prepuce. if the prepuce or tip of the penis is swollen, a small incision can be made in the preputial opening to exteriorize the penis. 7 the tissue covering the os penis is very thin. a surgicalloupe aids visualization of the small penile urethral opening lying on the ventral surface of the penis several millimeters proximal to the tip of the os. a 24-gauge pediatric catheter with the stylet removed can be used to localize the urethral orifice (fig. 2) . flush the catheter with sterile saline if resistance is met while catheterizing the urethra. in some cases, a 3.5-fr rubber feeding tube can be used as an indwelling catheter, but this will be too large in many ferrets. the author most often uses a 20-or 22-gauge, 8-in. jugular catheter with the stylet removed (see the section on iv catheterization). the stylet can be left in place with the end retracted to provide support around the pelvic flexure, but exercise extreme caution to avoid perforation of the urethra. suture the catheter to the skin using tape butterflies, and tape the administration port of the catheter to a tongue depressor to prevent kinking. place a padded bandage taped to the skin, and attach the catheter to a closed urinary collection system. monitor the ferret carefully, because many animals attempt to remove the bandage and catheter. the ferret with an indwelling urinary catheter should be treated empirically with a broad spectrum antibiotic (see the section on cystitis). for catheterization of the female ferret, place the anesthetized patient in ventral recumbency, elevate the hindquarters, and aseptically prepare the vulva and perivulvar area. use a small vaginal speculum or otoscope cone to locate the urethral opening on the ventral floor of the vaginal vestivule, approximately 1 to 1.5 cm cranial to the clitoral fossa?' 42 use a catheter as described above for the male ferret. in some instances of pneumothorax or pleural effusion, placement of a chest tube may be required. the author has performed this technique as described for a cat by using, as a chest tube, an 8-fr rubber feeding tube with a kirschner wire stylet. once sutured in place, the tube is capped for intermittent suction. the author has successfully used continuous suction at 13 to 15 cm h20-negative pressure in one case of a ferret with a continuous spontaneous pneumothorax. isoflurane provides the safest means of anesthesia in the critically ill ferret. avoid the use of injectable agents in debilitated animals. 35 ideally, a ferret should be fasted for 4 to 6 hours before induction. an induction chamber is recommended, because ferrets often violently resist induction by face mask. many ferrets hypersalivate during isoflurane induction, but because this is usually a transient behavior, atropine is generally not necessary? ferrets are not prone to laryngospasm, and intubation is relatively easy with a 2.0 to 4.0 mm endotracheal tube. 35 , 37 deliver anesthesia via a nonrebreathing circuit. to prevent hypothermia, place the ferret on a circulating warm water heating pad for any procedure lasting more than a few minutes. administer iv fluids to debilitated patients, taking care to avoid overhydration, especially in patients suspected of having cardiac disease, administer 2.5% to 5% dextrose-containing fluids to hypoglycemic ferrets during prolonged surgical procedures?' 35 monitor anesthesia with a pulse oximeter or a doppler ultrasound blood pressure monitor. 35 a rectal probe may increase the usefulness of the pulse oximeter in small animals ,35 clinical signs of pain in ferrets include lethargy, anorexia, vocalization, stiff movements, reluctance to curl up in a sleeping position, and squinting? buprenorphine (0,01 to 0.03 mg/kg iv, 1m, or sc q8 to 12 h) or butorphanol (0.1 to 0.5 mg/kg 1m or sc q12 h) is well-tolerated by ferrets ?' 13 monitor carefully for signs of significant depression, hypothermia, or hyperthermia. if necessary, naloxone (0.04 mg/kg iv, 1m, or sc) can be administered as a reversal agent? the pcv of ferrets is high relative to that of other species, and normal values have been reported to range from 46% to 61 %. 42 the normal leukocyte range of 2.8 to 8 x 10 3 , with clinically normal ferrets often having white blood cell counts of 3 to 4 x 10 3 , is generally lower than that of other species,29 there is no evidence that ferrets demonstrate a leukocytosis with stress? the ferret's normal differential parallels that of the cat. isoflurane administered for 15 minutes has been shown to result in significant decreases in hematocrit, hemoglobin, and plasma protein as well as red and white blood cell counts. 34 coagulation profiles have not been standardized for ferrets, but the normal mean prothrombin time has been reported to be 14.4 to 165 seconds, 42 in contrast to the cat and the dog, bun values in the ferret appear to be more sensitive than creatinine in the evaluation of renal status. in cases of renal failure, bun levels are often drastically elevated compared with relatively less significant increases in creatinine. 24 , 29 however, an elevated bun is not specific for a renal disorder. other significant clinicopathologic findings with renal disease in the ferret include hyperphosphatemia, hypocalcemia, hyperkalemia, and acidosis. 29 the cardiac silhouette of a normal ferret may appear slightly elevated from the sternum on lateral radiographs as a result of fat accumulation surrounding the ligament extending from the heart to the sternum,6 and this finding alone should not be interpreted as a pneumothorax. normal ferrets should have only a small amount of gas in the gastrointestinal tract. 55 variations from this norm may indicate an obstructive pattern or ileus. the spleen is often pronounced in radiographs of even clinically normal ferrets. cardiopulmonary resuscitation in the ferret is based on protocols published for cats. establish an airway by intubation with a 2.0 to 4.0 mm endotracheal tube, and ventilate the ferret at a rate of 20 to 30 breaths/min taking care not to overinflate the lungs. evaluate the cardiac rhythm with an electrocardiogram. in cases of asystole, place the ferret in lateral recumbency, and gently perform cardiac massage at a rate of 100/min. evaluate the femoral pulse frequently. if external massage alone does not stimulate a heart beat, administer epinephrine (0.2 to 0.4 mg/kg diluted with sterile water delivered intratracheally, or 0.2 mg/ kg via an intracardiac, iv, or 10 route). 16 establish an iv line, and administer fluids at a rate of 70 ml/kg/h. if mechanical systole returns but the ferret is bradycardic, administer atropine at a dose of 0.05 mg/ kg intravenously or 0.10 mg/kg intratracheally.16 continue to monitor the electrocardiogram for treatment of specific arrhythmias. differential diagnoses for the ferret in respiratory distress include pleural effusion (cardiac disease, neoplasia, infection, heartworm disease, hypoproteinemia, metabolic disease); pulmonary edema (cardiac disease, hypoproteinemia, metabolic disease, electrical cord bite); anterior mediastinal mass; pneumonia; pneumothorax; diaphragmatic hernia; tracheal obstruction; metabolic disease (acidosis); and profound weakness (circulatory collapse, hypoglycemia, anemia).21,36 hyperthermia or pain may also manifest as dyspnea in ferrets. the dyspneic ferret often requires oxygen supplementation before pursuing extensive diagnostics. evaluate the initial pcv, bg, tp, and bun if the ferret is insufficiently stable to collect blood for an initial complete blood count (cbq and serum chemistry. if cardiac disease is suspected, administer furosemide (see the section on cardiac disease). obtain whole body radiographs as soon as the animal tolerates further handling. perform thoracocentesis in cases of pronounced pleural effusion or pneumothorax, and submit fluid collected for cytology and a gram's stain as well as culture and sensitivity if indicated. cardiac disease is seen most frequently in middle-to older-aged ferrets. the most common form of cardiac disease in the ferret is dilated cardiomyopathy (dcm), with hypertrophic cardiomyopathy occurring less frequently.54 historical abnormalities include lethargy, anorexia, weight loss, exercise intolerance, and periods of dyspnea, tachypnea, or coughing. tachycardia, murmurs, irregular arrhythmias, moist rales, muffled heart and lung sounds, rear limb weakness, and hypothermia are possible physical examination findings. 54 radiographs may reveal an enlarged cardiac silhouette, pleural effusion, pulmonary edema, ascites, hepatomegaly, or splenomegaly. 54 electrocardiography is recommended if abnormal arrhythmias are ausculted. isoflurane anesthesia is usually needed to obtain an electrocardiogram (ecg). the ferret ecg normally contains small p waves but large r waves. 54 echocardiography is ultimately the most informative aspect of the cardiac workup in the ferret and should be performed as soon as possible. use feline dosages on a per kilogram basis for cardiac drugs when uncertain of specific dosing in the ferreu 4 furosemide for treatment of pulmonary edema is dosed at 1 to 4 mg/kg iv, sc, 1m, or po q8 to 12hy,54 although the iv route is most direct in cases of fulminant heart failure, the ferret in severe respiratory distress may only tolerate 1m injection. digoxin is used as a positive inotrope in cases of dcm or to depress av nodal conduction of supraventricular tachyarrhythmias at a dose of 0.01 mg/kg po q24h dosed on lean body weight (estimating a normal 15% body fat).54 balanced vasodilators used to decrease afterload in cases of dcm include enalapril (0.5 mg/kg po q48h) or captopril (1/8 of a 12.5-mg tablet/ferret po q48h),b, 54 monitor the ferret closely for hypotension, decreasing the frequency of dosing to once every third day if the ferret becomes lethargic. 54 alternatively, during the initial management of heart failure, one can use a venous vasodilator such as 2% nitroglycerin ointment (1/8 in. applied to the skin or inner pinna q12 to 24h).54 monitor the patient for signs of hypotension. beta-adrenergic blockers (atenolol, 6.25 mg/ferret po q24h) or calcium channel antagonists (diltiazem, 3.75 to 7.5 mg/ferret po q12h) are most commonly used to relax the myocardium and to improve diastolic filling in cases of hypertrophic cardiomyopathy.l3, 54 administer fluid therapy judiciously in ferrets with cardiac disease to avoid fluid overload and subsequent development of pulmonary edema. prednisone and diazoxide, medications often used to treat insulinoma, can also increase preload on the heart. 49 lymphosarcoma (lsa) is one of the most common forms of neoplasia reported in the ferreu dyspnea can result from an anterior mediastinal mass, pleural effusion, pulmonary metastasis, or profound weakness. mediastinal masses and pleural effusion are most common in ferrets less than 1 year of agey in young animals, acute onset of clinical signs or sudden death are typical with the rapidly progressive mediastinal form of lsa.2. 17 other clinical signs may include anorexia, weight loss, vomiting, depression, dehydration, or signs associated with secondary infections. accompanying physical examination findings may be referable to any body system but most commonly include hepatomegaly, splenomegaly, and lymphadenopathy. lo diagnosis of mediastinal lymphoma is generally made by cytologic examination of pleural fluid. ultrasound-guided aspiration of an anterior mediastinal mass can be used to collect samples for cytology.j5 ferrets that have mediastinal lsa have been reported by some authors to respond favorably to chemotherapeutic protocols described in the references.9. 10. 15 infection with the canine heartworm (dirofilaria immitis) is uncommon in ferrets, but the risk increases in heartworm endemic areas and when the ferret is housed outside. 25 signs include lethargy, coughing, dyspnea, ascites, heart murmur, pulmonary congestion, or sudden death. 9 . 54 the presence of even one adult worm in the small heart of a ferret can be fatal, 54 peripheral microfilaremia is uncommon in ferrets with dirofilariasis, so testing for the dirofilaria antigen may be inconclusive. 9 • 25. 54 antibody tests used for feline patients are species specific and are not applicable to ferrets (karen rosenthal, ms, dvm, abvp-avian, personal communication, october 1997). radiographic changes with dirofilariasis include cardiomegaly, pulmonary congestion, pleural effusion, and ascitesy· 25 a peripheral eosinophilia may be evident on the cbc. 9 the test of choice for diagnosing dirofilariasis in ferrets is echo cardiography, which images the parasite in the heart (karen rosenthal, ms, dvm, abvp-avian, personal communication, october 1997). in some cases, angiography has been used to confirm the presence of adult worms. 56 treatment protocols have been reported, but results are inconsistent. 9 • 54 pneumothorax pneumothorax can occur as a result of trauma, primary pulmonary disease, or esophageal perforation. the author has treated two ferrets with spontaneous pneumothorax. thoracocentesis is performed using protocols described for other small animals. if pleural air continues to accumulate, perform a tube thoracostomy. in uncommon cases of severe pneumothorax, resolution can be rapid when managed by continuous underwater suction if pressure is carefully monitored. 4o perform diagnostics as indicated to reveal the primary pathology, and treat as indicated. pneumonia is uncommon in ferrets and is most commonly associated with severe influenza virus or canine distemper virus (cdv) infections. 50 coughing is not as prominent a sign of pneumonia in ferrets as in dogs. 9 clinical signs of influenza include sneezing, epiphora, serous ocular discharge, rhinitis, anorexia, lethargy, and pyrexia. 9 ,5o influenza is usually mild and self-limiting in adult ferrets but can be fatal in neonates. 9 because ferrets are susceptible to human influenza viruses, affected caretakers should avoid contact with ferrets. ferrets with cdv may initially present with clinical signs similar to influenza but go on to develop a severe crusting dermatitis on the face and other areas of the body as well as marked hyperkeratosis of the footpads. 52 ferrets that have advanced cdv infection may also present with neurologic signs. 53 bacterial pneumonia is occasionally seen in ferrets. pulmonary mycosis is uncommonly reported. 9 , 50 radiographs and a cbc are often the most helpful initial diagnostic tests in cases of pneumonia. 9 a neutrophilic leukocytosis with a left shift may be pronounced. 9 ,5o radiographs demonstrate an interstitial pattern progressing to an alveolar pattern if the pneumonia worsens. 50 aspiration pneumonia primarily involves the dependent lung lobes. if the ferret is stable, a tracheal wash to collect samples for cytology as well as bacterial and fungal cultures is important in guiding treatment. diagnosis of cdv is generally made on the basis of clinical signs, but viral inclusion bodies may be visualized or fluorescent antibody tests performed on conjunctival or mucus membrane scrapings. 52 treatment for influenza consists of supportive care. antihistamines used to control sneezing or to improve appetite include chlorpheniramine (1 to 2 mg / kg po q8 to 12h) or diphenhydramine (0.5 to 2.0 mg/ kg po q8 to 12h).9, 13 use antibiotics only if a secondary bacterial infection is suspected. euthanasia is recommended for ferrets with cdy'9,62 antibiotic treatment of bacterial pneumonia is based on culture and sensitivity results. empirical broad-spectrum antibiotics to consider while awaiting culture results include trimethoprim / sulfonamide (30 mg/kg of combined drug po qi2h), cephalexin (15 mg / kg po qi2h), or amoxicillin / clavulanate (22 mg/kg po q8h).9, 13, 50 parenteral antibiotics used with cats more rapidly achieve therapeutic blood levels in severe cases. nebulization with gentocin may be useful in young animals. 9 clinical signs of gastrointestinal (gi) discomfort in ferrets are often not specific: collapse; lethargy; anorexia; dehydration; nausea evident by bruxism, ptyalism, pawing at the mouth, or frequent swallowing; retching or gagging; diarrhea; or vomiting. a number of the gi diseases affecting ferrets can have similar clinical presentations. profuse diarrhea can rapidly produce severe dehydration in the ferret. in contrast to the canine patient, diarrhea in the ferret is difficult to classify as being small intestinal or large intestinal in character.26 differential diagnoses for diarrhea include gi foreign body or trichobezoar, dietary indiscretion, helicobacter mustelae gastritis, eosinophilic gastroenteritis or other inflammatory bowel disease, neoplasia, metabolic disease (i.e., hepatopathy), clostridial overgrowth subsequent to prolonged antibiotic administration, influenza, rotavirus (usually in very young, unweaned ferrets), edv (generally accompanied by respiratory signs and a crusting dermatitis), epizootic catarrhal enteritis ("green slime disease"), gi parasitism (i.e., coccidiosis, giardiasis), and proliferative bowel disease. 21 , 26, 36 less common entities associated with diarrhea in pet ferrets include salmonellosis, aleutian disease, and mycobacteriosis. 21 , 26, 36 frank blood in the stool may be evident with proliferative bowel disease, salmonellosis, or clostridial overgrowth. 3 ,26 differential diagnoses for melena in the ferret include h. mustelae gastritis, toxin ingestion, gi foreign body or trichobezoar, hemorrhaging gi polyps, neoplasia, uremic ulceration, iatrogenic causes (i.e., nonsteroidal inflammatory drug induced), and aleutian disease (uncommon in pet ferrets).3, 36 . vomiting is not as common in ferrets as in dogs or cats and must be distinguished from regurgitation. 26 the most common differential diagnoses for true vomiting in . ferrets include gi foreign body, h. mustelae gastritis, or inflammatory gastroenteritis (including eosinophilic gastroenteritis).25 vomiting is not commonly seen with metabolic disease (i.e., azotemia or hepatic disease) in ferrets.25 regurgitation or gagging may be caused by esophageal foreign body or megaesophagus. initial treatment of dehydration and electrolyte imbalances is critical while pursuing diagnostics. evaluate the pev, bg, tp, and bun before treatment, and submit a ebe and serum chemistry. obtain initial survey radiographs. additional imaging diagnostics may include ultrasonography, endoscopy, or colonoscopy. evaluate a fecal sample wet mount and flotation as well as a fecal gram's stain. in cases of hematochezia or if the ferret is febrile, culture the stool for campylobacter jejuni or salmonella spp.26 rubber, latex, or cloth objects are the most common gi foreign bodies in ferrets under 1 to 2 years of age, whereas trichobezoars are more common in older ferrets. 9 , 26, 38 in contrast to cats, linear foreign bodies are uncommonly ingested by ferrets. 26, 38 clinical signs of gi foreign bodies vary greatly. in cases of total gastric or intestinal obstruction, the ferret may present recumbent, dehydrated, and in circulatory collapse. in general, vomiting is an infrequent finding with gi foreign bodies in ferrets. 9 , 26, 38 diarrhea or melena may be a part of the history with trichobezoars or other foreign bodies. in some cases, gastric foreign bodies may not be totally obstructive and instead act as chronic ball valves, causing intermittent nausea, vomiting, melena, gagging, anorexia, and weight loss. esophageal foreign bodies, though uncommon, can elicit regurgitation, gagging, or pawing at the mouth. 26 on physical examination, the ferret may be dehydrated with tacky mucus membranes and a delayed capillary refill time. ferrets often resist abdominal palpation or grind their teeth as a sign of gi pain, especially with small intestinal foreign bodies. 26 a gas-or fluid-dilated stomach or bowel may be palpable, and in some cases, the foreign body may be localized as well. radiographs are often diagnostic with abnormal findings including segmental ileus, gaseous distension of the stomach, and occasionally a visible object or trichobezoar. 26 the ferret's normal gi transit time is 3 to 4 hours? include the esophagus in the film for evaluation. barium studies are rarely needed to confirm the diagnosis of a gi foreign body. 26 obtain a pcv, tp, bg, and bun as a minimum database on admission, and submit blood for a cbc and serum chemistry. with ferrets in circulatory collapse, begin fluid therapy immediately via an iv or 10 catheter. administer a broad-spectrum antibiotic with severely compromised animals or those with melena. consider removal of a gi foreign body a surgical emergency when accompanied with signs of obstruction. 38 an esophageal foreign body, if devoid of sharp edges, may be removable with a small diameter endoscope, or alternatively, can be pushed into the stomach where it can be retrieved by way of gastrotomy.l1 if surgery is not an option in cases of suspected obstruction, fluid therapy and administration of a cat hairball remedy may facilitate passage of small objects. 25 h. mustelae naturally colonizes the stomach and the pyloric duodenum of many ferrets and may be clinically silent or can cause mild to severe gastritis, gastric and duodenal ulceration, and gastric adenocarcinoma. 3 , 18, 19, 27, 41 pathology may be exacerbated by environmental stress owing to environment or additional disease. 3 , 27 clinical signs include bruxism, ptyalism, abdominal pain, chronic vomiting, anorexia, weight loss, and melena, which can cause significant anemia. while clinical disease is reported to be most common in ferrets 12 to 20 weeks of age,3 this author has seen ferrets of all ages affected. assessment of the initial pcv and tp is especially important in cases of severe melena. a cbc, serum chemistry, and radiographs are part of the initial database. once the patient is stabilized, definitive diagnosis includes biopsy of the gastric mucosa via an endoscopic or surgical approach. administer fluids to correct hydration. supportive care includes parenteral nutrition in cases of severe ulcerative gastroenteritis. treatment for h. mustelae gastritis has centered on a three-drug combination: amoxicillin (20 mg/kg po q12h), metronidazole (20 mg/kg po q12h), and bismuth subsalicylate (1 ml/kg po q12h) administered for 3 to 4 weeks.3, 28 a combination of omeprazole (a proton-pump inhibitor), amoxicillin, and clarithromycin has shown a notable success rate in the eradication of helicobacter pyloru 8 however, omeprazole capsules supplied in timed-release form should not be opened before administration, therefore dosing is difficult in the ferret. epizootic catarrhal enteritis (ece) is a highly infectious diarrheal disease that affects ferrets and that can rapidly cause severe dehydration. this syndrome has been called /i green slime disease" by the lay community because of the profuse green diarrhea that is its most pronounced clinical manifestation. however, green mucoid diarrhea can be seen in a variety of malabsorptive syndromes affecting ferrets and is not pathognomonic for ece. although the etiology of ece has not been identified, histologic findings appear to be similar to those caused by rotavirus or corona virus. 26 ferrets most commonly affected are those recently exposed to new ferrets, that is, at fairs, pet stores, or as recent additions to the household. incubation appears to be only several days before the onset of clinical signs that may include a brief period of vomiting followed by anorexia, profuse diarrhea that often lasts from days to weeks, and subsequent pronounced dehydration. morbidity is high, but mortality is low with the most severely affected individuals being those having underlying disease. diagnosis is presumptive based on the ferret's history and clinical signs. the pcv and tp may be drastically elevated due to dehydration. profound leukopenia is generally not evident. radiographs may demonstrate segmental ileus, which can be confused with an obstructive pattern. hospitalized ferrets must be isolated from other ferrets, and personnel should be made aware of possible viral transmission by fomites. treatment consists of aggressive supportive care aimed at correcting dehydration and providing nutritional support. antibiotic use appears to be of little use unless indicated with accompanying disease. the author has noted significant improvement in persistent cases after administration of prednisone at anti-inflammatory doses. eosinophilic gastroenteritis (ege) is an uncommon disease of ferrets generally seen in animals older than 6 months. 3 the cause is unknown, but some authors suggest an allergic or immunologic response to foods or parasites as an initiating factor.3 clinical signs include vomiting, diarrhea, anorexia, melena, lethargy, and weight loss. thickened abdominalloops or enlarged mesenteric lymph nodes may be evident on palpation. ege is suspected when clinical signs are accompanied by a peripheral eosinophilia (equal to or greater than 1000/mm3); however, not all cases demonstrate an abnormal differentia1. 9 histopathologic demonstration of an eosinophilic infiltration of intestine, lymph node, or other tissues provides the definitive diagnosisy, 31 initiate supportive care with fluids and nutritional supplementation. although this author has successfully treated the majority of diagnosed cases with prednisone, treatment may be unreliable because the underlying cause is unknown. 27 adjust the dose of prednisone (0.25 to 1.0 mg/ kg sq or po ql2h) to control clinical signs. 9 resolution of signs is most often evident within 7 days, but treatment is recommended for an additional month before tapering the dose of corticosteroid. 9 in some cases, treatment may be lifelong. lymphoplasmacytic gastroenteritis is a similar clinical syndrome. histopathology provides the definitive diagnosis, and treatment is similar to that for ege. primary neoplasia of the gi tract is uncommon in ferrets. 26 lsa can affect any area of the ferret's digestive tract. 1o ,26 pyloric adenocarcinoma has been associated with chronic h. mustelae-associated gastritis in the ferreu 8 affected animals may have clinical signs similar to those seen with a gi foreign body. palpation and radiographs may demonstrate a fluid-distended stomach. lo , 18, 45 definitive diagnosis is made on biopsy, but abdominal ultrasonography may aid localization of pathology. critical care is supportive until the animal is stable for abdominal exploratory. surgical resection of the pyloric mass in a ferret followed by a bilroth i gastroduodenostomy was reported to be successful in one case of gastric adenocarcinoma. 18 lsa involving the intestine or liver typically responds poorly to chemotherapy.9, 10, 17 hepatopathies can present as acute or chronic conditions in the ferret. differential diagnoses include hepatic lipidosis, toxin ingestion, primary or metastatic neoplasia, bacterial infection, and inflammatory disease. other than neoplasia, primary hepatopathies appear to be rare in ferrets. 26 hepatic lipidosis is a common result of chronic anorexia. steroid hepatopathies are very rare, and to date, vascular shunts have not been reported in the ferret. 26 clinical signs of hepatic disease include lethargy, anorexia, weight loss, diarrhea, vomiting, melena, anemia, and icterus (in advanced stages).9. 26 the most consistent diagnostic abnormality with hepatic disease is an often drastic elevation of alanine aminotransferase (alt).9.29 alkaline phosphatase may be elevated as well, and in severe cases, total bilirubin levels may increase. abdominal ultrasound is recommended, but this author does not encourage ultrasound-guided liver biopsy, because coagulation panels, useful to assess risk of hemorrhage, have not been standardized for ferrets. definitive diagnosis requires biopsy of the liver. although bacterial hepatitis is not commonly reported for the ferret, in cases of persistent elevation of liver enzymes, submission of tissue for culture and sensitivity is recommended. institute supportive care while determining the definitive diagnosis of hepatopathy. nutritional supplementation is important in the treatment of hepatic lipidosis. once anorexia has resolved, hepatic lipidosis commonly requires no further treatment in the ferret. in cases of suspected bacterial hepatitis, empirical antibiotic choices pending culture and sensitivity include enrofloxacin (5 mg/kg po ql2h) or amoxicillin (20 mg/kg po ql2h) in combination with metronidazole (20 mg/kg po qi2h).9 administer vitamin k at the feline dosage on a per kilogram basis in cases of suspected coagulopathy. lactulose (0.15 to 0.75 mg/ kg po q8-12h)13 is indicated with hepatic encephalopathy. definitive treatment of hepatic neoplasia is unrewarding unless a small portion of the liver is involved and can be resected. although acquired megaesophagus is a chronic condition in the ferret, complications of the disease can lead to a critical situation. the cause of megaesophagus in the ferret is unknown. clinical signs include lethargy, anorexia, dysphagia, regurgitation, coughing or gagging, and weight 10ss.26 aspiration pneumonia is a common complication. diagnosis relies on history, clinical signs, and radiographic evidence of a dilated esophagus. aerophagia or aspiration pneumonia may also be apparent on radiographs. an esophagram may delineate esophageal abnormalities, and fluoroscopy can illustrate abnormal esophageal motility. 9 management of megaesophagus is generally less successful in fer-rets than in dogs, and the prognosis is very poor. 26 motility modifiers (metoclopramide at a dose of 0.2 to 1 mg/kg po or sc q6 to 8h, or cisapride at a dose of 0.5 mg/kg po q8 to 24h) may be useful.2 6 treat aspiration pneumonia with protocols described in the section on cardiorespiratory emergencies. rectal prolapse is not common in ferrets but can occur in some cases of proliferative bowel disease (pbd), coccidiosis, or neoplasia. 3 ,9, 10,26 pbd, uncommonly seen in the author's practice, generally affects ferrets less than 14 months of age. 3 , 9 clinical signs, in addition to rectal prolapse, include chronic liquid or mucoid diarrhea sometimes with frank blood, tenesmus, anorexia, and cachexia, pbd may be exacerbated by environmental stress,9 and affected ferrets may be more susceptible to other diseases, an affected ferret may be moderately to severely dehydrated with thickened bowel loops evident on abdominal palpation. initial diagnostics include a fecal direct wet mount and flotation, radiographs, a cbc, and serum chemistry. definitive diagnosis is made on biopsy of intestinal mucosa, rectal prolapse can initially be repaired with a pursestring suture. chloramphenicol (50 mg/kg 1m, sc, or po q12h for 14 to 21 days) is the treatment of choice for pbd.3,9 resolution of the prolapse may occur spontaneously as the colon heals. 3 coccidiosis is treated with feline protocols, urethral obstruction most commonly affects male ferrets, clinical signs include stranguria, lethargy, and anorexia. male ferrets may have a palpable mass caudodorsal to the bladder.37 ferrets with accompanying adrenal disease may also be pruritic with bilaterally symmetrical or diffuse alopecia. in the author's experience, the most common differential diagnoses for urinary obstruction include prostatic enlargement associated with adrenocortical disease in males, urolithiasis, and cystitis. less frequently, bladder neoplasia has also been reported as a cause of dysuria. 19 initial treatment for urinary obstruction is urethral catheterization. place an indwelling catheter while determining the origin of the obstruction. if a urinary catheter cannot be passed, a 25-or 28-gauge needle can be used to perform cystocentesis as an emergency measure. submit urine collected for urinalysis and culture and sensitivity. a cystostomy allows catheterization of the bladder directly from the body wall if passage of a catheter via the urethra is obstructed. submit blood for a cbc and serum chemistry. place an iv catheter and begin fluid diuresis concentrating on correction of dehydration and electrolyte imbalances. empirical broad-spectrum antibiotic therapy is recommended once a urine culture has been submitted and a urinary catheter placed (refer to the section on cystitis). further diagnostics to determine the cause of the obstruction include radiographs and abdominal ultrasound. along with ultrasonographic evaluation of the bladder, areas of special interest include the urethra, periurethral region, and adrenal glands. partial or complete urethral obstruction by hypertrophied prostatic tissue accompanies some cases of adrenocortical disease in male ferrets, and prostatic disease may be the most common cause of stranguria in male ferrets over 3 years of age. s , 47, 53 androgens of adrenal gland origin are hypothesized to cause hyperplasia or cystic changes of prostatic tissue, which subsequently presses on the urethra. 47 ,53 in some cases, prostatic tissue may become abscessed with a resultant thick purulent discharge from the prepuce. abdominal ultrasound may reveal unilateral or bilateral adrenomegaly, but not all cases of adrenal disease can be reliably diagnosed with this method. although studies of adrenal gland ultrasonography in ferrets have been published,39 diagnosis of adrenomegaly may be difficult for the ultrasonographer with little experience imaging ferrets. elevated plasma androgen and estrogen levels have been shown to be useful in diagnosing adrenal disease in ferrets,4s,53 but test results are often not available soon enough to be useful in an acute situation. removal of the diseased adrenal gland generally results in rapid dissipation of prostatic hypertrophy.37,53 cystotomy may be necessary to remove accumulation of purulent material,b and if a prostatic abscess is involved, surgical resection of the involved tissue is recommended after a sample has been retrieved for culture and sensitivity.9 this author maintains an indwelling urethral catheter for at least 2 to 3 days after surgery. persistent obstruction by prostatic tissue may be a result of infection or neoplastic changes or may be stimulated by remaining abnormal adrenal tissue. s , 53 the prognosis with prostatic abscessation may be poor because it is difficult to remove all affected tissue, and response to antibiotics is uncertain.s,9 magnesium ammonium phosphate (struvite) uroliths are the most common type of calculi reported in ferrets. 24 the incidence of struvite urolithiasis in ferrets has been less frequent with the introduction of higher-quality, animal protein-based diets that produce a more acidic urine than do plant protein-based foods. s ,24 diagnosis and treatment are similar to procedures used with cats and dogs. the practitioner can treat cases of severe or persistent urolithiasis with prescrotal urethrostomy using a technique similar to that described for the dog. 37 although primary cystitis is reported to be rare in the ferret,8, 24 purulent material in the bladder can be thick enough to cause a complete urethral obstructiony what appears to be cystitis in some male ferrets may actually be discharge from a prostatic abscess. 8 submit a urinalysis and a urinary culture and sensitivity obtained by cystocentesis. in cases of urethral obstruction, place a urinary catheter and stabilize the ferret. obtain radiographs and an abdominal ultrasound to evaluate the bladder, urethra, and adrenal glands. while awaiting culture and sensitivity results, begin treatment with a broad-spectrum antibiotic: trimethoprim/sulfonamide (30 mg/kg of combined drug po q12h), amoxicillin (20 mg/kg po q12h), or amoxicillin/ clavulanate (22 mg/kg po q8h).13 severe cases of cystitis may require treatment for 2 months or longer. clinical signs of renal failure may be nonspecific and include collapse, depression, lethargy, dehydration, anorexia, weight loss, rear limb weakness, polydipsia or polyuria, anemia, melena, oral ulcerations, or an azotemic odor to the breathy, 24 differential diagnoses for renal failure in the ferret include chronic interstitial nephritis, pyelonephritis, neoplasia, toxins, and glomerulonephropathy secondary to other causes such as aleutian disease (rarely seen in the clinical setting with pet ferrets). 24 diagnostics include a cbc, serum chemistries, urinalysis, urine culture and sensitivity, and radiographs. abdominal ultrasound is helpful in evaluating renal cysts, urinary tract calculi, or abnormal renal parenchyma. an ultrasound-guided renal aspirate may yield diagnostic information. initial treatment consists of iv fluid support geared toward correction of dehydration and electrolyte imbalances. an active urinary sediment indicates the need for a broad-spectrum antibiotic while awaiting results of a urine culture. the most common cause of hypoglycemia in pet ferrets in the united states is insulinoma (pancreatic islet cell neoplasia). additional differential diagnoses for hypoglycemia include anorexia or starvation, sepsis, neoplasia, hepatopathy, and other metabolic disease. 1 insulinoma affects ferrets ranging in age from 2 to 7 yearsy initial signs may be insidious and attributed by the owner to geriatric changes in their ferret. 49 the history may include episodes of collapse with hypersalivation or extreme weakness lasting from minutes to hours that typically resolve after administration of sugar-containing solutions. 43 other common clinical signs include depression, rear limb weakness (which may be apparent as posterior paresis or ataxia), gagging, pawing at the mouth, or a dazed appearance. occasionally severely hypoglycemic ferrets appear dyspneic. signs are often intermittent and may not be apparent on clinical presentation. although hypoglycemic seizures are the most common clinical sign of insulinoma in the dog, they are rare in ferretsy when a ferret over the age of 2 years presents collapsed or exhibiting any of the clinical signs described, immediately evaluate the bg level. initial evaluation can be performed with glucose measurement strips or a digital glucometer. 43 for more accurate evaluation of bg levels, submit blood in a sodium fluoride (gray top) tube. a bg glucose level less than 70 mg/ dl with accompanying clinical signs is suspicious for insulinoma, and ferrets presenting collapsed or comatose often have bg levels lower than 40 mg/dl.9, 43 an elevated serum insulin concentration (using an assay that has been validated for ferrets) concurrent with hypoglycemia is diagnostic for insulinoma, in cases of hypoglycemic collapse, administer a slow iv bolus of 50% dextrose (0.5 to 2 ml) to response, 43 the goal of treatment is stabilization and not the complete reversal of hypoglycemia. if dextrose is administered too rapidly, the tumor will be stimulated to release large amounts of insulin, resulting in rebound hypoglycemia, place an iv catheter and begin infusion of fluids supplemented with 5% dextrose, control rare persistent seizures with diazepam (refer to the section on seizures). administer prednisone (0.5 to 2 mg/kg sq) to inhibit peripheral tissue uptake of glucose and stimulate gluconeogenesis. 43 begin with the lowest dose needed to maintain adequate bg levels (i,e" > 70 mg/ dl), continue to monitor clinical signs along with bg concentrations q12h, and increase dextrose supplementation if necessary. once the ferret is stable, administer prednisone orally. in cases of persistent hypoglycemia, concurrently administer diazoxide, which acts to inhibit insulin release from pancreatic beta cells, beginning at the low end of the dose range (5 to 30 mg/kg po q12h).43 once clinical signs have resolved, gradually discontinue dextrose supplementation while monitoring the bg level, and adjust medications accordingly, prednisone doses can often be lowered with concurrent administration of diazoxide. offer a meatbased, high-protein ferret or feline diet while avoiding foods high in sugar or carbohydrate. in a minority of cases, hypoglycemic seizures or collapse may recur in spite of medications once iv dextrose supplementation is discontinued, these ferrets, once stabilized, may require surgical debulking of the tumors to allow further management. ongoing medical and surgical management of insulinoma is well described in the references. 9 , 43, 49 spontaneous diabetes mellitus (dm) is uncommon in ferrets. 47 most cases of hyperglycemia develop secondary to surgery for insulinoma. 47 clinical signs are similar to those seen in other small animals with dm. dm is suspected when the bg concentration is consistently greater than 400 mg/ dl in conjunction with glycosuria. 47 in cases of ketoacidosis, ketones are detected in the urine. a low insulin concentration concurrent with hyperglycemia confirms the diagnosis of dm. 47 for the initial treatment of ketoacidotic dm, follow protocols used with cats to stabilize metabolic derangements. tight regulation of bg levels in ferrets with dm can be difficult. one protocol suggests initiation of treatment with insulin when the bg level is consistently greater than 300 mg/ dl.47 neutral protamine hagedorn (nph) insulin is administered at an empirical dose of 0.1 unit per ferret twice daily with serial bg concentrations to dictate dosagey once the ferret is stabilized with a bg concentration less than 200 mg / dl, treatment is continued with either nph or ultralente insulin (which only requires once daily dosing). the owner is instructed to use dipsticks to check for the presence of glucose and ketones in the ferret's urine. the same dose of insulin is administered if only trace amounts of glucose are present in the urine. if no glucose is detected, insulin is not administered, and if the glucose concentration is elevated, the insulin dose is increasedy in cases of transient dm secondary to debulking of pancreatic tumors, hyperglycemia usually resolves within several days to 2 weeks after surgery.9, 47 ferrets with ongoing hyperglycemia are often very difficult to regulate. the differential diagnoses for anemia are broadly divided on the basis of regenerative versus nonregenerative processes. differentials for regenerative anemia in ferrets include blood loss (i.e. gi bleeding, trauma, flea infestation) and hemolytic anemia potentially caused by heavy metal toxicosis or immune-mediated disease. 22 causes of nonregenerative anemia include estrogen toxicosis of the bone marrow secondary to persistent estrus in an intact female, ovarian remnant(s), or adrenocortical disease; neoplastic infiltration of the bone marrow; or anemia of chronic disease. 22 immune-mediated hemolytic anemia has not been identified in ferrets owing to the lack of reagents specific for ferret antibodies. 22 aleutian disease, an immune-mediated disease caused by a parvovirus is uncommon in pet ferrets. although estrogen-induced bone marrow suppression was once a serious and common cause of nonregenerative anemia involving intact female ferrets, this form of pancytopenia is rarely seen owing to the current practice of spaying females at 5 to 6 weeks of age. 22 rarely, an ovarian remnant may secrete estrogen with similar results. 20 increased levels of estrogen may infrequently be associated with adrenocortical disease either as a result of estradiol secretion from an adrenocortical tumor or subsequent to conversion of tumor-secreted androgens to estrogen in peripheral tissues. 48 , 57 resultant pancytopenia has been reported,57 but this syndrome is uncommon in spite of markedly increased serum estrogen concentrations in some ferrets with adrenocortical disease. 32 clinical signs observed with pancytopenia of any cause include petechiation, ecchymosis, gi bleeding, infection, and sepsis. obtain an initial pcv from the critically anemic ferret before collecting a larger volume of blood for a cbc, platelet count, reticulocyte count, and serum chemistries. avoid anterior vena cava venipuncture in cases of suspected pancytopenia. normal mean reticulocyte counts for female and male albino ferrets have been reported as 5.3% and 4%, respectively.22 pursue further diagnostics as indicated. if the pcv is less than 15%, transfuse whole blood. if an 10 catheter is used, obtain a bone marrow sample before transfusion. if an ovarian remnant is suspected, administer human chorionic gonadotropin (hcg) (100 iu per ferret) to stimulate ovulation. 24 administration of hcg has no effect on hyperestrogenism secondary to adrenocortical disease. if the pcv remains below 15%, the prognosis is poor, and multiple transfusions along with b complex vitamins, an anabolic steroid (stanazolol, 0.5 mg/kg po, sc q12h),b iron supplementation, fluid therapy, and nutritional support may be required. 9 , 24 pursue surgical treatment for adrenocortical disease, a retained ovary, or bleeding gi masses once the patient is stabilized. anecdotal reports of reactions following canine distemper vaccination in ferrets describe clinical signs ranging from diarrhea, gagging, vomiting, fever, or erythematous skin to circulatory collapsey although many of these reactions happen within 30 minutes after vaccination, less severe signs can be noted up to several hours later. in cases of postvaccination collapse, administer diphenydramine hydrochloride (0.5 to 2.0 mg/kg iv or im),42 epinephrine (20 j-lg/kg iv, 1m, sc, or intratracheally),42 and a slow iv bolus of a short-acting corticosteroid (22 mg/kg of prednisolone sodium succinate or 6 to 8 mg/kg of dexamethasone sodium phosphate).b administer iv fluids, and provide supportive care following treatment protocols for small animals. seizures are uncommon in ferrets with the most common cause arguably being insulinoma-induced hypoglycemia. l additional differential diagnoses include hypoglycemia from other causes; trauma; toxin ingestion; ens infection (including rabies or canine distemper), inflammation, or neoplasia; renal failure; hepatopathy; or other metabolic derangements. 1 evaluate the bg level on presentation, and treat hypoglycemia as described above (refer to the section on hypoglycemia). administer diazepam (1 to 2 mg iv to effect), and treat supportively using protocols applied to other small animals. pursue diagnostics as indicated. lumbar cerebrospinal fluid tap has been described in the ferret. l gastrointestinal disease, neoplasia, cardiac disease, and endocrinopathy are among the most common syndromes affecting the ferret that presents in an emergency situation. knowledge of these and other disease processes, indicated diagnostic testing, and immediate treatment protocols are critical to provide efficient and effective care to the ferret in crisis. musculoskeletal and neurologic diseases a cluster of cases of juvenile mediastinal lymphoma in a ferret colony helicobacter muste/ae gastritis, proliferative bowel disease, and eosinophilic gastroenteritis intraosseous catheters in small mammals biology and medicine of the ferret ferrets: basic anatomy, physiology, and husbandry (of the ferret) clinical techniques in domestic ferrets stranguria in a castrated male ferret a practitioner's guide to rabbits and ferrets ferrets, rabbits, and rodents: clinical medicine and surgery. philadelphia, wb saunders medical and surgical management of esophageal foreign body in a ferret diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: 57 cases (1986-1994) exotic animal formulary s. pet ownership and pet populations chemotherapeutical remission of multicentric lymphosarcoma in a ferret (mustela putorius juro) cardiac emergencies clinical and pathologic findings in ferrets with lymphoma: 60 cases (1982-1994) helicobacter mustelae-associated gastric adenocarcinoma in ferrets (mustela putorius jura) gastric colonization of the ferret with helicobacter species: natural and experimental infections cystic ovarian remnant in a ferret critical care of ferrets, rabbits, and rodents ferrets, rabbits, and rodents gastrointestinal diseases of ferrets (mustela putorius juro! urogenital diseases cardiac disease in ferrets gastrointestinal diseases gastrointestinal diseases of ferrets helicobacter infection normal parameters and laboratory interpretation of disease states in the domestic ferret blood transfusions multisystemic eosinophilic complex in a ferret (mustela putorius juro) estradiol-17j3-secreting adrenocortical tumor in a ferret lack of detectable blood groups in domestic ferrets: implications for transfusion effect of isoflurane on hematologic variables in ferrets anesthesia, analgesia, and sedation for small mammals differential diagnoses for common clinical problems in ferrets soft tissue surgery gastrointestinal foreign body in ferrets: 25 cases ultrasonography of adrenal glands in normal ferrets pleura and pleural space helicobacter mustelae-associated hypergastrinemia in ferrets (mustela putorius jura) basic approach to veterinary care endocrine diseases: insulinoma use of a vascular access system for administration of chemotherapeutic agents to a ferret with lymphoma pyloric adenocarcinoma in a ferret the collapsed ferret endocrine diseases: adrenal gland disease, d iabetes mellitus, and thyroid disease evaluation of plasma androgen and estrogen concentrations in ferrets with hyperadrenocorticism proceedings of the north american veterinary conference ferret respiratory disease diagnosis new therapeutics in small mammals respiratory diseases stranguria in a castrated male ferret cardiovascular diseases: cardiac disease small mammal radiology radiographic and angiographic evaluations of ferrets experimentally infected with dirofilaria immitis estrogen induced bone marrow depression in a ferret (mus tela putorius juro) with adrenal gland tumors key: cord-262413-jm4qmpeg authors: mao, suling; huang, ting; yuan, heng; li, min; huang, xiaomei; yang, changxiao; zhou, xingyu; cheng, xiuwei; su, qian; wu, xianping title: epidemiological analysis of 67 local covid-19 clusters in sichuan province, china date: 2020-10-08 journal: bmc public health doi: 10.1186/s12889-020-09606-4 sha: doc_id: 262413 cord_uid: jm4qmpeg background: this study was intended to investigate the epidemiological characteristics of covid-19 clusters and the severity distribution of clinical symptoms of involved cases in sichuan province, so as to provide information support for the development and adjustment of strategies for the prevention and control of local clusters. methods: the epidemiological characteristics of 67 local clusters of covid-19 cases in sichuan province reported as of march 17, 2020 were described and analyzed. information about all covid-19 clusters and involved cases was acquired from the china information system for disease control and prevention and analyzed with the epidemiological investigation results taken into account. results: the clusters were temporally and regionally concentrated. clusters caused by imported cases from other provinces accounted for 73.13%; familial clusters accounted for 68.66%; the average attack rate was 8.54%, and the average secondary attack rate was 6.11%; the median incubation period was 8.5 d; a total of 28 cases met the criteria for incubation period determination, and in the 28 cases, the incubation period was > 14 d in 21.43% (6/28). a total of 226 confirmed cases were reported in the 67 clusters. ten cases were exposed before the confirmed cases they contacted with developed clinical symptoms, and the possibility of exposure to other infection sources was ruled out; two clusters were caused by asymptomatic carriers; confirmed cases mainly presented with fever, respiratory and systemic symptoms; a gradual decline in the severity of clinical symptoms was noted with the increase of the case generation. conclusions: population movement and gathering restrictions and strict close contact management measures will significantly contribute to the identification and control of cases. transmission during the incubation period and asymptomatic infections have been noted. studies on the pathogenicity and transmissibility in these populations and on covid-19 antibody levels and protective effects in healthy people and cases are required. so absence of stringent and effective prevention and control could have resulted in serious social consequences. we analyzed the epidemiological characteristics of covid-19 clusters in sichuan, intended to provide information support for the development and adjustment of local prevention and control strategies. the epidemiological characteristics of 67 local covid-19 clusters in sichuan reported as of march 17, 2020 and confirmed cases involved in these clusters were described and analyzed using a cross-sectional study design. in accordance with requirements in the guidance for corona virus disease 2019: prevention, control, diagnosis and management [2] , information about all covid-19 clusters and cases should be reported to the china information system for disease control and prevention. in this study, information about all local clusters was exported from sub-module "emergency public reporting system" and data about related confirmed cases and asymptomatic carriers from sub-module "infectious disease management information system". based on epidemiological investigation results, transmission chains were constructed and case generations were determined, followed by a comprehensive analysis. this study has covered all local clusters and all confirmed cases and asymptomatic carriers in the clusters, so sampling was not involved. confirmed cases with missing clinical information were not included into the analysis. confirmed cases, asymptomatic carriers and clusters were identified according to the definitions in the guidance for corona virus disease 2019: prevention, control, diagnosis and management [2] . a cluster outbreak indicated that more than two confirmed cases or asymptomatic carriers were found within 14 days in a small area (such as a family, a building site, a work unit), and there was a possibility of human-to-human transmission caused by close contact or by exposure to infectious source together. all confirmed cases were divided into mild cases (the clinical symptoms are mild and no pneumonia manifestation can be found in imaging); ordinary cases (with symptoms like fever and respiratory tract symptoms, and pneumonia manifestation can be seen in imaging); and severe cases (respiratory distress, rr ≥ 30 breaths/min; pulse oxygen saturation (spo2) ≤ 93% on room air at rest state; arterial partial pressure of oxygen (pao2)/oxygen concentration (fio2) ≤ 300 mmhg. the date of onset for a confirmed case was defined as the date of first appearance of clinical symptoms self-reported in field epidemiological investigation. the date of onset for an asymptomatic carrier was defined as the date on which a positive covid-19 pathological test was obtained with respiratory tract or other feasible samples. the attack rate was defined as the number of cases divided by the number of exposed persons, where the number of cases was the total number of confirmed cases involved in a transmission chain. as both secondgeneration (g2) and third-generation (g3) cases were identified from close contacts of cases of the previous generations, if any g2 case developed in a cluster, the number of exposed persons was the number of close contacts of first-generation (g1) cases plus the number of g1 cases; if any g3 case developed in a cluster, the number of exposed persons was the number of close contacts of g1 cases plus the number of close contacts of g2 cases resulting in the g3 cases plus the number of g1 cases; and so forth. the secondary attack rate was defined as the number of second-and later-generation cases divided by the number of exposed persons, where the number of exposed persons was calculated using the aforementioned algorithm except that the number of g1 cases was not included. the incubation period was determined based on confirmed cases that had been exposed for a single time in transmission chains, whose exposure and onset time were clearly known and in whom other factors potentially responsible for their infection were ruled out. cases for the determination of the attack rate, the secondary attack rate and the incubation period included confirmed cases and asymptomatic carriers. a family dinner was defined as a dinner attended by two or more families. epidemiological characteristics of clusters and demographic and clinical characteristics of confirmed cases were descriptively analyzed. attack rates, secondary attack rates and clinical types of cases and constituent ratios of symptoms were compared using χ2 test for constituent differences. the regional distribution map in fig. 2 was created by geocoding all covid-19 clusters and matching them to the city-level layers of polygon and point by administrative codes with the use of arc-gis software. on january 16, 2020, the first local covid-19 cluster was reported in sichuan. on january 24, 2020, a level-1 public health emergency response was launched, followed by the successive execution of multiple measures with respect to infection sources, routes of transmission and susceptible populations to effectively reduce population movement and gatherings and strengthen the management of close contacts. main measures and the timeline are shown in fig. 1 . on january 16, 2020, the first local covid-19 cluster was reported. a total of 31 clusters were reported in january and 36 in february. the daily number of clusters peaked on january 30, 2020 (n = 7) and gradually declined after february 06, 2020. a total of 50 (74.63%) clusters were reported as of february 06, 2020. temporal distribution of cluster reports is shown in fig. 1 . among the clusters, 49 (73.13%) were caused by imported cases, and in 18 the infection source of the first case was unknown. the 67 clusters had involved 16 prefectures and 48 districts/counties (fig. 2) . chengdu had the largest number of clusters (n = 17), followed by dazhou (n = 12), nanchong (n = 7) and guang'an (n = 6). clusters in the four prefectures accounted for 62.69% (42/67) of the total in the province. households were the primary exposure place in the reported clusters, accounting for 68.66% (46/67); living in the same household was the primary form of exposure, accounting for 60.87% (28/46). clusters caused by exposure in multiple places and in multiple forms accounted for 11.94% (fig. 3) . significantly more clusters were caused by family gatherings in february than in january (p = 0.002) ( table 1) . in the 67 clusters, a total of 226 confirmed cases were reported, accounting for 42.01% (226/538) of the total in the whole province; among these confirmed cases, 164 were local confirmed cases resulting from clusters, accounting for 72.57% (164/226); the male/female ratio was 1:1, the mean age was 47.56 years (1 month-87 years), and patients 30 years of age or above accounted for 87.17% (197/226); involved were 75 (33.19%) g1 cases, 131 (57.96%) g2 cases and 20 (8.85%) g3 cases ( table 2 ); 23.01% (52/226) had a history of travel or residence in wuhan, and 25.66% (58/226) had a history of travel or residence in other provinces except wuhan. the constituent ratios of cases with a history of travel or residence in both wuhan (p < 0.001) and other provinces except wuhan (p = 0.006) were higher in g1 cases were higher in g2 and g3 cases, and the differences were statistically significant ( table 3) . the 226 confirmed cases were clinically typed as follows: 65.04% (147/226) were ordinary, 26.55% (60/226) were mild, and 8.41% (19/226) were severe; no death occurred. with the increase of the transmission generation, the number of ordinary cases gradually decreased, with a significant difference noted overall (p = 0.045); a significant difference was found between g1 and g3 cases in the constituent ratio of ordinary cases (p = 0.013). no apparent between-generation difference was found in the constituent ratios of mild or severe cases (fig. 4) . among .the number of cases with fever showed a declining trend overall with the increase of transmission generation (p = 0.019), and significant differences were noted between g1 and g2 cases (p = 0.009) as well as g3 cases (p = 0.027) in terms of the constituent ratio of cases with fever. no statistically significant difference was found between generations in other symptoms (fig. 5) . among the 67 clusters, g2 cases were identified in 57 (85.07%) and g3 cases in 10 (14.93%); a total of 267 confirmed cases (including 41 asymptomatic carriers) were reported, and on average each cluster resulted in 3.99 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) cases; the average attack rate was 8.54% (1.02-100%), and the average secondary attack rate was 6.11% (0.51-66.67%). starting from february 02, 2020, we expanded the time window of close contact tracing to cover 3 days prior to the appearance of clinical symptoms in primary confirmed cases or prior to the obtainment of a positive nucleic acid test in primary asymptomatic carriers. attack rates and secondary attack rates before and after this time point were compared, and it was found that, following the expansion of the time window of close contact tracing, both the attack rate and the secondary attack rate increased, with statistical differences identified ( table 4) . among the 226 cases, 78.76% (178/226) had been exposed to confirmed cases. in 82.02% (146/178) of such cases, the exposure to the confirmed cases occurred at or after onset of the disease in them. in 10 cases (involved in 9 clusters), the exposure occurred before the confirmed cases they contacted with developed clinical symptoms, and they had been exposed only once while the possibility of exposure to other infection sources could be ruled out. the median time of exposure was 3 d (1-4 d) prior to disease onset in the confirmed cases they contacted with, and the mean was 2.5 d; 90.00% (9/ 10) of the cases were exposed within 3 d prior to disease onset in the confirmed cases they contacted with. of 226 confirmed cases, the median interval between hospital visit and onset was 1 d (0-23 d), the median interval between confirmation and onset was 5 d (0-27 d), and the median interval between isolation and confirmation was − 2 d (− 19-0 d). twenty-eight cases met the criteria for incubation period determination (involved in 17 clusters). the average incubation period was 10.3 d, the median incubation period was 8.5 d (1-24 d) and the incubation period was > 14 d in 21.43% (6/ 28). in the 67 clusters, a total of 41 asymptomatic carriers (all were identified during medical observation of close contacts) were reported, including 23 males and 18 females. the mean age was 35.31 years (8 months-81 years); and patients 30 years of age or above accounted for 70.73% (29/41). involved were 8 (19.51%) g1 cases, 31 (75.61%) g2 cases and 2 (4.88%) g3 cases. in one cluster, one g1 case (severe) was found to have spread the disease to one g2 case (mild) and one g3 case (asymptomatic). two clusters were caused by asymptomatic carriers and, in both clusters, only g2 cases developed, including one (asymptomatic) to two (confirmed) transmission in one cluster and one (asymptomatic) to one (confirmed) transmission in another cluster. the possibility of other infections was ruled out in all g2 cases. by the end of the two clusters, the g1 asymptomatic carriers had not presented with clinical symptoms or imaging abnormalities. our analysis of covid-19 clusters in sichuan reveals that the majority of clusters were reported before february 06 and occurred in four regions of sichuan, and three-fourth of the clusters were caused by importations from other provinces including wuhan. the study also found that the proportion of cases with a history of travel or residence in wuhan was significantly higher in g1 cases than in secondary cases and the clusters mainly occurred in households with a greater proportion in february than in january. these characteristics were similar to clusters in other provinces in china [3] . the above characteristics of covid-19 clusters were supposed to be related with several factors. firstly, the epidemic happened during the spring festival in china, when large numbers of people in china returned home for family reunion or visited their friends or relatives, forming a population shift peak, particularly in the midlate january. secondly, chengdu, the capital of sichuan province, as a rapidly growing metropolis in west china, has well-developed transport infrastructure including highways, railways and airlines, which facilitated the spread of the disease [4] ; big data analysis also showed that chengdu was ranked the top in terms of inflow of people from wuhan [5] [6] [7] ; dazhou and guang'an, located in the northeast of sichuan, are adjacent to chongqing, which was relatively hardly hit by the epidemic among provinces other than hubei in china [8] . finally, on january 24, 2020, sichuan launched a level-1 public health emergency response [9, 10], followed by the successive execution of multiple measures with respect to infection sources, routes of transmission and susceptible populations, effectively reducing population movement and gatherings and strengthening the management of close contacts. specifically, on january 25, 2020, the administration for market regulation, the health commission and the department of commerce of sichuan province jointly issued a document [11] for strengthening the supervision of dinner gatherings; on january 28, 2020, the people's government of sichuan province issued an order [12] requiring suspension of massive gatherings and business of recreational places and banning dinner gatherings. on february 8, 2020, the provincial headquarters for emergency response issued a notice on strengthening the supervision of dinner gatherings in rural areas [13] , banning dinner gatherings involving two or more families. the above measures have effectively reduced people's gatherings including festival celebrations and company dinner parties. however, still a minority of people, following traditional customs, organized and participated in family dinners and entertainment activities with inadequate protection during the chinese new year, resulting in the familial clustering characteristic of the epidemic. the attack rate of covid-19 clusters in our study was slightly lower than 9.6% in a study located in shenzhen [14] . the interval between hospital visit and onset was shorter than that reported in hunan [15] ; related data were not available from other provinces. the study also showed that the attack rate and the secondary attack rate were significantly higher after february 02, 2020 than before; cases were mainly confirmed during the isolation period and asymptomatic carriers were identified in the management of close contacts, which suggested that the intensive close contact management measures in sichuan including expansion of the close contact tracing time window to cover 3 d prior to the presence of clinical symptoms (or positive test for asymptomatic carriers) in the confirmed cases they contacted with, implemented from february 02, 2020, and the conduct of nucleic acid testing at both the start and the end of quarantine of close contacts, implemented from february 11, 2020, might have contributed to the discovery and control of cases and the reduction of spread. the median incubation period in the limited cases in sichuan was corresponding to the finding in other studies in china that the incubation period in the majority of cases was < 14 d, but being longer than the median incubation period reported in other studies (3-7 d) [16] [17] [18] [19] [20] . it was also found that the proportion of cases with an incubation period > 14 d was higher than that reported in a study in henan (7.45%) [21] . ten cases exposed before the confirmed cases they contacted with presented with clinical symptoms, of which most cases exposed within 3 d prior to disease onset, and these cases had been exposed only once and other potential sources of infection could be ruled out, suggesting covid-19 is possibly transmittable during the incubation period, similar to a report in zhejiang [22] . at the same time, the majority of cases involved in the clusters exposed within 3 days prior to onset of the disease in confirmed cases they contacted with, suggesting covid-19, like influenza, is possibly contagious at the end of the incubation period or tracing back to 3 d prior to disease onset in seeking close contact could miss earlier infections in the incubation period. what's more, two clusters caused by asymptomatic carriers indicated covid-19 could be transmitted by asymptomatic infected persons, as is similar to the report in henan [21] . nevertheless, the asymptomatic carriers had only caused a small number of g2 cases, indicating the transmissibility in this population may be limited. the above two populations cannot be effectively identified and thus are of special public health significance for sustained transmission of covid-19 [23] ; due to limited available data, the transmissibility of the disease cannot be analyzed, and studies on its pathogenicity and transmissibility in these populations are awaited. main presentations of confirmed cases involved in the clusters in sichuan included fever, respiratory and systemic symptoms, basically corresponding to the reported clinical characteristics of covid-19 cases, and no characteristic clinical symptom was seen [18, [24] [25] [26] [27] . in clusters, the proportion of ordinary cases and cases with fever decreased with the increase of the case generation, suggesting that the severity of clinical symptoms might gradually lighten with the development of the epidemic. this corresponded to reports that, in the initial period of the epidemic, cases were mainly severe and the case fatality rate was high [18] and, in the later period of the epidemic, cases were mainly mild [26] . this is possibly because the virus will maintain moderate virulence during passage and evolution for the purpose of long-time reproduction in human and the infection was limited to the upper respiratory tract [28] . in addition, it was reported that antibody levels would not last long, and repeated infection was common [29, 30] . however, limited data are available and further studies on covid-19 antibody levels and protective effects are awaited for investigating whether post-infection antibody levels will decline following the mitigation of covid-19 clinical symptoms or whether repeated infection will occur as a result of the short-term maintenance of antibody levels. the findings in the study are subject to several limitations. on the one hand, as only cases exposed only once were included, the cases calculated for the incubation period was limited. on the other hand, pcr detection of respiratory tract specimens was carried out once each time when close contacts were isolated and released, and the isolation period was 14 days. if an asymptomatic carrier recovered spontaneously during the isolation period, the attack rate might be underrated due to the missing count of cases. in conclusion, our study demonstrated the comprehensive epidemic situation of covid-19 in sichuan province and confirmed the infectivity during the incubation period and asymptomatic infection, providing a reference for decision makers to formulate and adjust control measures. abbreviations who: world health organization; covid-19: coronavirus disease; rr: respiratory rate; spo2: pulse oxygen saturation; pao2: arterial partial pressure of oxygen; fio2: fraction of inspiration o2; g1: first generation; g2: second generation; g3: third generation; pcr: polymerase chain reaction funding this study was supported by the science and technology department of sichuan province (grant no. 2020yfs0015). the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. this study was consulted to the ethics committees of sichuan center for disease control and prevention. ethics approval was not available because all data were collected as part of public health emergency surveillance following the laws of the people's republic of china on the prevention and treatment of infectious diseases. we also did not include any data of patients' personal information and therefore ethical approval was not required and waived off written informed consent. not applicable. director-general's opening remarks at the media briefing on covid-19 national health commission (nhc) of the prc, national administration of traditional chinese medicine of the prc. guidance for corona virus disease 2019: prevention, control, diagnosis and management people's medical publishing house special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association. an update on the epidemiological characteristics of novel coronavirus pneumonia (covid-19) analysis on the epidemic factors for the corona virus disease how internet of things big data aid in epidemic surveillance: where did wuhan people go the day before the city "lockdown risk assessment of exported risk of novel coronavirus pneumonia from hubei province chinese center for disease control and prevention. distribution of covid-19 cases notice of general office of the people's government of sichuan province on issuing level i emergency measures against pneumonia associated with the novel coronavirus emergency notice of 3 departments including administration for market regulation of sichuan province on strengthening the supervision of massive dinner gatherings during the covid-19 prevention and control period announcement of sichuan headquarters for emergency response to the covid-19 epidemic sichuan headquarters for emergency response to the covid-19 epidemic. notice on issuing eight measures for strengthening massive dinner gatherings in rural areas during the covid-19 prevention and control period epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts. medrxiv diagnosis and treatment of 697 confirmed cases of coronavirus disease 2019 in hunan province early transmission dynamics in wuhan, china, of novel coronavirus-infected 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molecular virology. 3th ed. chaoyang district: people's medical publishing house medical microbiology. 2th ed. chaoyang district: people's medical publishing house clinical and immunological assessment of asymptomatic sars-cov-2 infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we salute and thank all personnel engaged in the prevention, control, diagnosis and treatment of covid-19 in sichuan province. the authors declare that they have no competing interests. 1 key: cord-119576-8qp8o2g2 authors: xu, liyan; zhang, hongmou; deng, yuqiao; wang, keli; li, fu; lu, qing; yin, jie; di, qian; liu, tao; yin, hang; zhang, zijiao; du, qingyang; yu, hongbin; liu, aihan; jiang, hezhishi; guo, jing; yuan, xiumei; zhang, yun; liu, liu; liu, yu title: cost-effectiveness analysis of antiepidemic policies and global situation assessment of covid-19 date: 2020-04-16 journal: nan doi: nan sha: doc_id: 119576 cord_uid: 8qp8o2g2 with a two-layer contact-dispersion model and data in china, we analyze the cost-effectiveness of three types of antiepidemic measures for covid-19: regular epidemiological control, local social interaction control, and inter-city travel restriction. we find that: 1) intercity travel restriction has minimal or even negative effect compared to the other two at the national level; 2) the time of reaching turning point is independent of the current number of cases, and only related to the enforcement stringency of epidemiological control and social interaction control measures; 3) strong enforcement at the early stage is the only opportunity to maximize both antiepidemic effectiveness and cost-effectiveness; 4) mediocre stringency of social interaction measures is the worst choice. subsequently, we cluster countries/regions into four groups based on their control measures and provide situation assessment and policy suggestions for each group. coronavirus disease 2019 (covid -19) has been recognized as a pandemic by the world health organization in march 2020 (1) . nonetheless, on march 18, mainland china observed their first day with zero increase of local cases since the outbreak (2) . this indicates that, aside from the imported cases, the pandemic has been locally close to the end. many experiences and lessons can be shared by the rest of the world from the trajectories of the outbreak and the control strategies in mainland china. one question that is of particular importance is the costeffectiveness of different antiepidemic measures. drawn from the chinese experiences and lessons, three categories of measures have been implemented: a) "regular" epidemiological control and prevention measures, including identification of infected cases, tracing their close contacts, and quarantines for both; b) in-city activity restrictions, including work-from-home, shutdown of schools and public spaces, cancellation of events, and lock-down of residential neighborhoods; c) inter-city travel restrictions, including temperature screening at all transportation terminals, cancellation of flights and trains, and eventually travel bans from/to certain cities. specifically, (b) and (c) are considered "irregular," which contain the spread of disease in an aggressive manner through the suppression of all possible social interactions. however, these measures lead to enormous economic loss, which may mean higher unemployment rates, and shortage of food, medical services, and other necessities. those chain effects may also endanger the lives of certain social groups. fundamentally this is a trolley dilemma, and the life and health of human beings can hardly be evaluated using monetary values. nevertheless, a comprehensive understanding of the cost of each measure-including the opportunity cost of the shutdown economic activities-and the effectiveness of lifesaving can still help policy makers to compare different antiepidemic strategies in a more operable way. further, with these insights, we can then perform a cross-sectional assessment of the situation of the global antiepidemic campaign regarding the aforementioned measures in different countries/regions, such that typical policies can be clustered and prescriptive policy suggestions can be provided accordingly, which also echoes the argument in (3) . in prior research, scholars have used deductive models to find that the timing of lockdown, including both inter-city travel restrictions and social distancing, significantly changes the size of infected population and the spatial extent of spread (4) (5) (6) (7) . nonetheless, all prior research only focused on specific instances of policies without discussing generalizable impacts of different measures and lacked a cost-effectiveness assessment of each measure (8) (9) (10) . in this article, based on the transmission pattern of covid-19 in china, we build a two-layer contactspread model ( fig. 1) to recover the whole spatio-temporal transmission process, especially the early-stage numbers and distribution of cases at the prefecture level (see sm 1, 2, and 4 ). with this model as a generalizable baseline, we conducted a comprehensive sensitivity analysis for all antiepidemic measures (fig. 2) and concluded the following assessment on the effectiveness of the measures, in terms of number of infected cases, and number of infected cities. 1. containing daily social interaction, parameterized in the model as ! , " for the infected and the exposed populations, is the most effective measure for controlling both the number of infected cases and the spatial extent of the spread. more specifically, we find that a) the controls of ! and " show comparable and substitutable effects in containing the spread of disease, with different elasticity in different stages of the epidemic. controlling " is more effective when the number of cases is small (e.g., fewer than 10 in a city), and ! is more effective when the number of cases is sufficiently large. the implication is that at the early stage of transmission, comprehensive epidemic surveys and contact tracing, alongside with strict quarantine and social distancing, should be used to prioritize the reduction of the social interaction levels of the exposed population ( " ), while after the number of cases has increased to a sufficiently high level, comprehensive testing and identification of all infected cases should be prioritized in order to reduce the social interaction levels of the infected population ( ! ). b) stricter control of social interaction for both ! and " have diminishing returns in reducing the number of cases, but increasing returns in limiting the spatial extent of spread. for example, in one month of simulation, when daily social interaction drops from 100% to 90% of the normal level, the number of infected cases drops by 10%-25% (depending on the stage of epidemic), while the number of cities with infected cases drops only by 0-1%; when daily social interaction drops from 10% to 0, however, the number of cases drops only by 0.05%-0.5% for infected cases versus 2%-35% for cities with infected cases. an exception is at the ending stage of the epidemic, when controlling social interaction has increasing returns in both effects. c) the effects of the epidemiological and social interaction control measures are monotonic for the reduction of infected cases and the spatial extent of spread: the stricter they are enforced, the lower number of infected cases and the narrower spatial extent of spread can be observed. 2. the time of reaching turning point is independent of the current number of infected cases but is only related to the stringency of epidemiological and social interaction control measures, i.e., the relative change of ! , " . when the two parameters are 1/4-1/3 of the normal everyday values, the turning point comes in two weeks and the clearance of cases happens in two to three months; when ! , " are larger than 1/2-2/3 of the normal values, the turning point will never come, i.e., the peak value of case numbers will remain the same as if there are no such measures, but they only delay the time of peak. 3. except at the early stage and the ending stage, inter-city travel restriction has only minimal effect on both the reduction of infected cases and control of disease spread in a city network. overall, compared with in-city epidemiological and social interaction control measures, the contribution of inter-city travel restrictions to the reduction of the number of infected cases and the spatial spread of disease is much smaller-lower by two orders of magnitudes. when the number of cases is sufficiently large, inter-city travel restriction even exacerbates the situation since it limits the social interaction of infected cases, and "condenses" # locally (11) . this finding is consistent with that in prior research (12, 13) . therefore, to national or regional governments who manage a city network, and to international antiepidemic collaboration, travel restriction should only be regarded as an auxiliary measure at the beginning and ending stage of the spread to protect cities which have not been infected at all, or only with a sufficiently small number of cases. in the latter case epidemiological and social interaction control measures should also be implemented simultaneously to get the health care system and other prevention measures prepared. the simulation-based formal analysis above is consistent with the empirical evidence in china. the lockdown of wuhan, and nationwide strict enforcement of epidemiological control and social distancing policies around january 23, including the cancellation of all chinese new near gatherings mark the key move of the antiepidemic campaign. at that time point, all other cities in china were at early stages of the epidemic, which guaranteed the effectiveness of the wuhan travel ban. in addition, with aggressive social interaction control in all cities the turning point of the number of cases arrived in two weeks outside of wuhan. in wuhan, the key move was the functioning of 16 fāngcāng hospitals (mobile cabin hospitals) in early february which enabled citywide comprehensive quarantine of the infected population (14) . this measure reduced the social interaction of infected cases to almost zero, and together with strict social distancing they effectively reversed the trend of spread after two weeks. in terms of inter-city travel restrictions, since they were during the chinese new year and the extended holidays, and overlapped with social distancing measures, the net effect could not be easily isolated empirically. nonetheless, since mid-february the economy had re-opened. by the end of march, the inter-city migration in southern and eastern chinese cities had recovered to the same level as in previous years (15) , but most cities still observed almost zero increase of infected cases. this further supports that the effectiveness of travel restriction is very limited for the cities with small numbers of cases. another evidence to support this point is the 300,000 people who left wuhan right before the lockdown night (16) . this "escaped" population did not significantly change the effectiveness of the national antiepidemic effort. further analysis on the cost-effectiveness of the measures shows more irregularity and non-linearity, leading to more nuanced relationships (detailed in sm 5). here we summarize the most critical general patterns as follows: 1. the measures which can achieve both high antiepidemic effectiveness (low number of cases and narrow spatial spread) and high cost-effectiveness (smaller loss of economic outputs) only exist at the early stage of transmission. at the early stage, if epidemiological and social interaction control measures can be strictly enforced (sufficiently low ! and " ), it is possible to keep the spread at a low level, with a loss of economic outputs only up to 4%. the intuition is as follows: based on the assumptions of this article, the early-stage measures only include comprehensive testing, close contact tracing, and quarantine, but do not include indiscriminate restrictions of in-city social interaction and inter-city travel, which incurs high costs. the policy implication is straightforward: for early-stage cities and regions, it is critical to practice epidemiological control interventions, but not to necessarily mobilize the whole society into social interaction reduction. this finding is consistent with the suggestions in (6). 2. except for the early stage, it is impossible to simultaneously achieve both high antiepidemic effectiveness and high cost-effectiveness. except for a few "plateaus," the effectiveness of epidemiological and social interaction control measures monotonically increases with the stringency of control measures. however, the cost and cost-effectiveness functions are non-monotonic and there usually exists more than one peak (see details in sm 5), which in most cases do not coincide with the effectiveness peak. typically, the costs are the lowest when the control measures are at sufficiently low or sufficiently high levels. while the latter case has been explained in the last point, the sufficiently low control measure scenario basically leaves the whole population to be infected. therefore, the tradeoff between sufficiently low and sufficiently high levels of control measures depend on many technological factors, including the short-term and long-term capacity of healthcare systems, long-term uncertainty of virus mutation, and development of vaccines, as well as many non-technological factors, including the risk averse attitudes for the short term and the long term, the mental discounting between short-term and long-term tradeoffs, and the fundamental value judgement on the "value of lives," the discussion of which are beyond the scope of this article, and will be left for discussion at the end of this article. 3. lastly, although it is difficult to choose the optimal control strategy, the worst choice is explicit: mediocre control of social interaction, e.g., social distancing with leakage. this choice still incurs 20-60% loss of economic outputs, but only achieves 30-40% reduction in the number of cases, an extent which is insufficient to overturn the epidemic curve. except for moderately delaying the spread of disease which may be taken advantage of to get the healthcare system prepared, this strategy is the worst choice in all other dimensions. with the formal results above, we can now perform a cross-sectional assessment of the global situation of the antiepidemic campaign from a transmission-prevention policy perspective. among the three types of measures (epidemiological control measures, social distancing, and travel restriction), we disregard the travel restriction measure as our results clearly show that it is ineffective for most countries/regions under the current situation (we will discuss the exceptions later). rather, we use two datasets (17, 18) which codified the antiepidemic measures chosen by countries/regions as of april 7 (due to the lack of testing data, we use wuhan as a proxy for mainland china), and for the countries/regions analyze the relationship of their two stringency indices, $ and % , i.e., the activity levels of the infected and exposed populations, and the respective effectiveness on the reduction of infected cases. based on the stringency of the two dimensions of antiepidemic measures, we can divide all countries/regions into three groups (fig. 3) , each with a different antiepidemic "strategy": elimination, control, and delay. more than 100 countries/regions are not included because of the lack of data. we will also discuss the implications of this fact. 1. the "elimination" group: this group (up right corner of fig. 3 ) consists of only a few countries/regions, including mainland china (represented by wuhan), hong kong sar, vietnam, uae, bahrain, etc., all with # ≪ 1, such that the epidemic could be expected to dwarf within a reasonably short time period. mainland china is the most prominent example of this group, where aggressive measures have been taken on both dimensions to reduce the activity of the infected population as well as the exposed population. the measures include effective epidemiological control interventions, such as comprehensive testing and close contact tracing, and also aggressive social distancing measures, such as shutdown of schools, workplaces, and public transport, cancellation of events, and mass disease control education. these measures incur 40%-90% loss of economic outcome in a month, and the loss accumulates as the epidemic is not completely "eliminated". obviously, the underlying value judgment of the elimination strategy is an overwhelmingly high weight on health and lives over any cost-control or costeffectiveness reckoning. although the treasuring for lives is always respectable, long-lasting economic tightening also constitutes a threat to society, especially to the disadvantaged social groups. due to the existence of asymptomatic carriers, false-negative test results, and international imports of cases, a complete elimination of the epidemic is extremely difficult. thus, if the aim is to literally eliminate all cases, the economic losses are highly likely to accumulate to an unbearable level. therefore, we suggest that countries/regions which have followed the elimination strategy consider turning to the "control" strategy (elaborated below) to avoid excess economic losses on the condition that the active number of infected cases has been reduced to a sufficiently low level. we also suggest that these countries/regions keep the travel restriction measures-the most effective measure at this stage of the epidemic indicated by our simulation results. 2. the "control" group: this group includes south korea, singapore, qatar, norway, slovenia, russia, and new zealand, etc., all with # < 1, but still not sufficiently small, such that the epidemic can be reduced to a lower level (but not eliminated), depending on the stringency of intervention measures. the singapore in february was the most prominent example within this group, where antiepidemic measures have been mild enough not to affect everyday life by aggressive social distancing. through regular epidemiological control practices, they were managed to maintain a daily increase of infected cases fewer than 10, and only suffered 0.5%-4% loss of economic outcome in a month. the control strategy requires a highly capable epidemic control system. given the aforementioned long-term uncertainties, even with such a capable system, the strategy is still a tightrope-walking game with the risk of abrupt system overload by accidentally untracked surges of infection, which, unfortunately, appears to be the case in singapore in early april. under such circumstances, a timely turn to the "elimination" strategy may be necessary. 3. the "delay" group: all other countries/regions in fig. 3 belong to the third group, which appears to follow the "delay" strategy, with # > 1, such that the epidemic will continue to grow. this is often referred to as the "flatten the curve" strategy, which aims not to reduce the epidemic to an as-low-as-possible level within a short period of time, but only to delay its growth through mediocre epidemiological control and social distancing measures. our results show that this is usually the worst scenario in terms of cost-effectiveness. a country/region may opt to this strategy because their tradeoff between short-term certainty (economic loss avoidance) and longterm uncertainty (possible disappearance of the epidemic in the summer, development of vaccines, etc.) leans towards the former. unless they have strong evidence to justify the tradeoff, we strongly suggest they reconsider. moreover, our results show possible directions to improve-enhancing the social interaction control for the infected population through more comprehensive testing or enhancing the social interaction for the exposed population through stricter social distancing measures, whichever sees fit based on the location of the country/region on fig. 3 . rest of the world: more than 100 countries/regions do not appear in fig. 3 due to the lack of data, most of which are third-world countries/regions. although little information is available to us about the situations in these places, we conjecture that they may at this moment be pursuing cost-effectiveness of their antiepidemic interventions because of their limited availability of resources, which we call the "worth every penny" strategy. as our results show that the most cost-effective measures are usually neither the most effective one (actually they are usually very ineffective), nor the least costive ones, the "worth every penny" strategy is not a good option either. if a country/region opts to this scenario solely because of the lack of resources, it should be viewed as a humanitarian disaster, and we call for international aid in this situation. at the end, we acknowledge the extreme difficulty of even trying to lay out the comparison between human lives and economic activities, or the tradeoffs of lives between different social groups. we believe that the ethical discussion should be open to the whole society and hope that this article can contribute to the discussion. (19) and a network transmission layer based on inter-city migration. through inter-city travel, the numbers of exposed and infected populations are adjusted daily. the model is calibrated using the migration data and the number of reported cases in china. see materials and methods for model specifications. (a) gradient of the first effectiveness function (with the number of infected cases as the metric for effectiveness) at the peak/inflecting stage, displaying decreasing margins, as well as the reverse of the effect of the inter-city travel level as ! and " decreases. (b) gradient of the second effectiveness function (with the number of cities with infected cases as the metric for effectiveness) at the accelerating stage, displaying firstly increasing and then decreasing margins as ! and " decreases. (c) gradient of the second effectiveness function (with the number of cities with infected cases as the metric for effectiveness) at the ending stage, displaying monotonically decreasing margins as each dot represents a country/region. the sizes of the dots indicate the number of infected cases on april 7, 2020. classical seir model characterizes the dynamics of the susceptible, exposed, infected, and removed population, assuming a certain population size and transmission rate. in closer scrutiny, the fixed transmission rate assumption requires contact-based transmission process (19) . from the perspective of spatial interaction, this requirement is contradictory to the prior assumption of fixed population. due to the high mobility of modern society, fixed population requires a large spatial scale of the model, e.g., the national scale. moreover, contact-based assumption is valid only at the scale of human daily activities-such scale should not exceed the urban scale to incorporate multiday intercity travels. at a larger spatial scale, human activity is featured by the "return-explorer" dichotomy (20) . the former spreads outbreaks through daily contact, while the latter "diffuses" the epidemic through long-distance travels. the order of magnitude of the intercity long-distance migration can be massive in china, and related studies have also discussed the effect of this population flow on the spread of covid-19 outbreak (8, 9, 21, 22) . thus, the covid-19 outbreak is actually subject to a "contact-spread" two-step transmission process. therefore, the classic seir model is insufficient to accurately describe the epidemic dynamics in the urban network and needs to be expanded. thereupon, we constructed a series of seir models for all cities, coupled with an intercity network. the node-edge structure of the model corresponds to the above-mentioned contactspread process; and the required data (epidemic parameters of each city, and intercity spatial interaction) are relatively simple. here, the choice of appropriate spatial resolution is critical. on the one hand, while prior research have adopted similar model design, they conducted the analysis mostly at the provincial resolution (5, 23, 24) . according to the analysis above, the provincial level is not the best choice to reflect the intercity-spread mechanism. on the other hand, at too fine spatial scales, such as 1 km-grid, township, or census blocks, modeling results can hardly be verified due to the lack of data, and random errors can be large. attempting to make a compromise between simplicity and completeness, interpretability and prediction accuracy, data spatial accuracy and verifiability (25) (26) (27) , we took cities (administratively called prefectures in china) as the unit of analysis and built a model which we call the spatial-temporal explicit seir model (stex-seir). this model can not only be used to analyze the number of infected cases, spatial range of infection, and dynamics of the covid-19 outbreak, but can also be used to reveal the arrival time of the first case in each city. we calibrate the model with chinese case statistics. although it does not cover the entire globe, it is still informative to other countries since china has gone through the whole process of the epidemic. the stex-seir model relies on the following assumptions: 1) intra-city population is homogeneous, i.e., it is a "meta-population," which is a common practice in the absence of real demographic data; 2) omitting the impact of foreign imports and exports of covid-19 cases: the proportions of imported and exported cases during the covid-19 outbreak in china are extremely small and can be omitted over the study period. 3) virus remains unmuted: although a very large number of mutations have been recorded (28) , the transmission patterns of the virus have not noticeably changed so far. first step: update the immigrated and emigrated populations, the exposed population, and the infected population (assuming that the inputs and outputs of n, e, and i across cities are completed instantly at the beginning of time t). • ,-./01 : lockdown on january 23; • 0-*(2-. : measures taken on january 20; human-to-human transmission was confirmed on that day. : the proportion of the susceptible turning into the exposed status. second step: seir model in each city: in the above equations: s (unimmune and susceptible population) may turn into infected by contacting infected individuals; e (exposed) is the population who are in the incubation period after being infected; i (infected) is the population being symptomatic and infectious; r (removed) is the population who died or have been cured-they can neither infect others nor be infected again. n: total population in the city, subject to = + + + . other notations: • " : probability that a susceptible is infected and becomes an exposed person; • ! : probability that a susceptible is infected by an exposed person; • : probability that an exposed person turns into infectious status; • : probability of being cured; • : probability that an infected person dies; • " : probability that an exposed person is infectious; • ! : the number of susceptible persons who an infectious individual contact during each step of simulation; • " : the number of susceptible persons who an exposed individual contact during every step of simulation. the case data used in this article come from the national and provincial health commissions in china (29) . wuhan is the city hit hardest by the epidemic, accounted for 61.87% of the total number of cases. the number of cases in hubei province other than wuhan accounted for another 22.03%. therefore, wuhan and hubei province have very different medical conditions, prevention and control measures from other cities and provinces in china. thus, we assigned different initial parameters for wuhan, other cities in hubei, and cities in other provinces. the initial number of cases in wuhan was set to be 1, and the initial numbers in other cities in hubei and in other provinces was set to be 0. according to the national report, the first confirmed infected case in wuhan can be traced back to december 8, 2019. thus, we chose december 8, 2019 as the starting point for the simulation. according to other studies (30) , this date is as close as possible to the real starting point of the pandemic, and epidemiological data are only available from this point to calibrate model. the endpoint of model calibration is february 26, when the epidemic in china was almost over and the model has converged by that time. we will simulate the spread of the covid-19 epidemic from that date until the end of april as verification and prediction (by the time of writing this article). we used chinese national, provincial, and municipal reports as the baseline to calibrate our model, so that the fitted number of cases would match with the surveyed data. on the one hand, the assumption, based on case statistics, is reliable. reports are released separately by more than 300 cities nationwide and updated daily. panel data from different provinces are inter-validated with other and are also supported by data from other sources (e.g., total mortality rate of the population, or mortality data of similar symptoms such as influenza, pneumonia, etc.). thus, it is extremely unlikely that the data are fabricated continuously without being noticed. on the other hand, research shows that due to delayed diagnosis at the early stage of outbreak, the actual onset date of most confirmed cases is actually one week ahead of the report date (30, 31) . considering the incubation period, we pushed the outbreak curve from the reported dates backwards by a few days, thus yielding the baseline for model calibration. the calculation and initial values of , , 3 , 4 , , , , " , ! , " , and ∆ were articulated in section 4 of the supplementary material. among the three basic dimensions of epidemic control (control of the source of infection, cutting off transmission, and protection of the susceptible), we focus on the transmission cuttingoff dimension. specific measures belonging to this dimension can be further divided into three categories: 1) "regular" epidemiological control measures; 2) in-city social interaction control and 3) intercity travel restrictions. these measures can be directly mapped to the three key parameters ! , " , and ∆ (denoted as tl henceforth) in the stex-seir model (table s1) , and thus empower the quantitatively evaluation of the effectiveness and cost-effectiveness of various scenarios. regular epidemiological control measures include thorough epidemiological surveys, close contact tracing, and quarantines of all the infected individuals and their close contacts as early and as possible-especially the close contacts, who may be potential carriers. these measures are reflected in the model by tuning the values of ! and " . it is noteworthy that the limits of the effectiveness of regular epidemiological control measures: • it is very difficult for the close-contact tracing and quarantines to be exhaustive, so they cannot reduce " to 0, indicating an effectiveness "roof"; • close contact tracing is a tedious work and is heavily manpower dependent. given that the epidemiological control system in regular practice usually has limited capacity. under outbreaks the system could be overloaded and becomes dysfunctional. under these circumstances, more aggressive measures are needed to reduce " . in-city social interaction control measures include work-from-home, shutdown of schools, workplaces, public transit, and public spaces, cancelation of public events, lockdown of residential neighborhood, etc. the effects of these measures are to reduce the chance of close contact between the infected/exposed and susceptible individuals, and are represented in the model by the values of ! and " , especially " , with contributions of different measures aggregated to a final value. we collected the measures adopted in different prior papers and coded them to estimate the final values of ! and " . it needs to be noted that the effects of specific measures are clearly overlapping with each other. for example, when social distancing both at the work and home ends are effectively implemented, the marginal "contribution" of an additional control on public transit usage would be minimal. also, despite of the nominal public orders, the actual stringency of the measures could be flexible in practice. for simplicity, we utilize expert knowledge to estimate the overall efforts, i.e., the values of ! and " . intercity travel restriction includes suspending inter-provincial and intercity buses, trains and flights, closing highways and roads, etc. the purpose is to reduce the intensity of personnel exchanges across cities, thereby reducing the transmission of the infected/exposed individuals. this is represented in the model by the reduction of spatial interaction parameter tl, or ∆ " and ∆ ! . in this paper we use two indicators to evaluate the effectiveness of disease control measures: the number of infected cases, and the spatial extent (represented by the number of cities with infected cases). minimizing the total infected population is a self-explanatory goal from the perspective of epidemic control. since e will eventually be converted to i with a fixed probability, we only need to focus on the infected population i and use the relative change of i over the simulation period (one month) as one indicator of effectiveness. the total antiepidemic effect in all cities is the summation: aside from the total number of infected cases, the spatial extent of the epidemic is also an indicator worth noticing. if the epidemic can be contained within a narrow spatial extent, the medical resources in other cities can be diverted to the infected cities to help, which actually happened in china where medical teams across the country were sent to wuhan for support. we also use the relative change of the number of cities with infected cases during the simulation period (one month) to quantify the effect: the total effect in all cities is the summation: we used the opportunity cost of economic output as the indicator for the cost of the antiepidemic measures. for simplicity, we only used the work-hour loss as a proxy for the economic output loss and did not convert it to monetary values of goods and services. travel restriction leads to reduction in the total number of personnel exchanges between cities. in particular, the outbreak of covid-19 in china overlapped with the spring festival holidays, and travel restriction apparently caused the migrant workers who previously travelled back hometown not be able to return to cities where they had worked after the holidays. while the impact on economic output is affected by factors such as the productivity difference between the migrant workers and local workers, we can use wages as a proxy for productivity and estimate the differences (32) (33) (34) . calculations show that the average income of migrant workers in 2017 was 100.36% of that of the local workers, or roughly the same, so we do not need to adjust for the differences in the model 1 . therefore, this study ignores the role of the above factors. factors such as employment elasticity, which are more difficult to estimate, are also omitted for simplicity. therefore, the loss of economic output of city i at time t from travel restriction is: where _normal is the urban population in the normal status, approximated by the number of urban populations in the same period last year, and " _normal is the " in the normal status. for those who are not affected by the intercity travel restriction, we argue that the indiscriminately implemented in-city social interaction control measures reduce their intensity of social interaction, which in turn reduces their productivity and thus reduces the economic output. more specifically, we used " and ! as the proxy for productivity. it should be noted that lower " or ! is not necessarily the result of indiscriminate suppression of daily interactions but could also be the result of regular epidemiological control measures. a distinction needs to be made between these two situations. it can be reasonably assumed that when the number of the infected cases is small. for example, the daily increase of the number of infected cases does not exceed a certain threshold, the goal of reducing ! and " can be achieved solely by regular epidemiological control measures. therefore, a rational administration will choose not to opt to universal in-city social interaction control measures to avoid collateral loss. what is lost in this case is the output of only those who have been quarantined, i.e., the exposed and infected cases. we admit that it is extremely difficult to estimate such a threshold, which could be highly dependent on specific contexts such as governance capability, the resourcefulness of the epidemic control system, and cultural and geographic factors. although, a numerical estimation would still be of help. there was no opportunity to observe this threshold during the outbreak in china: in the early stage of the epidemic (before january 23, 2020), no effective epidemic control practices were taken outside of wuhan, and after january 23, the country had generally turned to aggressive and universal antiepidemic measures and it is difficult to separate the net effect of the each epidemic control measure. however, the singapore situation in january and february may constitute a good example, where in most cases daily reported new infected cases were under 10. another remark is that the chinese government sets a threshold of 3 cases for reporting a newly discovered epidemic to the national cdc. in light of these numerical example, we use 10 daily new infected cases as an estimate of the capacity threshold value. therefore, the loss of economic output of city i at time t from in-city social interaction control measures is set as follows. for straightforwardness, we convert the economic output loss into to a relative value: the proportion of the output with regard to that of the baseline (the normal situation). the total output loss ratio of city i during the entire simulation period is and the ratio of total output loss of all cities during the entire simulation period is: in summary, the comprehensive cost-effectiveness function of antiepidemic measures is: particularly, if cost = 0, it is stipulated that both cost/effectiveness ! , cost/effectiveness 6 equal to 0. for simplicity, we only calculated the cost and cost-effectiveness functions at the national level (the entire city network). to discover any initial value-dependence of the cost and the costeffectiveness functions, we ran the simulation at different stages of the epidemic. based on the calibrated model, five scenarios with the following starting and ending dates are set: the model is run for 30 days for each scenario, and five indicators are calculated: number of total infected cases, number of cities with infected cases, overall cost, effectiveness/cost ratio with respect to the number of infected cases, and effectiveness/cost ratio with respect to the number of cities with infected cases. we calculated the gradient function of the five indicators with respect to " , ! , and tl to analyze the structure of the solution space. for simplicity, we calculate the gradients discretely at the following points: ! = {0, 1, 2, 3, 4, 5, 6, 7, 8, 9}; " = {0, 2, 4, 6, 8, 10, 12, 14, 16}; tl = {1, 2, 3, 4, 5}, where the five levels of tl correspond to 0.2, 0.4, 0.6, 0.8, and 1 times of the normal intercity travel level. note that the lowest level of tl is not 0. this is because according to the situation in china (given by baidu migration data), when the tl is the lowest, the travel level is still about 20% of the normal level, and we believe that the stringency of intercity travel restriction in reality cannot be greater than this level. in contrast, aggressive in-city social interaction control measures can reduce the values of " and ! to levels very close to 0. we evaluated the antiepidemic situation of countries/regions based on the stringency of activity control policies of the infected and exposed, i.e., the degree of which ! and " are reduced. this takes 3 steps: 1) codifying policies and computation of the stringency indices; 2) mapping of the stringency indices into values of ! and " ; and 3) assessment of the antiepidemic situation based on the results of our analysis. for the exposed, effective activity control measures include close-contact tracing and social distancing. for the computation of the stringency of these measures, we used the data and method provided by the oxford covid-19 government response tracker (17) , and computed and compiled the exposed activity control stringency index with six indicators: school shutdown, workplace shutdown, public events cancelation, public transport shutdown, public information campaign, and close-contact tracing. for the infected population, quarantine is the most important measure to reduce their activities, which but also requires testing in the first place. we therefore used the comprehensiveness of testing as the proxy for the infected activity control stringency. as testing policy coding from the aforementioned source is rather coarse, we instead designed an alternative index based on two indicators: tests per 1 million population, and the ratio between cumulative cases and the number of total tests. the compiled index is gained through dividing the first indicator with the second, logarithmically transforming the quotient, and finally normalizing the result to the 0-100 range to match that of the exposed population's activity control stringency index. the logarithmic transformation is used only for visualization purposes, as very few countries/regions have much higher original indices. for the testing data, we employed sources from www.worldometers.info/coronavirus/. it should be noted that as mainland china does not publish testing data, we used the data from wuhan as a proxy. for both indices, we use the data as of april 7. a total of 99 countries/regions are present in the final dataset. analysis results show that there exist two threshold values for both ! and " that correspond to the "elimination" (r 0 << 1) and "control" (r 0 < 1) strategies, as shown in table s2 . it should be noted that the effects of the two indicators are substitutable, with the elasticity of ! to be larger. for the mapping from ! to the respective stringency index of a specific measure, it is clear that the mapping should not be linear because of the non-linear transformations we have done. we therefore employed an estimation method based on expert knowledge: to achieve 2/3 reduction of ! which is required to "eliminate" the epidemic, it is needed to quarantine as many infected cases as possible. considering that about 1/3 to 1/2 infected cases are asymptomatic (35) , this roughly means that all symptomatic cases should be tested. based on the statistics from wuhan and south korea, this requires the tests per 1 million population to be greater than 10,000, and the ratio between the number of cumulative cases and total tests to be smaller than 5%. converted from the above estimation to the respective stringency index, the value is around 70. we thus used this value as the boundary between "elimination" and "control". following similar methods, we estimated that the boundary between the "control" and "delay" policy groups to be around 60. for the mapping from " to the respective stringency index, because all six indicators' contribution to the actual stringency level are roughly equally weighted, we simply conducted a linear mapping between the composite stringency index and " . thus, the stringency index boundary between the "elimination" and "control" groups is 75, and that between "control" and "delay" is 50. epidemic parameter estimation is the first and fundamental step in the seir model. in previous studies, choices of these parameters usually adopted mathematical interpretation, such as the inverse of the incubation period or infectious period (7, 23) and thus the numbers are not physically interpretable. however, if we consider the actual characteristics of covid-19 as shown in epidemiological studies, the ambiguity of the model can be greatly reduced. we estimated these parameters based on real-world data, considering errors caused by possible concealment, omission, and late reports. we used close contact tracing data after january 28 to estimate ! , since statistical data in the early stage showed fluctuation due to the lag of reactions from the government. in the equation following, ( ) indicates the number of newly confirmed cases in a day. and ( ) indicates the number of close contacts who are still under medical observation in the same day. we averaged ! ( ) with regard to t and got 0.01986, so we set ! to 2%. in addition, we considered covid-19 to be infectious in the later stage of the incubation period: there is no evidence of weaker infectivity during the incubation period, and it is assumed that it can be as infectious as during the infected period, indicating " = ! = 2%. the incubation period is generally 2-14 days, and 3-7 days in most cases. the median incubation period is 4 days. assuming infectivity in the 1-3 days before the end of the incubation period, with an average of 2 days, the ratio of infectious exposed people ( " ) can be calculated as follows d is the average infectious day of the exposed, and e is the median of the incubation period. according to the new coronavirus pneumonia prevention and control program (second edition), suspected cases are those with similar clinical symptoms as infected people. if a suspected case tests positive for nucleic acid test or its viral gene sequencing is highly homologous with sars-cov-2, it is diagnosed as a confirmed case. considering the relationship between suspected and confirmed cases, the ratio between new confirmed cases to existing suspected cases is recorded as the ratio of the exposed cases becoming infected cases. this index is a characteristic of the disease itself and is generally the inverse of the incubation period from the mathematical interpretation. therefore, it should be uniform for all cities. this parameter has converged since january 28 and the average value is 0.1404 (14.04%). because of the presence of asymptomatic infections, only considering suspected cases can lead to overestimation. since asymptomatic infections accounted for 1.2% of confirmed cases, we scaled up the suspected cases and the revised to be 13.87%. according to the epidemiology working group of the chinese center for disease control, at least 104 cases existed in hubei province in december 2019, spread in fourteen counties. by january 10, 2020, there were 757 cases and 102 deaths in 113 cities of 20 provinces. however, in the national epidemic reporting system, the first death was not reported until january 20 in the whole country except wuhan. therefore, we took the results (table s3 ) published by the epidemiology working group and calculated the mortality rates in different regions at each stage (table s4) . we fitted the mortality rates with the following functions: wuhan: recovery rate is closely related to the capacity of local medical resources and city governments' responsiveness, so we conducted parameter fitting for each city. it is reasonable to believe that the number of cured cases published is reliable since there is no obvious reason for concealing recovery. therefore, the recovery rate is calculated as the ratio between newly cured cases and existed confirmed cases. the recovery rate has changed significantly by time. taking wuhan as an example, the abrupt outbreak led to a lack of medical resources at the beginning. then, aid resources from all over the world were sent to hubei province. in addition, huǒshénshān and léishénshān hospitals were built quickly to treat patients in severe conditions. these efforts contributed to the improvement of recovery rates (7) . to capture the time-wise changes, we fitted piecewise functions to the recovery rates based on the characteristics of each stage. among the different stages, the early recovery rate in wuhan fluctuated too heavily to fit, so we took the average value in the period as an indicator of the recovery rate. the specific fitted functions are as follows: mainland china except hubei province: close contact tracing is a common epidemiological control measure, especially when early treatment is not clear, and when vaccines are not available. this approach can identify potentially infected individuals, quickly isolate them before they turn to severe patients, and prevent the occurrence of secondary transmission (36) . social interaction intensities of the exposed and infected individuals are denoted as " and ! respectively in our model. these two parameters are highly related to local population densities. considering that the covid-19 outbreak occurred during the chinese new year, the intensity of social interaction was significantly higher than usual. wuhan, as china's large transportation hub, may have a higher per capita social interaction intensity than other cities. to address this, we used the contact rate of the infected population during sars (maximumly 12) as the initial value of ! (37). in general, the behavior of the exposed population is less restricted than the infected, so we assume that the maximum contact rate of the exposed is twice as high as that of the infected, which means that the maximum " is 24. according to the local epidemic reports, the initial values of ! and " in each area can be obtained (table s5) . then, based on the local control policies and response time, we adjusted the contact values at different stages. in january 20, 2020, zhong nanshan announced that there was a risk of human-to-human transmission of covid-19, and some people in wuhan began to consciously reduce their activities. until the citywide lockdown, the number of cases surged, and people began to realize the severity of the situation. then, strategies such as business shutdown, school shutdown, stay-at-home notices, and community shutdown were gradually implemented throughout the country to avoid contact between the susceptible and the infected population. in the late stage of the epidemic, numerous prevention and control strategies, including hospitalizing all confirmed cases and isolating all suspected cases, have greatly reduced the values of ! and " (figs.1 and 2 ). the initial value of each city's population in our model is that on december 8. then, the population changed after the new year migration, which can be calculated through the spatial interaction matrix. our research includes 368 prefectures in mainland china, with the time span from december 1, 2019 to april 30, 2020. the data collected included migration data from the baidu migration map (15), statistical yearbooks, and local covid-19 reports. the original baidu migration data is consisted of migration flows of 91,788 city pairs among 368 cities from january 1, 2020 to february 26, 2020. since there was no news of the covid-19 outbreak in december, the intensity of social activities was not affected and remained the same as normal workdays. in march and april after the outbreak, we assumed that due to the impact of the travel restrictions in most cities, residents' travel remained at a very low level. therefore, the migration data for december was set to be the same as an average workweek from january 6, 2020 to january 12, 2020, and the intercity migration after february 26 was set to be the same as between february 17 to february 23. in addition, the numbers of covid-19 cases in our model and population data for the 368 cities were collected from the national bureau of statistics of china (38) and the national health commission of china's reports. we collected the publicly announced epidemic control policies of all prefectures in china from january 23 to february 20. this dataset includes about 4,000 documents, among which 364 were issued at the provincial level. we codified the policies into 6 categories: one for longdistance travel restrictions, and the other five for in-city social interaction control measures (table s6) . we further translated the policies into their control stringency in terms of social interaction reduction based on expert evaluation on the policy terms as well as enforcement level inferred from media coverage. we lastly mapped the codified policies to the values of ! and " (figs. 1 and 2) . in the baseline (real-world) model, the initial number of cases at this stage was 53 and increased to 7,780 after 30 days. the initial number of infected cities were 23 and increased to 301 after 30 days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is 9 for ! and 16 for " ), and that of ! higher when the level of social interactions is low (as low as 0). however, the marginal contribution of ! and " are both 2-5 times higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ 3 and " ≤ 4, the number of infected cases will shrink, and will be reduced to 0 after approximately two months (table s7 ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high, and that of ! slightly higher when the level of social interactions is low. however, the marginal contribution of ! and " are both 1-2 times higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ 3 and " ≤ 4, the number of cities with infected cases will shrink, and will be reduced to 0 after approximately two months. since there was no baseline control at this stage, any change in the three coefficients means tighter control on social interactions, and hence a cost to economic output. first, the marginal contribution from the reduction of tl to the cost function is approximately 2-5 orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = 9, " = 12) and ( ! = 0, " = 16). the global gradient between the ridge and ( ! = 0, " = 0) is moderate, and a lowest plateau appears when ! ≤ 6 where the value of the cost function is 0. on the other side, the global gradient is much steeper, with a minimal value of the cost function that is also 0 at the corner. the global cost peak appears at ( ! = 9, " = 12), at which point the peak cost slightly exceeds 4% of the total output. in terms of cost-effectiveness, as the peaks and trends of the cost function and two effect functions are different, the two cost-effectiveness functions show greater non-linearity. in terms of the cost-effectiveness with respect to the reduction of the number of infected cases, two local peaks exist. one is at the "strictest control" point i.e., ( ! = 0, " = 0), when each 0.3% reduction in the number of infected cases is associated with 1% of economic output cost within a month; another peak occurs at the "non-control" point i.e., ( ! = 9, " = 16), when each 1.7% reduction in the number of infected cases is associated with 1% of economic output cost within a month. however, in the latter case, there will be no reduction in the number of infected cases at all according to the respective effectiveness function, while the former case coincides with the effectiveness peak. in terms of the cost-effectiveness with respect to the reduction of the number of cities with infected cases, situations are similar, only that a third peak appears at ( ! = 0, " = 16), which is also a hardly effective solution. overall, the best solution considering both effectiveness and cost-effectiveness in whichever terms at this stage of epidemic is at the "strictest control" point (fig. s3 ). stage: january 16, 2020 to february 15, 2020 5. in the baseline (real-world) model, the initial number of cases at this stage was 1,059 and increased to 8,920 after 30 days. the initial number of infected cities were 178 and increased to 310 after 30 days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is 9 for ! and 16 for " ), and that of ! higher when the level of social interactions is low (as low as 0). however, the marginal contribution of ! and " are both up to two orders of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ 3 and " ≤ 4, the number of infected cases will shrink, and will be reduced to 0 after approximately two months (table s8 ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the patterns of marginal contribution of the three coefficients are similar with the "reduction of infected cases" case and we do not elaborate here. first, the marginal contribution from the reduction of tl to the cost function is approximately 2-4 orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = 9, " = 4) and ( ! = 0, " = 16). the global gradient between the ridge and ( ! = 0, " = 0) is moderate and roughly linear, and a lowest plateau appears when ! ≤ 3 and " ≤ 4 (the "strictest control" scenario) where the cost is a small number (2%-4%). on the other side, the global gradient is much steeper with increasing margins, with a minimal value of the cost close to 0 at the corner (the "non-control scenario). one noteworthy character of the cost function is that the value of ! does not affect the position of the peak, but affects its value: keeping " the same, each 10% reduction in ! reduces the cost function by 2%-4%. the global cost peak appears at ( ! = 9, " = 4), or a "loose control" scenario, at which point the peak cost exceeds 40% of the total output. the intuition is that in this case, the control on social interactions is not sufficient to reverse the trend curve of the epidemic, and after the number of infected cases exceeding a certain threshold (the capacity "roof" of regular epidemiological measures to contain the epidemic), cities have to opt to a universal control on everyday social interactions to contain the epidemic, inflicting heavy loss on economic output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s4 . the baseline model witnessed the turning point of the epidemic, when the initial number of cases at this stage was 10,345 and reduced to 1,913 after 30 days. the initial number of infected cities were 314 and reduced to 253 after 30 days. in terms of the reduction of infected cases, the decreases of ! and " both have positive effects, but the effects are non-linear-with all returns diminishing marginally. the decrease of tl, however, have varying directions of effect dependent on the values of ! and " , with negative effect when both ! and " values are high and positive otherwise. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is 9 for ! and 16 for " ), and that of ! one order of magnitude higher when the level of social interactions is low (as low as 0). however, the marginal contribution of ! and " are both up to two orders of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ 3 and " ≤ 6, the number of infected cases will shrink, and will be reduced to 0 after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve. and at the non-control scenario, the number of infected cases will grow to approximately 200,000, or 20 times the number at the beginning of the baseline scenario (table s9 ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, but with increasing returns marginally. the patterns of marginal contribution of the three coefficients are similar with the "reduction of infected cases" case and we do not elaborate here. first, the marginal contribution from the reduction of tl to the cost function is approximately 2-4 orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = 9, " = 0) and ( ! = 2, " = 16). the global gradient between the ridge and ( ! = 0, " = 0) is moderate, and the lowest cost at the corner (the "strictest control" scenario) is a small number (2%-4%). on the other side, the global gradient is much steeper, with a minimal value of the cost function close to 0 at the corner (the "non-control scenario). again, the value of ! does not affect the position of the peak but affects its value: keeping " the same, each 10% reduction in ! reduces the cost function by 8%-9%. the global cost peak appears at ( ! = 9, " = 0), or a "loose control" scenario, at which point the peak cost exceeds 90% of the total output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s5 . in the baseline model, the initial number of cases at this stage was 3,886 and reduced to 22 after 30 days. the initial number of infected cities were 288 and reduced to 1 (wuhan) after 30 days. in terms of the reduction of infected cases, the decreases of ! and " both have positive effects, but the effects are non-linear-with all returns diminishing marginally. the decrease of tl, however, have varying directions of effect dependent on the values of ! and " , with negative effect when both ! and " values are high and positive otherwise. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " slightly higher when the level of social interactions is high (i.e., the values of ! and " are high, or close to the ordinary level, which is 9 for ! and 16 for " ), and that of ! 4 times higher when the level of social interactions is low (as low as 0). however, the marginal contribution of ! and " are both up to 1 order of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when ! ≤ 9 and " ≤ 10, or ! ≤ 6 and " ≤ 12, the number of infected cases will shrink, and will be reduced to 0 after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve. and at the non-control scenario, the number of infected cases will grow to approximately 10,000, or three times the number at the beginning of the baseline scenario (table s10 ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, with increasing returns marginally at most times except when the values of ! and " are both very low-a noteworthy character that is different with the symmetric accelerating stage. another noteworthy character of the effectiveness function is the occurrence of plateaus when ! ≤ 9, where the change of " and tl will not affect the spatial scope of the epidemic. first, the marginal contribution from the reduction of tl to the cost function is approximately 2-4 orders of magnitude lower than that of ! and " such that its impact is mostly negligible. second, the cost function has a ridge along the direction of ( ! = 9, " = 10) and ( ! = 0, " = 10). the global gradient at both sides of the ridge drops close to 0 with increasing margins and plateaus when ! is sufficiently low or high. again, the value of ! does not affect the position of the peak, but affects its value: keeping " the same, each 10% reduction in ! reduces the cost function by 5%-8%, and when ! < 6, a plateau occurs where cost is minimal. the global cost peak appears at ( ! = 9, " = 10), or a "loose control" scenario, at which point the peak cost exceeds 20% of the total output. the global gradients of the cost-effectiveness function for both effectiveness metrics show similar patterns with the previous stage, only with more non-linearity, which is too complicated to be elaborated here. for more details, refer to fig. s6 . in the baseline model, the initial number of cases at this stage was 267 and reduced to 1 after 30 days. the initial number of infected cities were 99 and reduced to 1 (wuhan) after 30 days. in terms of the reduction of infected cases, the decreases of the three coefficients, ! , " , and tl all have positive effects, but the effects are non-linear-with all returns diminishing marginally. the marginal contribution of ! and " are roughly comparable and also substitutable, with that of " about two times higher at all times. however, the marginal contribution of ! and " are both up to one order of magnitude higher than that of tl in most cases, and the latter is only significant when the values of ! and " are both very low. when " ≤ 12, or ! ≤ 6 and " ≤ 14, the number of infected cases will shrink, and will be reduced to 0 after approximately two months. higher values of ! and " (looser control on the level of social interactions) will not be sufficient to reverse the epidemic trend curve (table s11 ). in terms of the reduction of number of cities with infected cases, the decreases of the three coefficients, ! , " , and tl all have positive and non-linear effects, this time all with decreasing returns marginally -a noteworthy character that is different with the symmetric early stage. plateaus also exist at this stage, when ( ! , " ) is to the left (smaller) side of (6, 0)-(0, 8), where any further change of the coefficients will not affect the spatial scope of the epidemic. at this stage, the shape and value of the cost function are both dominated by " . they decrease monotonically and roughly linearly along with the direction of " 's decline, and the effects of ! and tl are very small. at this stage, the epidemic at the baseline has come to an end. although the initial number of cases is roughly equivalent to the initial stage of the epidemic, the strict control at the previous stages has led to a significant reduction in the number of the exposed population. therefore, the cost function at this time is dominated by " , and the peak value of the cost function is only 0.5% of the output, a great reduction compared to those in the previous three stages. since the lift of controls on social interactions will help restore economic output, the peak cost-effectiveness at this stage all occurs when all controls are lifted, at which time economic output will be very close to the normal level (when there is no epidemic). the second highest cost-effectiveness peak appears when the controls are maintained (given the number of infected cases, the controls now are realized predominately through effective epidemiological measures, rather than universal containment of social interactions), where the maximum loss of economic output is about 0.5%. while the former appears tempting, in the long run (about two months later) it is likely to cause a rebound in the epidemic (we only run the simulation for one month, so this effect cannot be directly observed in the model. however, the situation is similar to the initial period of the epidemic, and thus the lift of controls is just equivalent to starting the entire epidemic from the beginning, such that a second outbreak is inevitable if other conditions remain unchanged). the latter, on contrary, appears to achieve a good balance between cost and effectiveness within this study's analytical framework (fig. s7) . table s2 . threshold values for ! and " of the "elimination" and "control" strategies. elimination (r 0 << 1) control (r 0 < 1) ! 1/3 of the normal value while " is the normal value 2/3 of the normal value while " is the normal value " 1/4 of the normal value while ! is the normal value 1/2 of the normal value while ! is the normal value world health organization, who director-general's opening remarks at the media briefing on covid-19 national health commission of the people's republic of china a new twenty-first century science for effective epidemic response early phylogenetic estimate of the effective reproduction number of sars-cov-2 the effect of travel restrictions on the spread of the 2019 novel coronavirus modified seir and ai prediction of the epidemics trend of covid-19 in china under public health interventions the lockdown of hubei province causing different transmission dynamics of the novel coronavirus (2019-ncov) in wuhan and beijing the effect of human mobility and control measures on the covid-19 epidemic in china preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak estimating the value of containment strategies in delaying the arrival time of an influenza pandemic: a case study of travel restriction and patient isolation controlling pandemic flu: the value of international air travel restrictions fangcang shelter hospitals: a novel concept for responding to public health emergencies before wuhan lockdown, where did the 300,000 people go? variation in government responses to covid-19 version 3.0 cases and deaths by country, territory, or conveyance epidemic processes in complex networks returners and explorers dichotomy in human mobility covid-19 control in china during mass population movements at new year should internal migrants take full responsibility for spreading covid-19 modified seir and ai prediction of the epidemics trend of covid-19 in china under public health interventions effect of non-pharmaceutical interventions for containing the covid-19 outbreak in china a comprehensive framework for studying diffusion patterns of imported dengue with individual-based movement data impact of human mobility on the emergence of dengue epidemics in pakistan quantifying the impact of human mobility on malaria new crown pneumonia outbreak data sharing epidemiology working group for ncip epidemic response, the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china risk for transportation of 2019 novel coronavirus disease from wuhan to other cities in china why do wages increase with tenure ? on-the-job training and life-cycle wage growth observed within firms private-sector training and the earnings of young workers labor productivity: structural change and cyclical dynamics quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing key: cord-253256-909chgl0 authors: bajwa, sukhminder jit singh; sarna, rashi; bawa, chashamjot; mehdiratta, lalit title: peri-operative and critical care concerns in coronavirus pandemic date: 2020-03-28 journal: indian j anaesth doi: 10.4103/ija.ija_272_20 sha: doc_id: 253256 cord_uid: 909chgl0 world health organization (who) declared novel coronavirus outbreak a “pandemic” on march 11(th), 2020. india has already reached stage 2 (local transmission) and the indian government, in collaboration with the indian council of medical research (icmr), is taking all necessary steps to halt the community transmission(stage 3). anaesthesiologists and intensivists around the globe are making untiring efforts akin to soldiers at the final frontier during war. all efforts pertaining to adequate staffing, personal protective equipment (ppe) and strict adherence to hand hygiene measures are being stressed upon to prevent in-hospital transmission. in this article, all outbreak response measures including triaging, preparation of isolation rooms, decontamination and disinfection protocols as well as fundamental principles of critical care and anaesthetic management in covid-19 cases is being discussed. all the recommendations have been derived from the past experiences of sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome) outbreak as well as upcoming guidelines from the international health fraternity and indian health services. corona virus has created havoc amongst people of all nationalities with disturbingly rising count of affected people worldwide. a large database study of 72,314 affected chinese nationals has given a preliminary case-fatality rate of 2.3% with 81% experiencing mild flu symptoms, while rest of the 14% having severe symptoms and 5% being critically ill; death rate increasing exponentially with severity. [1] although the fatality has till now been reported lower than previous outbreaks of ebola, sarsandmersit is expected to rise if ipc (infection prevention and control) policies are not laid down and followed religiously. an estimated maximum likelihood (ml) of reproductive number of covid-19 infection was calculated to be 2.28 with a serial interval of 7 days which can only be curtailed by strict quarantine measures. [2] as anaesthesiologists and intensivists, our prime involvement in resuscitation, airway management, peri-surgical as well as critical care of such cases cannot be undermined. the need of the hour therefore is to update ourselves with clinicopathological spectrum of the deadly disease and provide evidence based medical services to all hospitalized cases with suspected covid-19 infection. all the recommendations below have been influenced by the past experiences of corona virus outbreaks and desire improved measures for personal protection of health care workers and consequent prevention of nosocomial transmission of infection. a systematic literature search was made using search engines including pubmed, google and google scholar with the use of the following single-text words and combinations: anaesthesia, corona virus, covid-19, intensivists from the year 2000 to 2020. the references of relevant articles were cross-checked and the articles containing all these keywords were thoroughly checked for imbibing the review. the data was also checked with national and international databases from internet and various newspapers. indian health authorities have submitted a total record of 283 lab-confirmed cases-2449 indians, 39 foreign nationalsand22 cured and discharged post-quarantine till the time of writing this article i.e., 21 th march, 2020. [3] the first three cases in india were students returning from china. they were successfully isolated in kerala and declared negative.till date,4 deaths have been reported from our country as a result of this deadly virus with old age (more than 60 years), hypertension and male gender being stated as independent risk factors. [4] the real number of patients carrying the virus is very difficult to establish as many people are avoiding the tests and many are not disclosing at all. the real danger to us as intensivists and anaesthesiologists is from these patients who have not been tested despite being potentially positive for harboring the virus. in this scenario, it is prudent to follow the national and international guidelines and treat any suspected patient as positive and to take necessary precautions during anaesthesia and critical care of such patients. screeningfor suspect cases n-cov is a positive sense, single-stranded rna corona virus belonging to sub-group sarbecovirus which closely resembles two bat-derived sars-like corona viruses but distant from sars-cov and mers-cov. preliminary phylogenetic analysis suggests bats as the original host of this virus with human to human transmission confirmed via droplet, contact and fomites and average incubation period of 2-10 days. [5] since a vast majority of indian institutions have anaesthesiologists as in charge of the emergency triaging and intensive care units, it becomes essential that our fellow colleagues should be aware of every minute details of this deadly pandemic. the following terminology has been standardized for isolation of a suspect case [ table 1 ]. although wuhan is considered the epicenter of outbreak, the increased number of cases being reported worldwide hasabolished the diagnostic link with ban on all tourist visas to india with effect from 13 th march 2020. for tracing contacts and categorizing risk of procuring infection, refer to table 2 . while the high and medium-risk contacts are advised immediate isolation with controlled local and air travel in medical transport vehicles with laboratory evaluation guided by symptomatology, low risk contacts are advised to avoid prolonged public exposure and if symptomatic get lab tests done when deemed appropriate. however, as anaesthesiologists and intensivists, the biggest challenge we will face in coming times is from the asymptomatic patients oncestage3 of the disease is crossed. it becomes imperative that during all these times, we should see only emergency and critical patients with an emphasis on personal safety as well as taking all the universal precautions against corona virus. for any emergency surgery in such patients, planning of anaesthesia becomes very crucial while taking all the necessary precautions. planning for anaesthesiaand surgery in covid-suspect cases anaesthesiologists and intensivists are the soldiers at the final frontier of corona disaster as all patients landing up in the hospital with severe respiratory distress will be primarily seen under their domain. while applying all precautionary measures pertaining to health care workers treating covid-infected individuals, necessary critical illness support needs to be provided using the current knowledge about the pathophysiology and management of covid infection. some patients with mild-moderate symptoms can also be encountered by the anaesthesiologists while being posted for surgeries. patients with mild symptoms resembling the symptomatology of other similar flu like illnesses are the real threat as chances of precautionary lapses are high in such cases. further,accidental chances of getting infection becomes highest when the positive patients without any symptomatology are being treated like normal patients. elective surgeries in these patients can be postponed till the resolution of symptoms while an emergency surgery undertaken with due risk consent as this is entirely a new clinical scenario for anaesthesia and surgery. indian journal of anaesthesia | volume 64 | issue 4 | april 2020 all suspect patients posted for surgery must be kept in isolation room till their shifting to or (operating room). for the operative procedure, negative pressure isolation rooms with minimum 12 air exchanges per hour. [7] temporary conversion of otherwise positive pressure ors into negative pressure with independent provision for air conditioning and humidification should be preferred. the negative pressure can be created by reducing the amount of air volume entering the inlet duct, while maintaining the air volume exiting through the other duct. two adjacent ors with one being utilized for the procedure and other as anteroom for the staff and doctors to wear and dispose of all ppes is ideal. the biggest challenge comes from the diversity of operation theater infrastructure set ups in our country which makes it difficultfor the implementation of universal guidelines meant for such outbreaks. therefore, such surgeries should be undertaken only in select centers with all the facilities. the policies for such clinical interventions have to be formulated by the appropriate health authorities at central governmentlevelwhich can be implemented uniformly across the nation. during such emergency surgeries, the following protocols should be followed religiously. this must be done in the isolation itself where the patient is kept. various personal protective equipment have been described for preventing aerosol and droplet transmission from patient to the anaesthesiologist. this includes-gown and shoe cover long length, full sleeved gown and shoe covers which are water resistant provide protection from any spillage of oral, nasal secretions or vomitus. it is essential for all suspect patients to wear surgical masks to prevent any droplet transmission while coughing or yawning. a plastic face shield to cover front as well as side of face, n-95 masks or pcm 2000 or powered air purifying respirators for mouth and nose, eye shields and head caps for peri-orbital mucous membranes and hair respectively must be worn to protect any transfer of virus emitted in patient's secretions to anaesthesiologist's body. all these protective equipment must be checked for proper-fit to prevent any uncovered zone. double-gloving is advised to all health care workers providing care to such patients to prevent any cross-contamination. once any procedure is done, the outer glove should be used to remove gown, shoe cover and face shield while the inner glove removed as soon as possible afterwards and disposed of with all other equipment in double-zip lock plastic container. any evidence of soiling of the glove also reprimands its removal and replacement with fresh pair of gloves. hand washing all the 9 steps of hand-washingwith alcohol based hand rubs must be adhered to strictly before wearing gloves for any procedure, before and after touching any contaminated area, anaesthesia trolley, airway cart or other instruments, and after removing gloves. protocol of keeping alcohol based hand wash at the entrance of every ward and icu helps in better compliance of health care workers.all ppes after exposure should be locked in a double zip lock plastic bag and discarded in a touch-free disposal. [8] after taking due risk for the procedure, along with nil-per-oral instructions of 2 hours for clear fluids, it is important to counsel the patient's relatives regarding need for strict isolation as well as their own testing. transportation to operation theatre during transportation, covid-19 infected patients must wear a surgical mask and be transported to the negative pressure or in an evacuated passageway and elevator preferably covered by plastic disposable material to be disposed of thereafter. all the ppes must be worn by the transporting nurse and doctor. these patients are highly anxious especially at seeing the level of protection worn by the team of doctors which gives them an impression of being doomed. adequate premedication in the form of benzodiazepines like midazolam and alprazolam or opioids like fentanylshould be prescribed before shifting to or. all ventilated patients must be intubated and transferred via special bain's circuit connected with the tube via hepa filter. the position of filter must be between the endotracheal tube connector and y-limb to prevent any aerosol transfer from the patient to portable ventilator and vice-versa. [8] hfnc or bi-pap therapy should be avoided due to increased generation of aerosols. [9] the patient must be suctioned by closed suction apparatus before transfer and given intravenous sedation and muscle relaxation to prevent any coughing and movement which might lead to disconnection of tube and dispersion of droplets. [10] adequate training regarding transport of such patients and mock drills should be done for proper implementation. after preparation of all drugs in disposable syringes, arranging for face mask, airway, endotracheal tube, stillete, bougie, laryngoscope blade, circuit, soda lime, etc. all in disposable form, the patient is taken in the or and monitors attached. as all the health care workers have to cover themselves from head to toe, there is decreased heat loss and sweating. this can be mitigated by using a lower temperature setting of 20-22 degree celsius in the operating room. [8] the leads for ecg, nibp, spo2 and etco2 should be single use or covered in sterile plastic cover to be discarded after use. in case of predicted difficult intubation, disposable blade and c-mac video laryngoscope should be kept standby while avoiding decision to conduct awakefibreoptic intubation due to potential of coughing during the procedure. [11] intravenous cannulation must be done with sterile drape and gloves to prevent any spillage. it is recommended to administer anti-emetics or prokinetics in every case as prophylaxis for vomiting. [12] the patient must be preoxygenated with 100% o2 for 3 minutes using the disposable circuit and hepa filter barrier for denitrogenation.it is suggested to use total intravenous anaesthesia in place of inhalational due to lack of isposablevaporizers and cost concern. rsi (rapid sequence induction) is the procedure of choice for intubation to abolish need for mask ventilation and regurgitation. after administering appropriate inductionagent in the form of propofol or thiopentone and muscle relaxant such as succinylcholine or rocuronium in desired doses, intubation is done by the most experienced anaesthesiologist in minimum possible time and confirmed using capnography. in cases of severely decreased po2/fio2 ratio, modified rsi can be done with low pressure ventilation before intubation. [13] it is important to reemphasize the use of "double-glove" technique during intubation. it is suggested to use total intravenous anaesthesia in place of inhalational for maintenance of anesthesia due to lack of disposable vaporizers and cost concern. the maintenance of anaesthesia can be done using disposable 50ml syringes and pmo lines. the depth of anaesthesiashould be maintained at all times to prevent any bucking in between the procedure. the setting of ventilatory parameters should be in accordance with the lung protective strategies with optimal peep and pplat<35 mmhg to achieve an spo2 >90% and admissible hypercapnia. [14] in case devices like point-of-care ultrasound (usg) have been used during the procedure, the machine and the probe along with the wire must be covered with a plastic sheath which is removed and discarded after use. any invasive procedures like epidurals or putting a central venous line should be done under usg guidance to prevent chances of failure. after the surgery, the decision to extubate should only be taken ideally in asymptomatic patients and threshold for keeping the patient intubated kept low in order to avoid any procedure with assumed dispersion of infective material in the environment. [15] in case of decision to extubate, the bucking on the tube should be avoided by "deep plane of extubation" along with anti-aspiration prophylaxis. after the patient is transported back to isolation, or should be kept closed with adequate air exchanges before allowing for subsequent patient care. all the ppes and materials used by the operating team and anaesthesiologists must be removed and sent for disposal. the left-over drugs and plastic sheets must be discarded. the pregnant, elderly and patients with co-morbid conditions have been implicated much more than general healthy population in terms of risk of infection and severity. considerations for all these high-risk populations are similar to those advocated during sars and mers outbreak. the individuals must be vaccinated against influenza and pneumonia causing bacteria to prevent secondary lrtis. [15] the patients with cardiovascular disorders are advised guideline-directed statins and diuretics to prevent potential risk of acute coronary syndrome and heart failure. other co-morbidities must be managed with respective treatment guidelines aggressively to improve outcome. in pregnant females, there is an additional risk of premature rupture of membranes, preterm birth, low birth weight and fetal distress in case of confirmed infection especially in the third trimester. as a precautionary measure, pregnant females should refrain from unnecessary travel and public transport together with practicing good personal hygiene. hence, any confirmed case coming in pregnant state must be screened for fetal well-being and advised betamethasone therapy for fetal lung maturity if anticipating pre-term delivery. the timing of delivery is entirely dependent on the condition of the mother and the fetus, wherein stable cases can be delivered at term vaginally, while critically ill mothers delivered earliest possible. as no shedding of virus has been found in the amniotic fluid, vaginal delivery can be performed but for early cord clamping and maternal separation for 2 weeks postnatally to prevent direct contact transmission. [16] till date, no guidelines have been issued regarding the anesthetic management of infected pregnant patients for caesarean section. however,spinal anaesthesia have been reported to be safe in one patient undergoing emergency c-section in wuhan. [17] there is no advisory issued against breastfeeding, but direct suckling from mother's breast is not advocated. rather expression of mother's milk and palade feeding is suitable. after prompt diagnosis based on aforementioned criterion, case must be reported by the provisional public health authorities to the national body within 24 hours in their own jurisdiction and transferred to negative pressure isolation cabin in the icu. as most of the icus are not equipped with negative and positive pressure regulations in india, an alternative approach is using hepa-carbon-photo-catalysis air purification systems as alternate means of source control. [14] the financial considerations for current icu models running in india to meet international standards in terms of quality assurance and workforce is humongous which can only be met by public-private partnership in health sector and generous gdp devoted to health. the clinical spectrum of covid-19 suspected patients can range from mild flu like symptoms to those mimicking community acquired pneumonia. in very severe cases, patients can present as ards (acute respiratory distress syndrome) with refractory hypoxemia requiring mechanical ventilation. the history taking, sampling for detection of all viral, bacterial pathogens involved in upper respiratory tract infection in the form of oropharyngeal or nasopharyngeal swabs and lower respiratory tract by sputum, endotracheal aspirate, or broncho-alveolar lavage must be sent for rt-pcr or nucleic acid detection. [14] throat swab to be collected for all virological examination should be stored in three-shelled receptacle for avoiding any spillage. more than one specimen is found to yield better results. viral rna has also been found in stools, urine and serum of infected individuals, hence can be used as alternate specimens. [14] the nasopharyngeal specimen and suction tip specimen must be sent for other bacteriological examination to rule out any secondary infection. decreased lymphocyte count, cd4 and cd8 cells along with increased crp, esr, il-6 and normal pct have been most consistent findings with this viral etiology. [18] abg analysis, liver, cardiac and kidney functions must be monitored for overall assessment of severity of infection. the radiological findings reported in these cases range from diffuse segmental or sub-segmental ground-glass opacities signifying interstitial oedema seemingly like "paving-stone" with extensive exudation into alveolar cavities leading to patchy areas of consolidation. the recent reports confirm more sensitivity of ct-scan than rt-pcr (98% versus 71%) for diagnosis of covid infection. [19] supportive therapy in the form of supplemental oxygen and antipyretics should be immediately started in patients with sari. liberal fluid administration should be avoided for risk of worsening oxygenation and periodic hemodynamic assessment used to guide goal-directed therapy.along with it, adequate nutritional support with balanced proportions of proteins, carbohydrates, vitamins and minerals boosts immunity to fight the infection. empirical antimicrobials must be given within one hour based on the clinical diagnosis, local epidemiology and susceptibility data to cover all likely pathogens causing community acquired pneumonia even if suspected to have covid. [20] a typical coverage would involve injection ceftriaxone 2gm i/v bd, azithromycin/levofloxacin 500mg od and doxycycline 100mg bd.de-escalation must be practiced after microbiological evidence.neuraminidase inhibitor like oseltemavir can be initiated for influenza when there is local circulation or other risk factors. systemic corticosteroids have controversial role in reducing mortality with viral pneumonia or ards. however, in rapidly progressing severe diseases, they provide symptomatic relief and faster resolution of pulmonary lesions, so advocated by many. [21] [22] [23] a landscape of therapeutic agents are being questioned and tested for use against 2019-cov with priority research on remdesivir and lopinavir/ritonavir + interferon beta. dgca body in india has put a nod on "restricted use" of chloroquine phosphate 500mg bd, lopinavir/ritonavir (400 mg/100 mg) for treating the virus. [24, 25] the samples must be repeated at least every 2-4 days for viral clearance until there are two consecutive negative results 24 hours apart in a clinically recovered patient. recognition of patients in respiratory distress not responsive to oxygen therapy must be swift and anaesthesiologiststrained in endotracheal intubation and all necessary equipment must be kept standby if need arises. post intubation by rsi, lung protective strategies involving use of lower tidal volumes (4-8 ml/kg predicted body weight), high peep and lower inspiratory pressures (plateau pressure <30 cmh 2 o) for meeting the ph goal of 7.30-7.45 have been postulated to prevent volutrauma, barotraumas, atelectrauma and biotrauma. [26, 27] deep sedation in the form of midazolam, propofol or fentanyl infusions are recommended to curb patient's respiratory drive and prevent dyssynchrony. the few indications of continuous neuromuscular blockade in the setting of severe ards has been agreed upon like ventilator dyssynchrony, inability to achieve target tidal volumes or refractory hypoxemia/hypercapnia. [28] in fulminating cases, prone ventilation for 12-18 hours per day and use of ino(inhaled nitric oxide) for bronchodilatation is recommended, failing which ecmo (extra corporeal membrane oxygenation) is the only modality for survival. it should only be offered in expert centres with a sufficient case volume to maintain expertise and that can apply the ipc measures required for covid patients. along with mechanical ventilation, periodic hemodynamic assessment by static procedures like passive leg raising and dynamic procedures like serial stroke volume measurements, variations in pulse pressure, inferior vena cava size, or stroke volume changes with intrathoracic pressure during mechanical ventilation must be done for guiding fluid therapy and achieving optimal response. in resuscitation of adults with septic shock, 30 ml/kg of isotonic crystalloid like ringer lactate must be administered in the first 3 hours for volume repletion. [29] if map target is not fulfilled on fluid loading and signs of volume overload appear, discontinuation of fluid administration is advised. vasopressors should be then initiated, norepinephrine being the first-line in adult patients. epinephrine or vasopressin can be added if first line drug fails. all these therapies must be instituted timely to prevent multi-organ dysfunction which carries grave prognosis. [30, 31] a term called "protected code blue "had come into existence during sars outbreak which emphasize on the use of paprs rather than n-95 masks and specialized ppes during resuscitation owing to the dynamic nature of the procedure with high risk of airborne transmission. the need for resuscitation should always be anticipated to allow for preparation of isolation room, disposable resuscitation packages instead of trolley and formation of four members team with designated roles. all the team members should wear ppes checked by infection control coach and then enter the isolation bringing the defibrillator and packages along with. [31] along with standard code blue protocol, each action should be considered in the lines of risk of aerosol transmission versus benefit to the patient. [32] future research and perspective beyond all these general measures, research is underway in different phases of completion would soon elaborate on candidate vaccines and therapeutic agents like hydroxychloroquine against covid. presently numerous false alarms are being raised from one city to another and from one country to another but nothing concrete or any definite vaccine has been developed till date. the laws of nature will definitely take their own course but till then human beings will be the worst sufferers. this is neither the first challenge nor the final one which humanshave to face when the natural ecological milieu is disturbed. at present best prophylaxis is conveyed by one sentence being used globally "stay home, stay safe" apart from taking other recommended precautions. financial support and sponsorship nil. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention serial interval of novel coronavirus (covid-19) infections all information related to novel coronavirus host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan transmission dynamics and evolutionary history of 2019-ncov coronavirus disease 2019 (covid-19) situation report -55 air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient perioperative care provider's considerations in managing patients with the covid-19 infections cross-contamination via anesthesia equipment? practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-ncov) patients outbreak of a new coronavirus: what anaesthetists should know staff safety during emergency airway management for covid-19 in hong kong 2020 features, evaluation and treatment coronavirus (covid-19) for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam). a rapid advice guideline for the diagnosis and treatment of 2019-novel coronavirus (2019-ncov) infected pneumonia (standard version) acc clinical bulletin cardiac implications of novel wuhan coronavirus (covid-19) american college of cardiology (acc) novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? emergency caesarean delivery in a patient with confirmed coronavirus disease 2019 under spinal anesthesia a family cluster of pneumonia associated with 2019 novel coronavirus indicating person to person transmission: a study of a family cluster correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases care for critically ill patients with covid-19 clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance 2020 use of glucocorticoid in the treatment of severe acute respiratory syndrome cases clinical analysis of 190 cases of outbreak of atypical pneumonia in guangzhou in spring the possibility of using lopinave/litonawe(lpv/r) as treatment of novel coronavirus 2019-ncov pneumonia: a quick systemic review based on earlier coronavirus clinical studies ministry of health & family welfare, directorate general of health services(emr division), government of india. guidelines on clinical management of covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: adescriptive study formal guidelines: management of acute respiratory distress syndrome preparing for the most critically ill patients with covid-19: the potential role of extracorporeal membrane oxygenation managing sepsis and septic shock. current guidelines and definitions endothelial biomarkers in human sepsis: pathogenesis and prognosis for pathological findings of covid-19 associated with acute respiratory distress syndrome mount sinai hospital critical care unit. sars resources. protected code blue isa family benevolent fund" • isa encourages members to join family benevolent fund of indian society of anaesthesiologists (isa-fbf) to help our colleagues' and our own families when they face the testing moments of their life. • become an isafbf member, not for you, but to help our colleague's families by donating rs.300/-per year /death. • to become an isafbf member kindly visit our website isafbf.com or contact your city branch/state/president/secretary • contact for details & application forms: dr. sugu varghese there are no conflicts of interest. key: cord-260180-kojb8efv authors: elsoukkary, sarah s.; mostyka, maria; dillard, alicia; berman, diana r.; ma, lucy x.; chadburn, amy; yantiss, rhonda k.; jessurun, jose; seshan, surya v.; borczuk, alain c.; salvatore, steven p. title: autopsy findings in 32 patients with covid-19: a single-institution experience date: 2020-09-17 journal: pathobiology doi: 10.1159/000511325 sha: doc_id: 260180 cord_uid: kojb8efv background: a novel coronavirus, sars-cov-2, was identified in wuhan, china in late 2019. this virus rapidly spread around the world causing disease ranging from minimal symptoms to severe pneumonia, which was termed coronavirus disease (i.e., covid). postmortem examination is a valuable tool for studying the pathobiology of this new infection. methods: we report the clinicopathologic findings from 32 autopsy studies conducted on patients who died of covid-19 including routine gross and microscopic examination with applicable special and immunohistochemical staining techniques. results: sars-cov-2 infection was confirmed by nasopharyngeal rt-pcr in 31 cases (97%) and by immunohistochemical staining for sars-cov-2 spike-protein in the lung in the remaining 1 case (3%). the ethnically diverse cohort consisted of 22 males and 10 females with a mean age of 68 years (range: 30–100). patients most commonly presented with cough (17 [55%]), shortness of breath (26 [81%]), and a low-grade fever (17 [55%]). thirty-one (97%) of the patients had at least 1 comorbidity (mean = 4). twenty-eight patients (88%) had widespread thromboembolic disease, as well as diffuse alveolar damage (30 [94%]), diabetic nephropathy (17 [57%]) and acute tubular injury. patterns of liver injury were heterogeneous, featuring 10 (36%) with frequent large basophilic structures in sinusoidal endothelium, and increased immunoblast-like cells in lymph nodes. conclusion: this series of autopsies from patients with covid-19 confirms the observation that the majority of severely affected patients have significant pulmonary pathology. however, many patients also have widespread microscopic thromboses, as well as characteristic findings in the liver and lymph nodes. coronaviruses (covs) are single-stranded, enveloped rna viruses that cause respiratory illnesses among humans and animals [1, 2] . these ubiquitous viruses cause variably severe diseases that range from the common cold to severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). several cases of elsoukkary et al. pathobiology 2 doi: 10.1159/000511325 pneumonia reported in wuhan, china in late 2019 led to identification of a novel coronavirus, later designated as sars coronavirus-2 (sars-cov-2), the etiologic agent of covid-19 [3, 4] . since that time, sars-cov-2 has spread rapidly across the world causing more than ten million infections and more than half a million deaths by early summer 2020 [5] . coronaviruses gain entry into cells via the angiotensin-converting enzyme 2 (ace2), which is expressed in multiple tissue types [6] . the virus disproportionately affects the respiratory system and, unlike sars and mers, disproportionately affects older patients with comorbidities such as hypertension, diabetes, obesity, and cardiovascular disease [4] . the reasons for this pattern of morbidity and mortality are yet to be elucidated. extrapulmonary manifestations of the disease have not yet been fully described. postmortem examination is an invaluable tool in understanding the pathobiology of disease. this study contributes to the growing data on this topic [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . the purpose of this study is to describe clinical and pathologic findings in major organ systems of patients who died from sars-cov-2 infection. written or verbal informed consent was obtained for postmortem examination from the next of kin of 32 patients with confirmed or presumed covid-19. cases were confirmed by sars-cov-2 rt-pcr testing in 31 (97%) patients and by tissuebased immunohistochemical staining for sars-cov-2 spike protein in 1 case (3%) of a patient who died at home and had not been tested previously, but had symptoms and positive family contacts. clinical information and laboratory data were obtained from the patients' electronic medical records. postmortem examination of the brain was not performed due to potential infectious risk related to extraction. two (6%) examinations were limited to the chest, one (3%) was confined to the right lung only, and one (3%) was limited to lung biopsies. the remaining 28 (88%) cases included examination of all thoracic and abdominal organs. the autopsies were performed from 5 to 382 h (median: 43) following death. photographs were taken, and representative sections submitted of all the organs and grossly visible lesions. the lungs were insufflated with formalin prior to sectioning. tissue specimens were fixed in 10% formalin for at least 24 h prior to processing. microscopic examination of all tissue sections was performed on hematoxylin and eosin-stained slides. immunohistochemical stains were performed on select cases and included cd61 (clone 2f2, leica ncl-cd61-308), cd163 (clone 10d6, leica ncl-cd163), erg (clone epr3864, ventana 6478450001), cd3 (clone sp7, fisher rm-9107-s), cd20 (clone l26, leica ncl-cd20-l26), and c5b-9 (clone ae11, quidel a239). special stains performed on various tissue types included pas, pas with diastase, iron, trichrome, reticulin, gms, afb, brown hopps gram (bhg), and warthin-starry. microscopic examination and interpretation of ancillary stains were performed by 5 pathologists (s.p.s., a.c.b., s.v.s., j.j., a.c.). the study was deemed exempt by the institutional review board. data normally distributed were presented as means and ranges. categorical variables were summarized as counts and percentages. statistical analysis was performed with microsoft excel. patient demographics and clinical information are summarized in table 1 and laboratory findings in table 2 . the ratio of males to females was 2.2: 1, with an average age of 68 years old (range = 30-100 years). thirty-seven percent of the patients were white, 31% were hispanic, 16% were asian, and 16% were black. most (72%) were admitted to the hospital from home or died at home. twenty-two percent were from a nursing home, and 6% were homeless. fifty-three percent of the patients had no history of smoking, 25% were former smokers (mean pack-year = 40, range = 10-120), and for 7 (22%) it was unknown. none of the patients were known to be current smokers. no correlation was seen for patients' blood types. the average oxygen saturation (spo 2 ) upon admission was 78% (range = 20-99%), with only 22% of patients having an spo 2 > 90%. the average temperature (t) of patients upon admission was 37.8 ° c (range = 37-41 ° c), with 41% of patients having a t > 37.5 ° c. most (81%) patients presented with shortness of breath and/or cough (53%); only 2 had anosmia (6%), and 1 (3%) had diarrhea recorded. a substantial number of patients had multiple comorbidities, particularly diabetes mellitus type 2 (63%), hypertension (53%), obesity (38%), and coronary artery disease (28%). fifty-three percent of admitted patients were transferred to the intensive care unit, and the average length of stay was 17 days (range = 1-28 days). fifteen (47%) patients required intubation, which continued for a mean of 17 days, 6% were maintained with bilevel positive airway pressure (bipap), and 31% required a non-rebreather mask. treatment included a combination of hydroxychloroquine (20, 63%), antibiotics (29, 91%), anticoagu-pathobiology doi: 10.1159/000511325 lation (25, 78%) and/or corticosteroids (2, 6%). the clinical course was complicated by acute kidney injury (aki) in 16 (50%) patients, with 6 (19%) requiring renal replacement therapy. five (16%) patients developed liver failure. the average overall duration of disease until time of death was 21 days (range: 1-58). twenty-seven (84%) of patients had macroscopic and/ or microscopic thrombi at autopsy (table 3) . twenty-two (69%) had elevated d-dimers (range: 2-12,200 ng/ml). coagulation factors were also prolonged, with a mean prothrombin time of 24 s and a mean activated partial data are presented as n (%) and mean (range) unless otherwise stated. spo2, oxygen saturation; na, not available; icu, intensive care unit; nrb, non-rebreather mask; bipap, bilevel positive airway pressure; ace, angiotensin converting enzyme; arb, angiotensin receptor blocker. a intubated on admission or information not available. b comorbidities included: hypertension, hyperlipidemia, diabetes, obesity, coronary artery disease, congestive heart failure, chronic obstructive pulmonary disease, asthma, obstructive sleep apnea, end stage renal disease, stroke, dementia, and cancer. elsoukkary et al. thromboplastin time of 51.6 s. thrombi were most frequently in respiratory (n = 25, 78%) and cardiovascular (n = 8, 25%) systems ( fig. 1 ). macroscopic pulmonary thrombi were detected in 11 (34%) cases. small intramyocardial vessels contained microthrombi in 6 (19%) and contained fibrin, platelets, or a mixture of both (fig. 1 ). thrombi were also observed in the prostatic venous plexus, trachea, lymph nodes, and kidney. several organs showed concurrent parenchymal infarction, including one case of acute myocardial infarction (mi). the clinicopathologic features of the respiratory system are summarized in table 4 . the average combined lung weight was 1,851 g (range = 1,000-3110 g; reference range = 685-1,050 g). most patients (n = 24, 75%) had both exudative and proliferative diffuse alveolar damage (dad). of those patients, 13 (54%) were intubated during their hospital course. three (9%) patients each showed only acute/exudative dad or organizing/proliferative dad. all 3 patients with exudative dad were never intubated and had a mean duration of disease of 9 days (range = 7-11), while 2 of the 3 patients with proliferative dad were intubated during their admission and had a mean duration of disease of 35 days (26-42 days). sixteen (50%) showed the presence of alveolar neutrophils and 14 (44%) showed evidence of organizing pneumonia. all cases had some degree of type ii pneumocyte hyperplasia with reactive atypia and bronchial squamous metaplasia. data are presented as n (%) or mean (range). wbc, white blood cells; pt, prothrombin time; ptt, partial thromboplastin time. clinicopathologic findings of 30 cases in which examination of the heart was performed are summarized in table 5 . hypertension was the most common cardiac comorbidity, present in 72% of cases (n = 23). clinically diagnosed coronary artery disease and chronic heart failure were present in 33% and 20% of patients, respectively. twelve patients (38%) had two or more concurrent cardiac comorbidities. the majority of patients had elevated troponin (i) (n = 23, 77%) which averaged 4.89 ng/ml. at autopsy, most patients (n = 28, 93%) were found to have mild cardiomegaly, average heart weight 494 (g) among women and 515 (g) among men. histologic examination showed myocyte hypertrophy in 24 cases (80%) and remote myocardial injury in 20 patients (67%) as patchy interstitial fibrosis. survey of the coronary tree showed at least moderate atherosclerosis (> 50%) in 17 patients (57%). acute myocardial ischemia was observed in a minority of patients (n = 5, 17%). of these patients, 1 had acute mi due to thrombosis or hemorrhage into an atherosclerotic plaque of a major coronary artery along with concurrent covid-19. one patient had persistent lymphocytic myocarditis, diagnosed 38 days prior to the onset of respiratory symptoms and prior to the diagnosis of covid-19. no other myocarditis or cardiac amyloid was seen in our cohort. histopathologic abnormalities were observed in the livers of 17 (61%) patients. steatosis was present in 9 (32%) cases and represented the most common finding and could be explained by concomitant obesity, diabetes mellitus, and/or hyperlipidemia. nonspecific portalbased mild lymphocytic inflammation was present in 6 (21%) cases. three cases showed evidence of chronic liver disease with bridging fibrosis and/or cirrhosis of unclear etiology. one patient had a poorly preserved intraductal trematode consistent with fasciola hepatica. large peculiar basophilic structures were found in the sinusoids of 10 (36%) cases (fig. 2) . these structures were surrounded by scant or no cytoplasm and were thought to represent nuclear remnants of altered cells. attempts to define their nature by immunohistochemistry using markers for megakaryocytes (cd61), histiocytes (cd163), and endothelial cells (erg) were unsuccessful. sections of the spleen and/or lymph nodes were examined from all 32 cases. due to safety concerns, bone marrow was not obtained except in 2 cases. lymph nodes submitted included hilar, mediastinal, paraesophageal, and paraaortic. microscopic examination showed variable degrees of autolysis in the spleen and lymph node tissues. thus, only a select group of lymph nodes (18 patients) and spleens (14 patients) from autopsies performed 1-7 days after death were reviewed. even with this restriction, the spleens showed significant autolysis, precluding accurate histologic evaluation. the lymph nodes demonstrated relatively preserved architecture, except for one patient with splenic marginal zone lymphoma (smzl), with morphologically intact lymphoid follicles containing centrally located germinal centers (15 cases), paracortical areas, and patent sinuses (fig. 3a) . this was confirmed by immu-nostaining with cd20 highlighting the intact follicles with central cd10-positive germinal center cells outlined by cd3-positive t cells (fig. 3b-d) . the subcapsular and intraparenchymal sinuses were frequently expanded and often contained a variable number of larger transformed cells with prominent nucleoli and amphophilic cytoplasm (fig. 3e-f ), which were variably cd20 positive by immunostaining (fig. 3e inset) . these transformed cells, some of which were immunoblast-like in appearance, were also seen scattered, next to vascular channels or sinuses and in loose groups in the paracortical region. in at least 2 cases, the loose groups of these larger cells were associated with apoptotic debris. these larger cells were present in all cases including the case of smzl. except for the scattered larger transformed cells, the cells in the paracortical areas were largely small lymphocytes or plasma cells. in some cases, the plasma cells were the prominent cell population. while most cases showed predominately cd3-positive t cells in the paracortical areas by immunostaining, a few cases had more cd20-positive b cells than usual. in addition, many lymph nodes showed prominent vascular congestion. the 3 cases where germinal centers were not identified were from patients who had smzl (1), were under treatment for acute myeloid leukemia (1), or had significant fibrosis associated with anthracotic pigment (1). however, the latter 2 cases showed otherwise normal architectural features including sinuses. clinical parameters for comorbidities and renal function tests on all 32 patients are summarized in table 6 . diabetes and hypertension were frequently present, and patients had subsequent associated chronic kidney disease. two patients had esrd at the time of autopsy, while 16 had aki corresponding to the onset of viral infection. five (17%) required new renal replacement therapy. presenting creatinine ranged from 0.66 to 9.6 mg/dl (mean 1.7 mg/ dl). proteinuria was present in 9 patients with 2+ to 3+ intensity (of 18 with urinalysis performed), but no quantification was undertaken in any patients in this cohort. histologic examination was available for 28 autopsies. the pathology correlated with their underlying comor-bidities, including diabetic nephropathy in 14 patients (50%), extensive tubulointerstitial scarring (> 25%) in 43% of cases, with moderate to severe vascular sclerosis in 79% of cases (fig. 4) . tubular autolysis precluded assessment of acute tubular injury in the majority of cases, which was likely the main etiology of aki on chronic kidney disease. the peritubular capillaries were dilated in nearly all cases with inflammatory margination frequently identified. rare other pathologic diagnoses were also seen ( table 6 ; fig. 4 ). two cases showed thrombotic microangiopathy of the glomeruli with acute thrombi focally present within the glomerular hilar vessels and/or peripheral capillary loops leading to endothelial swelling and luminal closure. evidence of complement-mediated endothelial injury was identified by c5b-9 staining in up to 67% of cases, including the 2 with frank thrombi (table 6 ; fig. 4e ). in this study, we described the unique and multisystem clinical and pathologic findings in 32 autopsies of patients who died from the novel coronavirus, sars-cov-2. clinically, our patient cohort was older with multiple underlying comorbidities, similar to those described in other publications [1, 4] . the majority of patients were male, on average in their late 60s, and had an average of 4 comorbidities, most commonly hypertension, coronary artery disease, obesity, and diabetes. more than half of the patients were caucasian or hispanic, but in distinction to studies from the chinese and european literature, ours was a diverse patient population with deaths seen across the ethnic spectrum [18] [19] [20] . the most common presenting symptoms were shortness of breath, cough, and a low-grade fever. on histologic examination, we observed findings secondary to the patients' preexisting conditions in the heart, lungs, liver, and kidneys, as well as changes secondary to sars-cov-2 infection such as various stages of dad and multiple thromboemboli in large and small vessels in multiple organs. as previously reported, lung injury was the main cause of death in our patients [4, 8] . the virus is believed to infect the epithelial lining cells of the respiratory tract using the ace2 enzyme as a viral receptor leading to dad, edema, and a marked increase in lung weights at autopsy. notably, the lung pathology was heterogeneous, corresponding to the radiographic findings of patchy ground glass opacifications, and on histology the pulmonary parenchyma demonstrated a range of findings from patchy exudative hyaline membrane disease to organizing pneumonia, while some areas were histologically normal. these features are most likely indicative of the spread of virus in the lung, but future studies are needed to investigate this in greater detail. multiple authors have reported clinical evidence of cardiac injury seen in the form of elevated troponins, and pathologically concurrent mi and myocarditis [1, 4, [21] [22] [23] [24] . similar to other studies, the majority of our patients had elevated troponin (i) [8] . the most significant histologic finding in our cohort was the presence of intramyocardial thrombi, identified in 19% of patients. currently, it is known that endothelial injury and thrombosis are seen in patients with sars-cov-2 infection as a part of microangiopathy [25] . thrombi are most frequently seen in the lungs [19, 25] . however, few reports address microthrombi in the myocardium, which we found in these patients when evaluating random heart sections. we believe that sars-cov-2-infected patients have a propensity to develop microthrombi in the myocardial vessels which correspond to microthrombi also seen in other organs. microthrombi are not typically seen in the microcirculation of the heart in conditions other than thrombotic thrombocytopenic purpura and hemolytic uremic syndrome [26, 27] . while frank myocardial ischemia was not frequently present, except in 1 case with well-documented mi, the elevated troponins and clinical myocar-dial dysfunction may be due to small-vessel thromboses in addition to general hypoxia. sars-cov-2 has also been reported to cause myocarditis, mostly reported based on imaging and electrocardiogram changes [22, 23, 28] . as more studies emerge, the link of covid-19 and viral-associated myocarditis does not appear to have a major role, if any, in the cardiac injury in these patients. in our series, one patient had lymphocytic myocarditis at autopsy. however, the patient had underlying congenital heart disease requiring multiple surgeries, and the myocarditis was temporally distant, arising earlier than viral symptoms. therefore, we believe it was unrelated to the viral infection. no other patient had any inflammatory infiltrates in the heart. no viral inclusion was observed in any cases. liver injury, in the form of moderately elevated aminotransferases, has been reported in up to 53% of patients [29] . rare patients have transaminases rise above a thousand [30] . in contrast to the cardiovascular pathology, few patients had preexisting chronic disease, with the exception of steatosis, similar to other studies [18, [29] [30] [31] . close to a half of our cohort exhibited moderately elevated aspartate and alanine aminotransferase enzymes, and a minority of patients (n = 5) developed acute liver failure of unclear etiology. the presence of ace-2 which is integral for viral entry into the cells is present more on cholangiocytes compared to hepatocytes; however, no significant elevation in bilirubin or histologic evidence of ductular injury was seen, similar to the study by fan et al. [32] . nuclear material of sars-cov has been detected in the liver by rt-pcr [33, 34] ; however, no specific histologic correlate of direct hepatocyte injury by sars-cov has been reported with the exception of nonspecific findings of acidophil bodies, ballooning hepatocytes, and atypical mitoses. drug-mediated liver damage may confound the interpretation of hepatocyte injury in these patients particularly when treated with lopinovir and ritonavir for covid-19 [32] . however, our patients have been treated predominately with hydroxychloroquine which is not known to cause direct liver injury [35] . a number of mechanisms have been proposed to induce clinically observed liver damage in covid patients such as locoregional viral replication, cytokine storm, hypoxia, hypovolemia, and acute on chronic injury of preexisting conditions [33, 36] . we have observed peculiar previously unreported basophilic structures of unknown origin, in 36% of patients. these structures are singly scattered and located predominately in sinusoids. while immunohistochemical staining failed to prove their identity as macrophage, megakaryocytic, or endothelial derived structures, there is a possibility that ihc staining was compromised on this autolyzed autopsy material. as one of these patients had blue-green crystals in circulating monocytes and neutrophils premortem, which have been ascribed to liver injury in the literature, it is possible that these structures are related [37] . by further identification and study of these structures, hopefully full characterization will be possible. the renal manifestations of aki in these patients are mostly due to acute tubular injury in addition to chronic diseases from the patients' underlying conditions, particularly diabetes and hypertension, confirming that seen in prior studies [3, 4] . acute tubular injury may be related to tubular ischemia alone such as hemodynamic changes or could be further exacerbated by direct viral infection, cytokine mediated injury, or drug-related tubular toxicity. considerable renovascular disease is present with moderate-to-severe sclerosis in 80% of cases, atheroemboli, papillary necrosis, and 2 cases of glomerular fibrin thrombi also detected. the latter corresponding to patients with more widespread multiorgan thromboses, but overall the majority (67%) had evidence of complement-mediated endothelial injury, by c5b-9 staining, potentially indicating a role of viral-associated endothelial pathology. although the primary target of coronaviruses is the respiratory system, coronaviruses are known to cause dis-seminated infection [33] . one of unusual sites is the hematopoietic system. clinically patients have lymphopenia and mild thrombocytopenia, as seen in this study. previously reported lymphocyte depletion in the lymph nodes and absent germinal centers have not been observed in this cohort [8] . we have seen preservation of lymph node architecture with addition of sinus dilatation and congestion, similar to that reported by menter et al. [18] . it is unclear if viral proteins are present within the lymph nodes and therefore whether morphologic changes in the lymph nodes are a direct consequence of viral replication or a nonspecific change to a viral infection. overall, the autopsies show 2 patterns of injury, chronic and acute. the patients had significant underlying comorbidities, and the more chronic pathologic changes reflect that, including the chronic kidney disease namely diabetic nephropathy and vascular sclerosis, the cardiac hypertrophy, patchy cardiac fibrosis, and coronary artery disease, as well as the hepatic steatosis and nonspecific fibrosing changes. on top of those, there are acute changes either directly due to viral infection or associated with the recent infection which include the pulmonary dad, widespread microscopic thrombi and associated organ infarcts, reactive lymph node changes with immunoblastic-type cells, atypical hepatic sinusoidal concretions/ cells, renal tma, and acute tubular injury in the kidneys. the autopsies all came from a 2-month period of time when the treatment the patients received was largely antibiotics plus or minus hydroxychloroquine in 78% of these cases. as the microthrombi became a more regarded complication, anticoagulation was also utilized, also in 78% (n = 25) of the cases. however, of those 25, 19 still were found to have thrombi in the lungs at autopsy and of the 6 without pulmonary thrombi, 2 had other organ involvement bringing the total to 21 of 25 patients. in comparison, 6 of the 7 patients who were not anti-coagulated also showed thrombi. steroids were not frequently utilized and only given to 2 patients, for 3 days each, 17 and 24 days prior to their death. larger studies are needed to further investigate the impact of anticoagulation and immunosuppression in this disease. in conclusion, this autopsy series of 32 ethnically diverse patients with sars-cov-2 infection from a single nyc autopsy service shows a multisystemic pathology which leads to death typically in those with comorbidities. while the lung findings are most significant for the majority of those infected, other organ systems are frequently involved including with widespread microscopic thromboses in numerous organs, as well as liver, kidney, and lymph node pathology. autopsy findings in 12 doi: 10.1159/000511325 these most severely affected patients can serve as a valuable source of information in studying this new disease process. the epidemiology and clinical information about covid-19 covid-19): a review of clinical features clinical and immunological features of severe and moderate coronavirus disease 2019 clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study who. coronavirus disease (covid-19) situation report -163 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis covid-19 autopsies pulmonary arterial thrombosis in 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autopsy findings and venous thromboembolism in patients with covid-19: a prospective cohort study endorsed by the isth, natf, esvm, and the iua, supported by the esc working group on pulmonary circulation and right ventricular function. covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review fatal eosinophilic myocarditis in a healthy 17-year-old male with severe acute respiratory syndrome coronavirus 2 (sars-cov-2c) myocarditis in a patient with covid-19: a cause of raised troponin and ecg changes clinical predictors of mortality due to cov-id-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 thrombotic thrombocytopenic purpura. nat rev dis primers cardiac implications of thrombotic thrombocytopenic purpura coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin liver injury in co-vid-19: management and challenges liver injury during highly pathogenic human coronavirus infections coronavirus disease 2019 and prevalence of chronic liver disease: a meta-analysis clinical features of covid-19-related liver functional abnormality fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus covid-19: gastrointestinal manifestations and potential fecal-oral transmission bethesda: national institute of diabetes and digestive and kidney diseases characteristics and mechanism of liver injury in 2019 coronavirus disease clinical significance of blue-green neutrophil and monocyte cytoplasmic inclusions in sars-cov-2 positive critically ill patients the authors wish to acknowledge nector garcia, alisa ramineni, dr. amelia baxter-stoltzfus, dr. mona farahi, dr. william towne, dr. kartik viswanathan, dr. elizabeth crowe, and dr. paul barone for their assistance in performing the autopsies. the study was exempt from institutional review board approval as it did not meet criteria for living human research; however, informed consent was obtained from the next of kin for all patients. the authors have no conflicts of interest to declare. none. key: cord-131678-rvg1ayp2 authors: ponce, marcelo; sandhel, amit title: covid19.analytics: an r package to obtain, analyze and visualize data from the corona virus disease pandemic date: 2020-09-02 journal: nan doi: nan sha: doc_id: 131678 cord_uid: rvg1ayp2 with the emergence of a new pandemic worldwide, a novel strategy to approach it has emerged. several initiatives under the umbrella of"open science"are contributing to tackle this unprecedented situation. in particular, the"r language and environment for statistical computing"offers an excellent tool and ecosystem for approaches focusing on open science and reproducible results. hence it is not surprising that with the onset of the pandemic, a large number of r packages and resources were made available for researches working in the pandemic. in this paper, we present an r package that allows users to access and analyze worldwide data from resources publicly available. we will introduce the covid19.analytics package, focusing in its capabilities and presenting a particular study case where we describe how to deploy the"covid19.analytics dashboard explorer". in 2019 a novel type of corona virus was first reported, originally in the province of hubei, china. in a time frame of months this new virus was capable of producing a global pandemic of the corona virus disease (covid19), which can end up in a severe acute respiratory syndrome (sars-cov-2). the origin of the virus is still unclear [4, 5, 6] , although some studies based on genetic evidence, suggest that it is quite unlikely that this virus was human made in a laboratory, but instead points towards cross-species transmission [7, 8] . although this is not the first time in the human history when humanity faces a pandemic, this pandemic has unique characteristics. for starting the virus is "peculiar" as not all the infected individuals experience the same symptoms. some individuals display symptoms that are similar to the ones of a common cold or flu while other individuals experience serious symptoms that can cause death or hospitalization with different levels of severity, including staying in intensive-care units (icu) for several weeks or even months. a recent medical survey shows that the disease can transcend pulmonary manifestations affecting several other organs [9] . studies also suggest that the level of severity of the disease can be linked to previous conditions [10] , gender [11] , or even blood type [12] but the fundamental and underlying reasons still remain unclear. some infected individuals are completely asymptomatic, which makes them ideal vectors for disseminating the virus. this also makes very difficult to precisely determine the transmission rate of the disease, and it is argued that in part due to the peculiar characteristics of the virus, that some initial estimates were underdetermining the actual value [13] . elderly are the most vulnerable to the disease and reported mortality rates vary from 5 to 15% depending on the geographical location. in addition to this, the high connectivity of our modern societies, make possible for a virus like this to widely spread around the world in a relatively short period of time. what is also unprecedented is the pace at which the scientific community has engaged in fighting this pandemic in different fronts [14] . technology and scientific knowledge are and will continue playing a fundamental role in how humanity is facing this pandemic and helping to reduce the risk of individuals to be exposed or suffer serious illness. techniques such as dna/rna sequencing, computer simulations, models generations and predictions, are nowadays widely accessible and can help in a great manner to evaluate and design the best course of action in a situation like this [15] . public health organizations are relying on mathematical and data-driven models (e.g. [16] ), to draw policies and protocols in order to try to mitigate the impact on societies by not suffocating their health institutions and resources [17] . specifically, mathematical models of the evolution of the virus spread, have been used to establish strategies, like social distancing, quarantines, self-isolation and staying at home, to reduce the chances of transmission among individuals. usually, vaccination is also another approach that emerges as a possible contention strategy, however this is still not a viable possibility in the case of covid19, as there is not vaccine developed yet [18, 19] . simulations of the spread of virus have also shown that among the most efficient ways to reduce the spread of the virus are [20] : increasing social distancing, which refers to staying apart from individuals so that the virus can not so easily disperse among individuals; improving hygiene routines, such as proper hand washing, use of hand sanitizer, etc. which would eventually reduce the chances of the virus to remain effective; quarantine or self-isolation, again to reduce unnecessary exposure to other potentially infected individuals. of course these recommendations based on simulations and models can be as accurate and useful as the simulations are, which ultimately depend on the value of the parameters used to set up the initial conditions of the models. moreover these parameters strongly depend on the actual data which can be also sensitive to many other factors, such as data collection or reporting protocols among others [21] . hence counting with accurate, reliable and up-to-date data is critical when trying to understand the conditions for spreading the virus but also for predicting possible outcomes of the epidemic, as well as, designing proper containment measurements. similarly, being able to access and process the huge amount of genetic information associated with the virus has proben to shred light into the disease's path [22, 23] . encompassing these unprecedented times, another interesting phenomenon has also occurred, in part related to a contemporaneous trend in how science can be done by emphasizing transparency, reproducibility and robustness: an open approach to the methods and the data; usually refer as open science. in particular, this approach has been part for quite sometime of the software developer community in the so-called open source projects or codes. this way of developing software, offers a lot of advantages in comparison to the more traditional and closed, proprietary approaches. for starting, it allows that any interested party can look at the actual implementation of the code, criticize, complement or even contribute to the project. it improves transparency, and at the same time, guarantees higher standards due to the public scrutiny; which at the end results in benefiting every one: the developers by increasing their reputation, reach and consolidating a widely validated product and the users by allowing direct access to the sources and details of the implementation. it also helps with reproducibility of results and bugs reports and fixes. several approaches and initiatives are taking the openness concepts and implementing in their platforms. specific examples of this have drown the internet, e.g. the surge of open source powered dashboards [24] , open data repositories, etc. another example of this is for instance the number of scientific papers related to covid19 published since the beginning of the pandemic [25] , the amount of data and tools developed to track the evolution of pandemic, etc. [26] . as a matter of fact, scientists are now drowning in publications related to the covid19 [27, 28] , and some collaborative and community initiatives are trying to use machine learning techniques to facilitate identify and digest the most relevant sources for a given topic [29, 30, 31] . the "r language and environment for statistical computing" [1, 2] is not exception here. moreover, promoting and based on the open source and open community principles, r has empowered scientists and researchers since its inception. not surprisingly then, the r community has contributed to the official cran [32] repository already with more than a dozen of packages related to the covid19 pandemic since the beginning of the crisis. in particular, in this paper we will introduce and discuss the covid19.analytics r package [3] , which is mainly designed and focus in an open and modular approach to provide researchers quick access to the latest reported worldwide data of the covid19 cases, as well as, analytical and visualization tools to process this data. this paper is organized as follow: in sec. 2 we describe the covid19.analytics , in sec. 3 we present some examples of data analysis and visualization, in sec. 4 we describe in detail how to deploy a web dashboard employing the capabilities of the covid19.analytics package providing full details on the implementation so that this procedure can be repeated and followed by interested users in developing their own dashboards. finally we summarize some conclusions in sec. 5. the covid19.analytics r package [3] allows users to obtain live 1 worldwide data from the novel covid19. it does this by accessing and retrieving the data publicly available and published by two main sources: the "covid-19 data repository by the center for systems science and engineering (csse) at johns hopkins university" [33] for the worldwide and us data, and the city of toronto for the toronto data [34] . the package also provides basic analysis and visualization tools and functions to investigate these datasets and other ones structured in a similar fashion. the covid19.analytics package is an open source tool, which its main implementation and api is the r package [3] . in addition to this, the package has a few more adds-on: • a central github repository, https://github.com/mponce0/covid19.analytics where the latest development version and source code of the package are available. users can also submit tickets for bugs, suggestions or comments using the "issues" tab. • a rendered version with live examples and documentation also hosted at github pages, https: //mponce0.github.io/covid19.analytics/; • a dashboard for interactive usage of the package with extended capabilities for users without any coding expertise, https://covid19analytics.scinet.utoronto.ca. we will discuss the details of the implementation in sec. 4. • a "backup" data repository hosted at github, https://github.com/mponce0/covid19analytics. datasets -where replicas of the live datasets are stored for redundancy and robust accesibility sake (see fig. 1 ). one of the main objectives of the covid19.analytics package is to make the latest data from the reported cases of the current covid19 pandemic promptly available to researchers and the scientific community in what follows we describe the main functionalities from the package regarding data accessibility. the covid19.data function allows users to obtain realtime data about the covid19 reported cases from the jhu's ccse repository, in the following modalities: • aggregated data for the latest day, with a great 'granularity' of geographical regions (ie. cities, provinces, states, countries) • time series data for larger accumulated geographical regions (provinces/countries) • deprecated : we also include the original data style in which these datasets were reported initially. the datasets also include information about the different categories (status) "confirmed"/"deaths"/"recovered" of the cases reported daily per country/region/city. this data-acquisition function, will first attempt to retrieve the data directly from the jhu repository with the latest updates. if for what ever reason this fails (eg. problems with the connection) the package will load a preserved "image" of the data which is not the latest one but it will still allow the user to explore this older dataset. in this way, the package offers a more robust and resilient approach to the quite dynamical situation with respect to data availability and integrity. in addition to the data of the reported cases of covid19, the covid19.analytics package also provides access to genomics data of the virus. the data is obtained from the national center for biotechnology information (ncbi) databases [35, 36] . table 1 shows the functions available in the covid19.analytics package for accessing the reported cases of the covid19 pandemic. the functions can be divided in different categories, depending on what data they provide access to. for instance, they are distinguished between agreggated and time series data sets. they are also grouped by specific geographical locations, i.e. worldwide, united states of america (us) and the city of toronto (ontario, canada) data. the time series data is structured in an specific manner with a given set of fields or columns, which resembles the following format: "province.state" | "country.region" | "lat" | "long" | ... sequence of dates ... one of the modular features this package offers is that if an user has data structured in a data.frame organized as described above, then most of the functions provided by the covid19.analytics package for analyzing time series data will just work with the user's defined data. in this way it is possible to add new data sets to the ones that can be loaded using the repositories predefined in this package and extend the analysis capabilities to these new datasets. sec. 3.5 presents an example of how external or synthetic data has to be structured so that can use the function from the covid19.analytics package. it is also recommended to check the compatibility of these datasets using the data integrity and consistency checks functions described in the following section. due to the ongoing and rapid changing situation with the covid-19 pandemic, sometimes the reported data has been detected to change its internal format or even show some anomalies or inconsistencies 1 . for instance, in some cumulative quantities reported in time series datasets, it has been observed that these quantities instead of continuously increase sometimes they decrease their values which is something that should not happen 2 . we refer to this as an inconsistency of "type ii". some negative values have been reported as well in the data, which also is not possible or valid; we call this inconsistency of "type i". when this occurs, it happens at the level of the origin of the dataset, in our case, the one obtained from the jhu/ccesgis repository [33] . in order to make the user aware of this, we implemented two consistency and integrity checking functions: • consistency.check: this function attempts to determine whether there are consistency issues within the data, such as, negative reported value (inconsistency of "type i") or anomalies in the cumulative quantities of the data (inconsistency of "type ii") • integrity.check: this determines whether there are integrity issues within the datasets or changes to the structure of the data alternatively we provide a data.checks function that will execute the previous described functions on an specified dataset. data integrity. it is highly unlikely that the user would face a situation where the internal structure of the data or its actual integrity may be compromised. however if there are any suspicious about this, it is possible to use the integrity.check function in order to verify this. if anything like this is detected we urge users to contact us about it, e.g. https://github.com/mponce0/covid19.analytics/issues. data consistency. data consistency issues and/or anomalies in the data have been reported several times 1 these are claimed, in most of the cases, to be missreported data and usually are just an insignificant number of the total cases. having said that, we believe that the user should be aware of these situations and we recommend using the consistency.check function to verify the dataset you will be working with. nullifying spurious data. in order to deal with the different scenarios arising from incomplete, inconsistent or missreported data, we provide the nullify.data function, which will remove any potential entry in the data that can be suspected of these incongruencies. in addition ot that, the function accepts an optional argument stringent=true, which will also prune any incomplete cases (e.g. with nas present). similarly to the rapid developments and updates in the reported cases of the disease, the sequencing of the virus is moving almost at equal pace. that's why the covid19.analytics package provides access to good number of the genomics data currently available. the covid19.genomic.data function allows users to obtain the covid19's genomics data from ncbi's databases [36] . the type of genomics data accessible from the package is described in table 2 . although the package attempts to provide the latest available genomic data, there are a few important details and differences with respect to the reported cases data. for starting, the amount of genomic information available is way larger than the data reporting the number of cases which adds some additional constraints when retrieving this data. in addition to that, the hosting servers for the genomic databases impose certain limits on the rate and amounts of downloads. in order to mitigate these factors, the covid19.analytics package employs a couple of different strategies as summarized below: • most of the data will be attempted to be retrieved live from ncbi databases -same as using src='livedata'. • if that is not possible, the package keeps a local version of some of the largest datasets (i.e. genomes, nucleotides and proteins) which might not be up-to-date -same as using src='repo'. • the package will attempt to obtain the data from a mirror server with the datasets updated on a regular basis but not necessarily with the latest updates -same as using src='local'. these sequence of steps are implemented in the package using trycath() exceptions in combination with recursivity, i.e. the retrieving data function calling itself with different variations indicating which data source to use. as the covid19.analytics package will try present the user with the latest data sets possible, different strategies (as described above) may be in place to achieve this. one way to improve the realiability of the access to and avialability of the data is to use a series of replicas of the datasets which are hosted in different locations. fig. 1 summarizes the different data sources and point of access that the package employs in order to retrieve the data and keeps the latest datasets available. genomic data as mentioned before is accessed from ncbi databases. this is implemented in the covid19.genomic.data function employing the ape [37] and rentrez [38] packages. in particular the proteins datasets, with more than 100k entries, is quite challenging to obtain "live". as a matter of fact, the covid19.genomic.data function accepts an argument to specify whether this should be the case or not. if the src argument is set to 'livedata' then the function will attempt to download the proteins list directly from ncbi databases. if this fail, we recommend using the argument src='local' which will provide an stagered copy of this dataset at the moment in which the package was submitted to the cran repository, meaning that is quite likely this dataset won't be complete and most likely outdated. additionaly, we offer a second replica of the datasets, located at https://github.com/mponce0/covid19analytics.datasets where all datasets are updated periodically, this can be accessed using the argument src='repo'. in addition to the access and retrieval of the data, the covid19.analytics package includes several functions to perform basic analysis and visualizations. table 3 shows the list of the main functions in the package. description main type of output data acquisition covid19.data obtain live* worldwide data for covid19 virus, from the jhu's ccse repository [33] return dataframes/list with the collected data covid19.toronto.data obtain live* data for covid19 cases in the city of toronto, on canada, from the city of toronto reports [34] return dataframe/list with the collected data covid19.us.data obtain live* us specific data for covid19 virus, from the jhu's ccse repository [33] return dataframe with the collected data genomics covid19.genomic.data c19.refgenome.data c19.fasta.data c19.ptree.data c19.nps.data c19.np_fasta.data obtain genomic data from ncbi databases -see table 2 in the reported data, this is mostly given by the province/city and/or country/region. in order to facilitate the processing of locations that are located geo-politically close, the covid19.analytics package provides a way to identify regions by indicating the corresponding continent's name where they are located. i.e. "south america", "north america", "central america", "america", "europe", "asia" and "oceania" can be used to process all the countries within each of these regions. the geographicalregions function is the one in charge of determining which countries are part of what continent and will display them when executing geographicalregions(). in this way, it is possible to specify a particular continent and all the countries in this continent will be processed without needing to explicitly specifying all of them. reports. as the amount of data available for the recorded cases of covid19 can be overwhelming, and in order to get a quick insight on the main statistical indicators, the covid19.analytics package includes the report.summary function, which will generate an overall report summarizing the main statistical estimators for the different datasets. it can summarize the "time series" data (when indicating cases.to.process="ts"), the "aggregated" data (cases.to.process="agg") or both (cases.to.process="all"). the default will display the top 10 entries in each category, or the number indicated in the nentries argument, for displaying all the records just set nentries=0. the function can also target specific geographical location(s) using the geo.loc argument. when a geographical location is indicated, the report will include an additional "rel.perc" column for the confirmed cases indicating the relative percentage among the locations indicated. similarly the totals displayed at the end of the report will be for the selected locations. in each case ("ts" or/and "agg") will present tables ordered by the different cases included, i.e. confirmed infected, deaths, recovered and active cases. the dates when the report is generated and the date of the recorded data will be included at the beginning of each table. it will also compute the totals, averages or mean values, standard deviations and percentages of various quantities, i.e. • it will determine the number of unique locations processed within the dataset • it will compute the total number of cases per case type • percentages -which are computed as follow: for the "confirmed" cases, as the ratio between the corresponding number of cases and the total number of cases, i.e. a sort of "global percentage" indicating the percentage of infected cases with respect to the rest of the world covid19.analytics "internal" rsync/git -when a new release is push to cran "internal" scripts src="livedata" src="repo" src="local" https://github.com/mponce0/covid19analytics.datasets figure 1 : schematic of the data acquision flows between the covid19.analytics package and the different sources of data. dark and solid/dashed lines represent api functions provided by the package accesible to the users. dotted lines are "internal" mechanisms employed by the package to synchronize and update replicas of the data. data acquisition from ncbi servers is mostly done utilizing the ape [37] and rentrez [38] packages. for "confirmed" cases, when geographical locations are specified, a "relative percentage" is given as the ratio of the confirmed cases over the total of the selected locations for the other categories, "deaths"/"recovered"/"active", the percentage of a given category is computed as the ratio between the number of cases in the corresponding category divided by the "confirmed" number of cases, i.e. a relative percentage with respect to the number of confirmed infected cases in the given region • for "time series" data: it will show the delta (change or variation) in the last day, daily changes day before that (t − 2), three days ago (t − 3), a week ago (t − 7), two weeks ago (t − 14) and a month ago (t − 30) when possible, it will also display the percentage of "recovered" and "deaths" with respect to the "confirmed" number of cases the column "globalperc" is computed as the ratio between the number of cases for a given country over the total of cases reported -the "global perc. average (sd: standard deviation)" is computed as the average (standard deviation) of the number of cases among all the records in the data -the "global perc. average (sd: standard deviation) in top x" is computed as the average (standard deviation) of the number of cases among the top x records a typical output of the summary.report for the "time series" data, is shown in the example 4 in sec. 3 . in addition to this, the function also generates some graphical outputs, including pie and bar charts representing the top regions in each category; see fig. 2 . totals per location & growth rate. it is possible to dive deeper into a particular location by using the tots.per.location and growth.rate functions. these functions are capable of processing different types of data, as far as these are "time series" data. it can either focus in one category (eg. "ts-confirmed", "ts-recovered", "ts-deaths",) or all ("ts-all"). when these functions detect different types of categories, each category will be processed separately. similarly the functions can take multiple locations, ie. just one, several ones or even "all" the locations within the data. the locations can either be countries, regions, provinces or cities. if an specified location includes multiple entries, eg. a country that has several cities reported, the functions will group them and process all these regions as the location requested. totals per location. the tots.per.location function will plot the number of cases as a function of time for the given locations and type of categories, in two plots: a log-scale scatter one a linear scale bar plot one. when the function is run with multiple locations or all the locations, the figures will be adjusted to display multiple plots in one figure in a mosaic type layout. additionally, the function will attempt to generate different fits to match the data: • an exponential model using a linear regression method • a poisson model using a general linear regression method • a gamma model using a general linear regression method the function will plot and add the values of the coefficients for the models to the plots and display a summary of the results in the console. it is also possible to instruct the function to draw a "confidence band" based on a moving average, so that the trend is also displayed including a region of higher confidence based on the mean value and standard deviation computed considering a time interval set to equally dividing the total range of time over 10 equally spaced intervals. the function will return a list combining the results for the totals for the different locations as a function of time. growth rate. the growth.rate function allows to compute daily changes and the growth rate defined as the ratio of the daily changes between two consecutive dates. the growth.rate function shares all the features of the tots.per.location function as described above, i.e. can process the different types of cases and multiple locations. the graphical output will display two plots per location: • a scatter plot with the number of changes between consecutive dates as a function of time, both in linear scale (left vertical axis) and log-scale (right vertical axis) combined • a bar plot displaying the growth rate for the particular region as a function of time. when the function is run with multiple locations or all the locations, the figures will be adjusted to display multiple plots in one figure in a mosaic type layout. in addition to that, when there is more than one location the function will also generate two different styles of heatmaps comparing the changes per day and growth rate among the different locations (vertical axis) and time (horizontal axis). furthermore, if the interactivefig=true argument is used, then interactive heatmaps and 3d-surface representations will be generated too. some of the arguments in this function, as well as in many of the other functions that generate both static and interactive visualizations, can be used to indicate the type of output to be generated. table 4 lists some of these arguments. in particular, the arguments controlling the interactive figures -interactivefig and interactive.display-can be used in combination to compose an interactive figure to be captured and used in another application. for instance, when interactive.display is turned off but interactivefig=true, the function will return the interactive figure, so that it can be captured and used for later purposes. this is the technique employed when capturing the resulting plots in the covid19.analytics dashboard explorer as presented in sec. 4.2. finally, the growth.rate function when not returning an interactive figure, will return a list combining the results for the "changes per day" and the "growth rate" as a function of time, i.e. when interactivefig is not specified or set to false (which its default value) or when interactive.display=true. when is turned off, but interactivefig=true, the function will return the interactive figure, so that it can be captured and used for later purposes. trends in daily changes. the covid19.analytics package provides three different functions to visualize the trends in daily changes of reported cases from time series data. • single.trend, allows to inspect one single location, this could be used with the worldwide data sliced by the corresponding location, the toronto data or the user's own data formatted as "time series" data. • mtrends, is very similar to the single.trend function, but accepts multiple or single locations generating one plot per location requested; it can also process multiple cases for a given location. • itrends function to generate an interactive plot of the trend in daily changes representing changes in number of cases vs total number of cases in log-scale using splines techniques to smooth the abrupt variations in the data the first two functions will generate "static" plots in a compose with different insets: • the main plot represents daily changes as a function of time • the inset figures in the top, from left to right: total number of cases (in linear and semi-log scales), changes in number of cases vs total number of cases changes in number of cases vs total number of cases in log-scale • the second row of insets, represent the "growth rate" (as defined above) and the normalized growth rate defined as the growth rate divided by the maximum growth rate reported for this location plotting totals. the function totals.plt will generate plots of the total number of cases as a function of time. it can be used for the total data or for a specific or multiple locations. the function can generate static plots and/or interactive ones, as well, as linear and/or semi-log plots. plotting cases in the world. the function live.map will display the different cases in each corresponding location all around the world in an interactive map of the world. it can be used with time series data or aggregated data, aggregated data offers a much more detailed information about the geographical distribution. the covid19.analytics package allows users to model the dispersion of the disease by implementing a simple susceptible-infected-recovered (sir) model [39, 40] . the model is implemented by a system of ordinary differential equations (ode), as the one shown by eq.(1). where s represents the number of susceptible individuals to be infected, i the number of infected individuals and r the number of recovered ones at a given moment in time. the coefficients β and γ are the parameters controlling the transition rate from s to i and from i to r respectively; n is the total number of individuals, i.e. n = s(t) + i(t) + r(t); which should remain constant, i.e. eq.(1) can be written in terms of the normalized quantities, although the ode sir model is non-linear, analytical solutions have been found [41] . however the approach we follow in the package implementation is to solve the ode system from eq.(1) numerically. the function generate.sir.model implements the sir model from eq.(1) using the actual data from the reported cases. the function will try to identify data points where the onset of the epidemic began and consider the following data points to generate proper guesses for the two parameters describing the sir ode system, i.e. β and γ. it does this by minimizing the residual sum of squares (rss) assuming one single explanatory variable, i.e. the sum of the squared differences between the number of infected cases i(t) and the quantity predicted by the modelĩ(t), the ode given by eq.(1) is solved numerically using the ode function from the desolve and the minimization is tackled using the optim function from base r. after the solution for eq.(1) is found, the function will provide details about the solution, as well as, plot the quantities s(t), i(t), r(t) in a static and interactive plot. the generate.sir.model function also estimates the value of the basic reproduction number or basic reproduction ratio, r 0 , defined as, which can be considered as a measure of the average expected number of new infections from a single infection in a population where all subjects can be susceptible to get infected. the function also computes and plots on demand, the force of infection, defined as, f inf ection = βi(t), which measures the transition rate from the compartment of susceptible individuals to the compartment of infectious ones. for exploring the parameter space of the sir model, it is possible to produce a series of models by varying the conditions, i.e. range of dates considered for optimizing the parameters of the sir equation, which will effectively "sweep" a range for the parameters β, γ and r 0 . this is implemented in the function sweep.sir.models, which takes a range of dates to be used as starting points for the number of cases used to feed into the generate.sir.model producing as many models as different ranges of dates are indicated. one could even use this in combination to other resampling or monte carlo techniques to estimate statistical variability of the parameters from the model. in this section we will present some basic examples of how to use the main functions from the covid19.analytics package. we will begin by installing the covid19.analytics package. this can be achieved in two alternative ways: 1. installing the latest stable version of the package directly from the cran repository. this can be done within an r session using the install.packages function, i.e. > install.packages("covid19.analytics") 2. installing the development version from the package's github repository, https://github.com/ mponce0/covid19.analytics using the devtools package [42] and its install_github function. i.e. # begin by installing devtools if not installed in your system > install.packages("devtools") # install the covid19.analytics packages from the github repo > devtools::install_github("mponce0/covid19.analytics") after having installed the covid19.analytics package, for accessing its functions, the package needs to be loaded using r's library function, i.e. the covid19.analytics uses a few additional packages which are installed automatically if they are not present in the system. in particular, readxl is used to access the data from the city of toronto [34] , ape is used for pulling the genomics data from ncbi; plotly and htmlwidgets are used to render the interactive plots and save them in html documents, desolve is used to solve the differential equations modelling the spread of the virus, and gplots, pheatmap are used to generate heatmaps. lst. 1 shows how to use the covid19.data function to obtain data in different cases. # obtain all the records combined for " confirmed " , " deaths " and " recovered " cases # for the global ( worldwide ) * aggregated * data covid19 . data . allcases <-covid19 . data () # obtain time series data for global " confirmed " cases covid19 . confirmed . cases <-covid19 . data ( " ts -confirmed " ) # reads all possible datasets , returning a list covid19 . all . datasets <-covid19 . data ( " all " ) # reads the latest aggregated data of the global cases covid19 . all . agg . cases <-covid19 . data ( " aggregated " ) # reads time series data for global casualties covid19 . ts . deaths <-covid19 . data ( " ts -deaths " ) # read " time series " data for the city of toronto toronto . ts . data <-covid19 . data ( " ts -toronto " ) # this can be also done using the covid19 . toronto . data () fn tor . ts . data <-covid19 . toronto . data () # or get the original data as reported by the city of toronto tor . df . data <-covid19 . toronto . data ( data . fmr = " orig " ) # retrieve us time series data of confirmed cases us . confirmed . cases <-covid19 . data ( " ts -confirmed -us " ) # retrieve us time series data of death cases us . deaths . cases <-covid19 . data ( " ts -deaths -us " ) # or both cases combined us . cases <-covid19 . us . data () listing 1: reading data from reported cases of covid19 using the covid19.analytics package. in general, the reading functions will return data frames. exceptions to this, are when the functions need to return a more complex output, e.g. when combining "all" type of data or when requested to obtain the original data from the city of toronto (see details in table 3 ). in these cases, the returning object will be a list containing in each element dataframes corresponding to the particular type of data. in either case, the structure and overall content can be quickly assessed by using r's str or summary functions. one useful information to look at after loading the datasets, would be to identify which locations/regions have reported cases. there are at least two main fields that can be used for that, the columns containing the keywords: 'country' or 'region' and 'province' or 'state'. lst. 2 show examples of how to achieve this using partial matches for column names, e.g. "country" and "province". # read a data set data <-covid19 . data ( " ts -confirmed " ) # look at the structure and column names str ( data ) names ( data ) # find ' country ' column country . col <-pmatch ( " country " , names ( data ) ) # slice the countries countries <-data [ , country . col ] # list of countries print ( unique ( countries ) ) # sorted table of countries , may include multiple entries print ( sort ( table ( countries ) ) ) # find ' province ' column prov . col <-pmatch ( " province " , names ( data ) ) # slice the provinces provinces <-data [ , prov . col ] # list of provinces print ( unique ( provinces ) ) # sorted table of provinces , may include multiple entries print ( sort ( table ( provinces ) ) ) listing 2: identifying geographical locations in the data sets. an overall view of the current situation at a global or local level can be obtained using the report.summary function. lst. 3 shows a few examples of how this function can be used. # a quick function to overview top cases per region for time series and aggregated records report . summary () # save the tables into a text file named ' covid19 -summaryreport _ currentdate . txt ' # where * currrentdate * is the actual date report . summary ( savereport = true ) # summary report for an specific location with default number of entries report . summary ( geo . loc = " canada " ) # summary report for an specific location with top 5 report . summary ( nentries =5 , geo . loc = " canada " ) # it can combine several locations report . summary ( nentries =30 , geo . loc = c ( " canada " ," us " ," italy " ," uruguay " ," argentina " ) ) a typical output of the report generation tool is presented in lst. 4. typical output of the report.summary function. this particular example was generated using report.summary(nentries=5,graphical.output=true,savereport=true), which indicates to consider just the top 5 entries, generate a graphical output as shown in fig. 2 and a to save text file including the report which is the one shown here.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~# #### ts -confirmed cases --data dated : 2020 -06 -25 :: 2020 -06 -26 13:10:01 # #### ts -deaths cases --data dated : 2020 -06 -25 :: 2020 -06 -26 13:10:02 # #### ts -recovered cases --data dated : 2020 -06 -25 :: 2020 -06 -26 13:10:02 # #### aggregated data --ordered by confirmed cases --data dated : 2020 -06 -26 :: 2020 -06 -26 13:10:03 # #### aggregated data --ordered by deaths cases --data dated : 2020 -06 -26 :: 2020 -06 -26 13:10:03 # #### aggregated data --ordered by recovered cases --data dated : 2020 -06 -26 :: 2020 -06 -26 13:10:03 # #### aggregated data --ordered by active cases --data dated : 2020 -06 -26 :: 2020 -06 -26 13:10:03 * statistical estimators computed considering 3781 independent reported entries * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * overall summary * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * statistical estimators computed considering 266 / 266 / 253 independent reported entries per case -type * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * a daily generated report is also available from the covid19.analytics documentation site, https: //mponce0.github.io/covid19.analytics/. the covid19.analytics package allows users to investigate total cumulative quantities per geographical location with the totals.per.location function. examples of this are shown in lst. 5. # totals for confirmed cases for " ontario " tots . per . location ( covid19 . confirmed . cases , geo . loc = " ontario " ) # total for confirmed cases for " canada " tots . per . location ( covid19 . confirmed . cases , geo . loc = " canada " ) # total nbr of confirmed cases in hubei including a confidence band based on moving average tots . per . location ( covid19 . confirmed . cases , geo . loc = " hubei " , confbnd = true ) # total nbr of deaths for " mainland china " tots . per . location ( covid19 . ts . deaths , geo . loc = " china " ) # ## # read the time series data for all the cases all . data <-covid19 . data ( 'ts -all ') # run on all the cases tots . per . location ( all . data , " japan " ) # ## # total for death cases for " all " the regions tots . per . location ( covid19 . ts . deaths ) # or just tots . per . location ( covid19 . data ( " ts -confirmed " ) ) listing 5: calculation of totals per country/region/province. in addition to the graphical output as shown in fig. 3 , the function will provide details of the models fitted to the data. similarly, utilizing the growth.rate function is possible to compute the actual growth rate and daily changes for specific locations, as defined in sec. 2.2. lst. 6 includes examples of these. # read time series data for confirmed cases ts . data <-covid19 . data ( " ts -confirmed " ) # compute changes and growth rates per location for all the countries growth . rate ( ts . data ) # compute changes and growth rates per location for ' italy ' growth . rate ( ts . data , geo . loc = " italy " ) # compute changes and growth rates per location for ' italy ' and ' germany ' growth . rate ( ts . data , geo . loc = c ( " italy " ," germany " ) ) # #### # combining multiple geographical locations : # obtain time series data tsconfirmed <-covid19 . data ( " ts -confirmed " ) # explore different combinations of regions / cities / countries # when combining different locations , heatmaps will also be generated comparing the trends among these locations growth . rate ( tsconfirmed , geo . loc = c ( " italy " ," canada " ," ontario " ," quebec " ," uruguay " ) ) growth . rate ( tsconfirmed , geo . loc = c ( " hubei " ," italy " ," spain " ," united ␣ states " ," canada " ," ontario " ," quebec " ," uruguay " ) ) growth . rate ( tsconfirmed , geo . loc = c ( " hubei " ," italy " ," spain " ," us " ," canada " ," ontario " , " quebec " ," uruguay " ) ) # turn off static plots and activate interactive figures growth . rate ( tsconfirmed , geo . loc = c ( " brazil " ," canada " ," ontario " ," us " ) , staticplt = # static and interactive figures growth . rate ( tsconfirmed , geo . loc = c ( " brazil " ," italy " ," india " ," us " ) , staticplt = true , interactivefig = true ) listing 6: calculation of growth rates and daily changes per country/region/province. in addition to the cumulative indicators described above, it is possible to estimate the global trends per location employing the functions single.trend, mtrends and itrends. the first two functions generate static plots of different quantities that can be used as indicators, while the third function generates an interactive representation of a normalized a-dimensional trend. the lst. 7 shows examples of the use of these functions. fig. 6 displays the graphical output produced by these functions. # single location trend , in this case using data from the city of toronto tor . data <-covid19 . toronto . data () single . trend ( tor . data [ tor . data $ status == " active ␣ cases " ,]) # or data from the province of ontario ts . data <-covid19 . data ( " ts -confirmed " ) ont . data <-ts . data [ ts . data $ province . state == " ontario " ,] single . trend ( ont . data ) # or from italy single . trend ( ts . data [ ts . data $ country . region == " italy " ,]) # multiple locations ts . data <-covid19 . data ( " ts -confirmed " ) mtrends ( ts . data , geo . loc = c ( " canada " ," ontario " ," uruguay " ," italy " ) ) # multiple cases single . trend ( tor . data ) # interactive plot of trends # for all locations and all type of cases itrends ( covid19 . data ( " ts -all " ) , geo . loc = " all " ) # or just for confirmed cases and some specific locations , saving the result in an html file named " itrends _ ex . html " itrends ( covid19 . data ( " ts -confirmed " ) , geo . loc = c ( " uruguay " ," argentina " ," ontario " ," us " ," italy " ," hubei " ) , filename = " itrends _ ex " ) listing 7: calculation of trends for different cases, utilizing the single.trend, mtrends and itrends functions. the typical representations can be seen in fig. 6. most of the analysis functions in the covid19.analytics package have already plotting and visualization capabilities. in addition to the previously described ones, the package has also specialized visualization functions as shown in lst.8. many of them will generate static and interactive figures, see table 3 for details of the type of output. in particular the live.map function is an utility function which allows to plot the location of the recorded cases around the world. this function in particular allows for several customizable features, such as, the type of projection used in the map or to select different types of projection operators in a pull down menu, displaying or not the legend of the regions, specify rescaling factors for the sizes representing the number of cases, among others. the function will generate a live representation of the cases, utilizing the plotly package and ultimately open the map in a browser, where the user can explore the map, drag the representation, zoom in/out, turn on/off legends, etc. # retrieve time series data ts . data <-covid19 . data ( " ts -all " ) # static and interactive plot totals . plt ( ts . data ) # totals for ontario and canada , without displaying totals and one plot per page totals . plt ( ts . data , c ( " canada " ," ontario " ) , with . totals = false , one . plt . per . page = true ) # totals for ontario , canada , italy and uruguay ; including global totals with the linear and semi -log plots arranged one next to the other totals . plt ( ts . data , c ( " canada " ," ontario " ," italy " ," uruguay " ) , with . totals = true , one . plt . per . page = false ) # totals for all the locations reported on the dataset , interactive plot will be saved as " totals -all . html " totals . plt ( ts . data , " all " , filename = " totals -all " ) # retrieve aggregated data data <-covid19 . data ( " aggregated " ) # interactive map of aggregated cases --with more spatial resolution live . map ( data ) # or live . map () # interactive map of the time series data of the confirmed cases with less spatial resolution , ie . aggregated by country live . map ( covid19 . data ( " ts -confirmed " ) ) listing 8: examples of some of the interactive and visualization capabilities of plotting functions. the typical representations can be seen in fig. 7 . last but not least, one the novel features added by the covid19.analytics package, is the ability of model the spread of the virus by incorporating real data. as described in sec. 2.2, the generate.sir.model function, implements a simple sir model employing the data reported from an specified dataset and a particular location. examples of this are shown in lst.9. the generate.sir.model function is complemented with the plt.sir.model function which can be used to generate static or interactive figures as shown in fig. 8 . the generate.sir.model function as described in sec.2 will attempt to obtain proper values for the parameters β and γ, by inferring the onset of the epidemic using the actual data. this is also listed in the output of the function (see lst.10), and it can be controlled by setting the parameters t0 and t1 or deltat, which are used to specify the range of dates to be considered for using when determining the values of β and γ. the fatality rate (constant) can also be indicated via the fatality.rate argument, as well, as the total population of the region with tot.population. # read time series data for confirmed cases data <-covid19 . data ( " ts -confirmed " ) # run a sir model for a given geographical location generate . sir . model ( data , " hubei " , t0 =1 , t1 =15) generate . sir . model ( data , " germany " , tot . population =83149300) generate . sir . model ( data , " uruguay " , tot . population =3500000) generate . sir . model ( data , " ontario " , tot . population =14570000 , add . extras = true ) # the function will aggregate data for a geographical location , like a country with multiple entries generate . sir . model ( data , " canada " , tot . population =37590000 , add . extras = true ) fig.(8) , also raises an interesting point regarding the accuracy of the sir model. we should recall that this is the simplest approach one could take in order to model the spread of diseases and usually more refined and complex models are used to incorporate several factors, such as, vaccination, quarantines, effects of social clusters, etc. however, in some cases, specially when the spread of the disease appears to have enter the so-called exponential growth rate, this simple sir model can capture the main trend of the dispersion (e.g. left plot from fig.8 ). while in other cases, when the rate of spread is slower than the freely exponential dispersion, the model clearly fails in tracking the actual evolution of cases (e.g. right plot from fig.8) . finally, lst. 11 shows an example of the generation of a sequence of values for r 0 , and actually any of the parameteres (β, γ) describing the sir model. in this case, the function takes a range of values for the initial date t0 and generates different date intervals, this allows the function to generate multiple sir models and return the corresponding parameters for each model. the results are then bundle in a "matrix"/"array" object which can be accessed by column for each model or by row for each paramter sets. # read timeseries data ts . data <-covid19 . data ( " ts -confirmed " ) # select a location of interest , eg . france # france has many entries , just pick " france " fr . data <-ts . data [ ( ts . data $ country . region == " france " ) & ( ts . data $ province . state == " " ) ,] # sweep values of r0 based on range of dates to consider for the model ranges <-15:25 deltat <-35 params _ sweep <-sweep . sir . models ( data = fr . data , geo . loc = " france " , t0 _ range = ranges , deltat = deltat ) # the parameters --beta , gamma , r0 --are returned in a " matrix " " array " object print ( params _ sweep ) as mentioned before, the functions from the covid19.analytics package also allow users to work with their own data, when the data is formated in the time series strucutre as discussed in sec.2.1.2. this opens a large range of possibilities for users to import their own data into r and use the functions already defined in the covid19.analytics package. a concrete example of how the data has to be formatted is shown in lst. 12. the example shows how to structure the data in a ts format from "synthetic" data generated from randomly sampling different distributions. however this could be actual data from other places or locations not accesible from the datasets provided by the package, or some researchers may have access to their own private sets of data too. the example also shows two cases, where the data can include the "status" column or not, and whether it could be more than one location. as a matter of fact, we left the "long" and "lat" fields empty but if one includes the actual coordinates, the maping function live.map can also be used with these structured data. # ts data structure : # " province . state " " country . region " " lat " " long " dates . . . # first let ' s create a ' fake ' location fake . locn <-c ( na , na , na , na ) # names for these columns names ( fake . locn ) <-c ( " province . state " ," country . region " ," lat " ," long " ) # let ' s set the dates dates . vec <-seq ( as . date ( " 2020 / 1 / 1 " ) , as . date ( " 2020 / 4 / 09 " ) , " days " ) # data . vecx would be the actual values / cases data . vec1 <-rpois ( length ( dates . vec ) , lambda =25) # can also add more cases data . vec2 <-abs ( rnorm ( length ( dates . vec ) , mean =135 , sd =15) ) data . vec3 <-abs ( rnorm ( length ( dates . vec ) , mean =35 , sd =5) ) # this will names the columns as your dates names ( data . vec1 ) <-dates . vec names ( data . vec2 ) <-dates . vec names ( data . vec3 ) <-dates . vec # merge them into a data frame with multiple entries synthetic . data <-as . data . frame ( rbind ( rbind ( c ( fake . locn , data . vec1 ) ) , rbind ( c ( fake . locn , data . vec2 ) ) , rbind ( c ( fake . locn , data . vec3 ) ) ) ) # finally set you locn to somethign unqiue , so you can use it in the generate . sir . model fn synthetic . data $ country . region <-" mylocn " # one could even add " status " synthetic . data $ status <-c ( " confirmed " ," death " ," recovered " ) # or just one case per locn synthetic . data2 <-synthetic . data [ , -ncol ( synthetic . data ) ] synthetic . data2 $ country . region <-c ( " mylocn " ," mylocn2 " ," mylocn3 " ) # now we can use this ' synthetic ' dataset with any of the ts functions # data checks integrity . check ( synthetic . data ) consistency . check ( synthetic . data ) data . checks ( synthetic . data ) # quantitative indicators tots . per . location ( synthetic . data ) growth . rate ( synthetic . data ) single . trend ( synthetic . data2 [3 ,] ) mtrends ( synthetic . data ) # sir models synthsir <-generate . sir . model ( synthetic . data2 , geo . loc = " mylocn " ) plt . sir . model ( synthsir , interactivefig = true ) sweep . sir . models ( synthetic . data2 , geo . loc = " mylocn " ) listing 12: example of structuring data in a ts format, so that it can be used with any of the ts functions from the covid19.analytics package. the covid19.analytics package provides access to genomics data available at the ncbi databases [35, 36] . the covid19.genomic.data is the master function for accesing the different variations of the genomics information available as shown in gtypes <-c ( " genome " ," fasta " ," tree " , " nucleotide " ," protein " , " nucleotide -fasta " ," protein -fasta " , " genomic " ) each of these functions return different objects, lst. 14 shows an example of the different structures for some of the objects. the most involved object is obtained from the covid19.genomic.data when combining different types of datasets. # str ( results ) list of 7 $ refgenome : list of 3 .. $ livedata : chr [1:29903] " a " " t " " t " " a " ... .. $ repo : chr [1:29903] " a " " t " " t " " a " ... .. $ local : chr [1:29903] " a " " t " " t " " a " . 29903] " a " " t " " t " " a " ... .. .. -attr ( * , " species " ) = chr " severe _ acute _ respiratory _ syndrome _ coronavirus _ 2 " .. $ local : list of 1 .. .. $ nc _ 045512.2: chr [1:29903] " a " " t " " t " " a " ... .. .. -attr ( * , " species " ) = chr " severe _ acute _ respiratory _ syndrome _ coronavirus _ 2 " $ ptns : list of 2 .. $ repo : chr [1:117619] " yp _ 009742608 " " yp _ 009742609 " " yp _ 009742610 " " yp _ 009742611 " ... .. $ local : chr [1:117619] " yp _ 009742608 " " yp _ 009742609 " " yp _ 009742610 " " yp _ 009742611 " ... : chr [1:11142] " 2020 -01 -13 t00 :00:00 z " " 2020 -07 -17 t00 :00:00 z " " 2020 -07 -17 t00 :00:00 z " " 2020 -07 -17 t00 :00:00 z " ... : chr [1:11142] " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " .. : chr [1:11142] " homo ␣ sapiens " " homo ␣ sapiens " " homo ␣ sapiens " " homo ␣ sapiens " ... .. $ isolation _ source : chr [1:11142] " " " " " " " " ... .. $ collection _ date : chr [1:11142] " 2019 -12 " " 2020 -06 -14 " " 2020 -06 -13 " " 2020 -06 -13 " ... .. $ biosample : chr [1:11142] " " " samn15488404 " " samn15488426 " " samn15488426 " ... .. $ genbank _ title : chr [1:11142] " severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2 ␣ isolate ␣ wuhan -hu -1 , ␣ complete ␣ genome " " severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2 ␣ isolate ␣ sars -cov -2 / human / ind / gbrc242 / 2020 , ␣ complete ␣ genome " " severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2 ␣ isolate ␣ sars -cov -2 / human / ind / gbrc264a / 2020 , ␣ complete ␣ genome " " severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2 ␣ isolate ␣ sars -cov -2 / human / ind / gbrc264b / 2020 , ␣ complete ␣ genome " ... $ proteins : ' data . frame ': 117619 obs . of 20 variables : .. $ accession : chr [1:117619] " yp _ 009742608 " " yp _ 009742609 " " yp _ 009742610 " " yp _ 009742611 " ... .. $ sra _ accession : chr [1:117619] " " " " " " " " ... .. $ release _ date : chr [1:117619] " 2020 -03 -30 t00 :00:00 z " " 2020 -03 -30 t00 :00:00 z " " 2020 -03 -30 t00 :00:00 z " " 2020 -03 -30 t00 :00:00 z " ... .. $ species : chr [1:117619] " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " " severe ␣ acute ␣ respiratory ␣ syndrome -related ␣ coronavirus " .. : chr [1:117619] " leader ␣ protein ␣ [ severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2] " " nsp2 ␣ [ severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2] " " nsp3 ␣ [ severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2] " " nsp4 ␣ [ severe ␣ acute ␣ respiratory ␣ syndrome ␣ coronavirus ␣ 2] " ... $ sra : list of 2 .. $ sra _ info : chr [1:6] " this ␣ download ␣ ( via ␣ ftp ) ␣ provides ␣ coronaviridae ␣ family -containing ␣ sra ␣ runs ␣ detected ␣ with ␣ ncbi ' s ␣ kmer ␣ analysis ␣ ( stat ) ␣ tool . ␣ " " it ␣ provides ␣ corresponding ␣ sra ␣ run ␣ ( srr ) ,␣ sample ␣ ( srs ) ,␣ and ␣ submission ␣ ( sra ) ␣ accessions , ␣ as ␣ well ␣ as ␣ biosample ␣ an " | _ _ truncated _ _ " the ␣ stat ␣ kmer ␣ analysis ␣ was ␣ performed ␣ via ␣ a ␣ two -step ␣ process ␣ with ␣ a ␣ 32 -mer ␣ coarse ␣ database ␣ and ␣ a ␣ 64 -mer ␣ fine ␣ database . ␣ " " the ␣ database ␣ is ␣ generated ␣ from ␣ refseq ␣ genomes ␣ and ␣ the ␣ viral ␣ genome ␣ set ␣ from ␣ nt ␣ using ␣ a ␣ minhash -based ␣ approach . ␣ " ... .. $ sra _ runs : ' data . frame ': 26776 obs . of 5 variables : .. .. $ acc : chr [1:26776] " err1857120 " " err1857132 " " err3955908 " " err3955909 " ... .. .. $ sample _ acc : chr [1:26776] " ers1572626 " " ers1572627 " " ers2783634 " " ers2783635 " ... .. .. $ biosample : chr [1:26776] " samea1 008836 68 " " same a10088 4418 " " samea4965217 " " samea4965218 " ... .. .. $ sra _ study : chr [1:26776] " erp021740 " " erp021740 " " erp111280 " " erp111280 " ... .. .. $ bioproject : chr [1:26776] " " " " " " " " ... $ references : list of 2 .. $ : chr " covid19 . analytics ␣ --␣ local ␣ data " .. $ : chr " / users / marcelo / library / r / 4.0 / library / covid19 . analytics / extdata / " listing 14: objects composition for the example presented in lst. 13 one aspect that should be mentioned with respect to the genomics data is that, in general, these are large datasets which are continuously being updated hence increasing theirs sizes even more. these would ultimately present pragmatical challenges, such as, long processing times or even starvation of memory resources. we will not dive into major interesting examples, like dna sequencing analysis or building phylogenetics trees; but packages such as ape, apegenet, phylocanvas, and others can be used for these and other analysis. one simple example we can present is the creation of dynamical categorization trees based on different elements of the sequencing data. we will consider for instance the data for the nucleotides as reported from ncbi. the example on lst. 15 shows how to retrieve either nucleotides (or proteins) data and generate categorization trees based on different elements, such as, hosting organism, geographical location, sequences length, etc. in the examples we employed the collapsibletree package, that generates interactive browsable trees through web browsers. # retrieve the nucleotides data nucx <-covid19 . genomic . data ( type = ' nucleotide ' , src = ' repo ') # identify specific fields to look at len . fld <-" length " acc . fld <-" accession " geoloc . fld <-" geo _ location " seq . fld <-" sequence _ type " host . fld <-" host " seq . limit1 <-25000 seq . limit1 <-29600 seq . limit2 <-31000 # selection criteria , nucleotides with seq . length between 29600 and 31000 selec . ctr .1 <-nucx $ length < seq . limit2 & nucx $ length > seq . limit1 # remove nucletoides without specifying a " host " listing 15: example of how to generate a dynamic browsable tree using some of information included in the nucleotides dataset. some of these trees representations are shown in fig. 9 . in this section we will present and discuss, how the covid19.analytics dashboard explorer is implemented. the main goal is to provide enough details about how the dashboard is implemented and works, so that users could modify it if/as they seem fit or even develop their own. for doing so, we will focus in three main points: • the front end implementation, also know as the user interface, mainly developed using the shiny package • the back end implementation, mostly using the covid19.analytics package • the web server installation and configuration where the dashboard is hosted the covid19.analytics dashboard explorer is built using the shiny package [43] in combination with the covid19.analytics package. shiny allows users to build interactive dashboards that can work through a web interface. the dashboard mimics the covid19.analytics package commands and features but enhances the commands as it allows users to use dropdowns and other control widgets to easily input the data rather than using a command terminal. in addition the dashboard offers some unique features, such as a personal protective equipment (ppe) model estimation, based on realistic projections developed by the us centers for disease control and prevention (cdc). the dashboard interface offers several features: 1. the dashboard can be run on the cloud/web allowing for multiple users to simultaneously analyze the data with no special software or hardware requirements. the shiny package makes the dashboard mobile and tablet compatible as well. 2. it aids researchers to share and discuss analytical findings. 3. the dashboard can be run locally or through the web server. 4. no programming or software expertise is required which reduces technical barriers to analyzing the data. users can interact and analyze the data without any software expertise therefore users can focus on the modeling and analysis. in these times the dashboard can be a monumental tool as it removes barriers and allows a wider and diverse set of users to have quick access to the data. 5. interactivity. one feature of shiny and other graphing packages, such as plotly, is interactivity, i.e. the ability to interact with the data. this allows one to display and show complex data in a concise manner and focus on specific points of interest. interactive options such as zoom, panning and mouse hover all help in making the user interaction enjoyable and informative. 6. fast and easy to compare. one advantage of a dashboard is that users can easily analyze and compare the data quickly and multiple times. for example users can change the slider or dropdown to select multiple countries to see the total daily count effortlessly. this allows the data to be displayed and changed as users analysis requirements change. the dashboard can be laucnhed locally in a machine with r, either through an interactive r session or in batch mode using rscript or r cmd batch or through the web server accessing the following url https://covid19analytics.scinet.utoronto.ca. for running the dashboard locally, the covid19.analytics package has also to be installed. for running the dashboard within an r session the package has to be loaded and then it should be invoked using the following sequence of commands, > library(covid19.analytics) > covid19explorer() the batch mode can be executed using an r script containing the commands listed above. when the dashboard is run locally the browser will open a port in the local machine -localhost:port-connection, i.e. http://127.0.0.1. it should be noted, that if the dashboard is launched interactively within an r session the port used is 5481 -http://127.0.0.1:5481-, while if this is done through an r script in batch mode the port used will be different. to implement the dashboard and enhance some of the basic functionalities offered, the following libraries were specifically used in the implementation of the dashboard: • shiny [43] : the main package that builds the dashboard. • shinydashboard [44] : this is a package that assists us to build the dashboard with respect to themes, layouts and structure. • shinycssloaders [45] : this package adds loader animations to shiny outputs such as plots and tables when they are loading or (re)-calculating. in general, this are wrappers around base css-style loaders. • plotly [46] : charting library to generate interactive charts and plots. although extensively used in the core functions of the covid19.analytics , we reiterate it here as it is great tool to develop interactive plots. • dt [47] : a datatable library to generate interactive table output. • dplyr [48] : a library that helps to apply functions and operations to data frames. this is important for calculations specifically in the ppe calculations. the r shiny package makes developing dashboards easy and seamless and removes challenges. for example setting the layout of a dashboard typically is challenging as it requires knowledge of frontend technologies such as html, css3 and bootstrap4 to be able to position elements and change there asthetic properties. shiny simplifies this problem by using a built in box controller widget which allows developers to easily group elements, tables, charts and widgets together. many of the css properties, such as, widths or colors are input parameters to the functions of interest. the sidebar feature is simple to implement and the shiny package makes it easy to be compatible across multiple devices such as tablets or cellphones. the shiny package also has built in layout properties such as fluidrow or columns making it easy to position elements on a page. the library does have some challenges as well. one challenge faced is theme design. shinydashboard does not make it easy to change the whole color theme of the dashboard outside of the white or blue theme that is provided by default. the issue is resolved by having the developer write custom css and change each of the various properties manually. the dashboard contains two main components a sidebar and a main body. the sidebar contains a list of all the menu options. options which are similar in nature are grouped in a nested format. for example the dashboard menu section called "datasets and reports", when selected, displays a nested list of further options the user can choose such as the world data or toronto data. grouping similar menu options together is important for making the user understand the data. the main body displays the content of a page. the content a main body displays depends on the sidebar and the selected menu option the user selects. there are three main generic elements needed to develop a dashboard: layouts, control widgets and output widgets. the layout options are components needed to layout the features or components on a page. in this dashboard the layout widgets used are the following: • box: the boxes are the main building blocks of a dashboard. this allows us to group content together. • tabpanels: tabpanels allow us to create tabs to divide one page into several sections. this allows for multiple charts or multiple types of data to be displayed in a single page. for example in the indicators page there are four tabs which display four different charts with mosaic tab displaying the charts in different configurations. • header and title: these are used to display text, title pages in the appropriate sizes and fonts. an example describing these elements and its implementation is shown in lst. 16 . ␣ table ' ) , h4 ( ' world ␣ data ␣ of ␣ all ␣ covid ␣ cases ␣ across ␣ the ␣ globe ') , column (4 , selectinput ( ns ( " category _ list3 " ) , label = h4 ( " category " ) , choices = category _ list ) ) , column (4 , downloadbutton ( ns ( ' downloaddata ') , " download " ) ) , withspinner ( dt :: datatableoutput ( ns ( " table _ contents " ) ) ) ) } listing 16: snippet of a code that describes the various features used in generating a dashboard. the ns(id) is a namespaced id for inputs/outputs. withspinner is the shiny cssloaders which generates a loading gif while the chart is being loaded. shiny modules are used when a shiny application gets larger and complicated, they can also be used to fix the namespacing problem. however shiny modules also allow for code reusability and code modularity as the code can be broken into several pieces called modules. each module can then be called in different applications or even in the same application multiple times. in this dashboard we break the code into two main groups user interface (ui) modules and server modules. each menu option has there own dedicated set of ui and associated server modules. this makes the code easy to build and expand. for each new menu option a new set of ui and sever module functions will be built. lst. 16 is also an example of an ui module, where it specifies the desing and look of the element and connect with the active parts of the application. lst. 20 shows an example of a server function called reportserver. this type of module can update and display charts, tables and valueboxes based on the user selections. this same scenario occurs for all menu options with the ui/server paradigm. another way to think about the ui/server separation, is that the ui modules are in charge of laying down the look of a particular element in the dahboard, while the sever is in charge of dynamically 'filling' the dynamical elements and data to populate this element. control widgets, also called input widgets, are widgets which users use to input data, information or settings to update charts, tables and other output widgets. the following control widgets were used in this dashboard: • numericalinput: a textbox that only allows for numerical input which is used to select a single numerical value. • selectinput: a dropdown that may be multi select for allowing users to select multiple options as in the case of the country dropdown. figure 10 : screenshot from the "covid19.analytics dashboard explorer", "mosaic" tab from the 'indicators' category. four interactive figures are shown in this case: the trends (generated using the itrends function), totals (genrated using the totals.plt function) and two world global representations of covid19 reported cases (generated using the live.map function). the two upper plots are adjusted and re-rendered according to the selection of the country, category of the data from the input boxes. • slider: the slider in our dashboard is purely numerical but used to select a single numerical value from a given range of a min and max range. • download button: this is a button which allows users to download and save data in various formats such as csv format. • radiobuttons: used to select only one from a limited number of choices. • checkbox: similar in purpose to radiobuttons that also allow users to select one option from a limited number of options. output control widgets are widgets that are used to display content/information back to the user. there are three main ouput widgets used in this dashboard: • plotlyouput: this widget output and creates plotly charts. plotly is a graphical package library used to generate interactive charts. • rendertable: is an output that generates the output as an interactive table with search, filter and sort capabilities provided out of the box. • valuebox: this is a fancy textbox with border colors and descriptive font text to generate descriptive text to users such as the total number of deaths. the dashboard contains the menus and elements shown in 17 and described below: • indicators: this menu section displays different covid19 indicators to analyze the pandemic. there are four notable indicators itrend, total plot, growth rate and live map, which are displayed in each of the various tabs. itrend displays the "trend" in a log-log plot, total plot shows a line graph of total number, growth rate displays the daily number of changes and growth rate (as defined in sec. 2.2), live map shows a world map of infections in an aggregated or timeseries format. these indicators are shown together in the "mosaic" tab. • models: this menu option contains a sub-menu presenting models related to the pandemic. the first model is the sir (susceptible infection recovery) which is implemented in the covid19.analytics package. sir is a compartmental model to model how a disease will infect a population. the second two models are used to estimate the amount of ppe needed due to infectious diseases, such as ebola and covid19. • datasets and reports: this section provides reporting capability which outputs reports as csv and text files for the data. the world data subsection displays all the world data as a table which can be filtered, sorted and searched. the data can also be saved as a csv file. the toronto data displays the toronto data in tabular format while also displaying the current pandemic numbers. data integrity section checks the integrity and consistency of the data set such as when the raw data contains negative numbers or if the cumulative quantities decrease. the report section is used to generate a report as a text file. • references: the reference section displays information on the github repo and documentation along with an external dashboards section which contains hyperlinks to other dashboards of interest. dashboards of interest are the vaccine tracker which tracks the progress of vaccines being tested for covid19, john hopkins university and the canada dashboard built by the dall lana school of epidemiology at the university of toronto. • about us: contact information and information about the developers. in addition to implementing some of the functionalities provided by the covid19.analytics package, the dashboard also includes a ppe calculator. the hospital ppe is a qualitative model which is designed to analyze the amount of ppe equipment needed for a single covid19 patient over a hospital duration. the ppe calculation implemented in the covid19.analytics dashboard explorer is derived from the cdc's studies for infectious diseases, such as ebola and covid19. the rationality is that ebola and covid19 are both contagious infections and ppe is used to protect staff and patients and prevent transmission for both of these contagious diseases. the hospital ppe calculation estimates and models the amount of ppe a hospital will need during the covid19 pandemic. there are two analysis methods a user can choose to determine hospital ppe requirement. the first method to analyze ppe is to determine the amount of ppe needed for a single hospitalized covid19 patient. this first model requires two major component: the size of the healthcare team needed to take care of a single covid19 patient and the amount of ppe equipment used by hospital staff per shift over a hosptialization duration. the model is based off the cdc ebola crisis calculation [49] . alhough ebola is a different disease compared to covid19, there is one major similarity. both covid19 and ebola are diseases which both require ppe for protection of healthcare staff and the infected patient. to compensate the user can change the amount of ppe a healthcare staff uses per shift. that information can be adjusted by changing the slider values in the advanced setting tab. the calculation is pretty straightforward as it takes the ppe amount used per shift and multiplies it by the number of healthcare staff and then by the hospitalization duration. the first model has two tabs. the first tab displays a stacked bar chart displaying the amount of ppe equipment user by each hospital staff over the total hospital duration of a single patient. it breaks each ppe equipment by stacks. the second tab panel called advanced settings has a series of sliders for each hospital staff example nurses where users can use the slider to change the amount of ppe that the hospital staff will user per shift. the second model is a more recent calculation developed by the cdc [50] . the model calculates the burn rate of ppe equipment for hospitals for a one week time period. this model is designed specifically for covid19. the cdc has created an excel file for hospital staff to input their information and also an android app as well which can be utilized. this model also implemented in our dashboard, is simplified to calculate the ppe for a one week setting. the one week limit was implemented for two reasons, first to limit the amount of input data a user has to enter into the system as too much data can overwhelm and confuse a user; second because the covid19 pandemic is a highly fluidic situation and for hospital staff to forecast their ppe and resource equipments greater than a one week period may not be accurate. note that this model is not accurate if the facilitiy recieves a resupply of ppe. for resupplied ppe start a new calculation. there are four tab panels to the burn rate calculation which displays charts and settings. the first tab daily usage displays a multi-line chart displaying the amount of ppe used daily, ∆p p e daily . the calculation for this is a simple subtraction between two consecutive days, i.e. the second day (j + 1) from the first day (j) as noted in eq. (6) . the tab panel called remaining supply shows a multi line chart the number of days the remaining ppe equipment will last in the facility. the duration of how long the ppe equipment can last in a given facility, inversely depends on the amount of covid19 patients admitted to the hospital. to calculate the remaining ppe one calculates the average amount of ppe used over the one week duration and then divides the amount of ppe at the beginning of the day by the average ppe usage, as shown in eq. (7), where t denotes the time average over a t period of time. the third panel called ppe per patient displays a multi line chart of the burn rate, i.e. the amount of ppe used per patient per day. eq.(8) represents the calculation as the remaining ppe supply divided by the number of covid19 patients in the hospital staff during that exact day. the fourth tab called advanced settings is a series of show and hide "accordians" where users can input the amount of ppe equpiment they have at the start of each day. there are six collapsed boxes for each ppe equipment type and for covid19 patient count. expanding a box displays seven numericalinput textboxes which allows users to input the number of ppe or patient count for each day. the equations describing the ppe needs, eqs. (6, 7, 8) are implemented in the shiny dashboard using the dplyr library. the dplyr library allows users to work with dataframe like objects in a quick and efficient manner. the three equations are implemented using a single dataframe. the advanced setting inputs of the burn rate analysis tab are saved into a dataframe. the ppe equations -eqs. ( the back-end implementation of the dashboard is achieved using the functions presented in sec.2 on the server module of the dashboard. the main strategy is to use a particular function and connect it with the input controls to feed the needed arguments into the function and then capture the output of the function and render it accordingly. let's consider the example of the globe map representation shown in the dashboard which is done using the live.map function. lst. 19 shows how this function connects with the other elements in the dashboard: the input elements are accessed using input$... which in this are used to control the particular options for the displaying the legends or projections based on checkboxes. the output returned from this function is captured through the renderplotly({...}) function, that is aimed to take plotly type of plots and integrate them into the dashboard. # livemap plot charts on the three possible commbinations output $ ts _ livemap <-output $ ts2 _ livemap <-output $ ts3 _ livemap <-output $ ts4 _ livemap <-renderplotly ({ legend <-input $ sel _ legend projections <-input $ sel _ projection live . map ( covid19 . data ( " ts -confirmed " ) , interactive . display = false , no . legend = legend , select . projctn = projections ) }) listing 19: example of how the live.map function is used to render the ineractive figures display on the dashboard. another example is the report generation capability using the report.summary function which is shown in lst. 20. as mentioned before, the input arguments of the function are obtained from the input controls. the output in this case is rendered usign the rendertext({...}) function, as the output of the original function is plain text. notice also that there are two implementations of the report.summary, one is for the rendering in the screen and the second one is for making the report available to be downloaded which is handled by the downloadhandler function. reportserver <-function ( input , output , session , result ) { output $ report _ output _ default <-rendertext ({ # extract the vairables of the inputs nentries <-input $ txtbox _ nentries geo _ loc <-input $ geo _ loc _ select6 ts <-input $ ddl _ ts capture . output ( report . summary ( graphical . output = false , nentries = nentries , geo . loc = geo _ loc , cases . to . process = ts ) ) } , sep = '\ n ') report <-reactive ({ nentries <-input $ txtbox _ nentries geo _ loc <-input $ geo _ loc _ select6 ts <-input $ ddl _ ts report <-capture . output ( report . summary ( graphical . output = false , nentries = nentries , geo . loc = geo _ loc , cases . to . process = ts ) ) return ( report ) }) output $ downloadreport <-downloadhandler ( filename = function () { paste ( " report -" , sys . date () ," . txt " , sep = " " ) } , content = function ( file ) { writelines ( paste ( report () ) , file ) } ) } listing 20: report capabilites implemented in the dashboard using the report.summary function. the final element in the deployment of the dashboard is the actual set up and configuration of the web server where the application will run. the actual implementation of our web dashboard, accessible through https://covid19analytics.scinet.utoronto.ca, relies on a virtual machine (vm) in a physical server located at scinet headquarters. we should also notice that there are other forms or ways to "publish" a dashboard, in particular for shiny based-dashboards, the most common way and perhaps straighforward one is to deploy the dashboard on https://www.shinyapps.io. alternatively one could also implement the dashboard in a cloud-based solution, e.g. https://aws.amazon.com/blogs/big-data/running-r-on-aws/. each approach has its own advantages and disadvantages, for instance, depending on a third party solution (like the previous mentioned) implies some cost to be paid to or dependency on the provider but will certainly eliminate some of the complexity and special attention one must take when using its own server. on the other hand, a self-deployed server will allow you for full control, in principle cost-effective or cost-controled expense and full integration with the end application. in our case, we opted for a self-controlled and configured server as mentioned above. moreover, it is quite a common practice to deploy (multiple) web services via vms or "containers". the vm for our web server runs on centos 7 and has installed r version 4.0 from sources and compiled on the vm. after that we proceeded to install the shiny server from sources, i.e. https://github.com/rstudio/ shiny-server/wiki/building-shiny-server-from-source. after the installation of the shiny-server is completed, we proceed by creating a new user in the vm from where the server is going to be run. for security reasons, we recommend to avoid running the server as root. in general, the shiny server can use a user named "shiny". hence a local account is created for this user, and then logged as this user, one can proceed with the installation of the required r packages in a local library for this user. all the neeeded packages for running the dashboard and the covid19.analytics package need to be installed. lst. 21 shows the commands used for creating the shiny user and finalizing the configuration and details of the log files. # place a shortcut to the shiny -server executable in / usr / bin sudo ln -s / usr / local / shiny -server / bin / shiny -server / usr / bin / shiny -server # create shiny user sudo useradd -r -m shiny # create log , config , and application directories sudo mkdir -p / var / log / shiny -server sudo mkdir -p / srv / shiny -server sudo mkdir -p / var / lib / shiny -server sudo chown shiny / var / log / shiny -server sudo mkdir -p / etc / shiny -server listing 21: list of commands used on the vm to finalize the setup of the shiny user and server. source: https: //github.com/rstudio/shiny-server. for dealing with the apache configuration on port 80, we added the file /etc/httpd/conf.d/rewrite.conf as shown in lst. 22. rewritecond %{ request _ scheme } = http rewriterule^https : / / %{ server _ name }%{ request _ uri } [ qsa , r = permanent ] listing 22: modifications to the apache configurations, specified in the file rewrite.conf. these three lines rewrite any incoming request from http to https. for handling the apache configuration on port 443, we added this file /etc/httpd/conf.d/shiny.conf, as shown in lst.23. this virtualhost receives the https requests from the internet on port 443, establishes the secure connection, and redirects all input to port 3838 using plain http. all requests to "/" are redirected to "http://0.0.0.0:3838/app1/", where app1 in this case is a subdirectory where a particular shiny app is located. there is an additional configuration file, /etc/httpd/conf.d/ssl.conf, which contains the configuration for establishing secure connections such as protocols, certificate paths, ciphers, etc. the main tool we use in order to communicate updates between the different elements we use in the development and mantainance of the covid19.analytics package and dashboard web interface is orchestrated via git repositories. in this way, we have in place version control systems but also offer decentralized with multiple replicas. fig. 11 shows an schematic of how our network of repositories and service is connected. the central hub for our package, is located at the github repo htttps: //github.com/mponce0/covid19.analytics; we then have (and users can too) our own clones of local copies of this repo -we usually use this for development and testing-. when a stable and substantial contribution to the package is reached, we submit this to the cran repository. similarly, when an update is done on the dashboard we can synchronize the vm via git pulls and deploy the updates on the server side. in this paper we have presented and discussed the r covid19.analytics package, which is an open source tool to obtain, analyze and visualize data of the covid19 pandemic. the package also incorporates a dashboard to facilitate the access to its functionalities to less experienced users. as today, there are a few dozen other packages also in the cran repository that allow users to gain access to different datasets of the covid19 pandemic. in some cases, some packages just provide access to data from specific geographical locations or the approach to the data structure in which the data is presented is different from the one presented here. nevertheless, having a variety of packages from which users can try and probably combine, is an important and crucial element in data analysis. moreover it has been reported different cases of data misuse/misinterpretation due to different issues, such as, erroneous metadata or data formats [51] and in some cases ending in articles' retractions [52] . therefore providing additional functionalities to check the integrity and consistency of the data, as our the covid19.analytics package github repo -central repository https://github.com/mponce0/covid19.analytics shiny server, running on vm https://covid19analytics.scinet.utoronto.ca local copies cran repo https://cran.r-project.org/package=covid19.analytics local copies local copies github io -web rendering https://mponce0.github.io/covid19.analytics/ private instances figure 11 : schematic of the different repositories and systems employed by the covid19.analytics package and dashboard interface. does is paramount. this is specially true in a situation where the unfolding of events and data availability is flowing so fast that sometimes is even hard to keep track of all the changes. moreover, the covid19.analytics package offers a modular and versatile approach to the data, by allowing users to input their own data for which most of the package functions can be applied when the data is structured using a time series format as described in this manuscript. the covid19.analytics is also capable of retrieving genomics data, and it does that by incorporating a novel, more reliable and robust way of accessing and designing different pathways to the data sources. another unique feature of this package is the ability of incorporating models to estimate the disease spread by using the actual data. although a simple model, it has shown some interesting results in agreement for certain cases. of course there are more sophisticated approaches to shred light in analyzing this pandemic; in particular novel "community" approaches have been catalyzed by this too [53] . however all of these approaches face new challenges as well [54] , and on that regards counting with a variety, in particular of open source tools and direct access to the data might help on this front. r: a language and environment for statistical computing, r foundation for statistical computing r: a language for data analysis and graphics covid19.analytics: load and analyze live data from the covid-19 pandemic the biggest mystery: what it will take to trace the coronavirus source animal source of the coronavirus continues to elude scientists a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 bat-borne virus diversity, spillover and emergence extrapulmonary manifestations of covid-19 opensafely: factors associated with covid-19 death in 17 million patients considering how biological sex impacts immune responses and covid-19 outcomes coronavirus blood-clot mystery intensifies using influenza surveillance networks to estimate state-specific prevalence of sars-cov-2 in the united states consolidation in a crisis: patterns of international collaboration in early covid-19 research critiqued coronavirus simulation gets thumbs up from code-checking efforts timing social distancing to avert unmanageable covid-19 hospital surges special report: the simulations driving the world's response to covid-19 covid-19 vaccine design: the janus face of immune enhancement covidep: a web-based platform for real-time reporting of vaccine target recommendations for sars-cov-2 social network-based distancing strategies to flatten the covid-19 curve in a post-lockdown world asymptotic estimates of sarscov-2 infection counts and their sensitivity to stochastic perturbation evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic an interactive web-based dashboard to track covid-19 in real time pandemic publishing poses a new covid-19 challenge will the pandemic permanently alter scientific publishing? how swamped preprint servers are blocking bad coronavirus research project, trainees, faculty, advancing scientific knowledge in times of pandemics covid-19 risk factors: literature database & meta-analysis coronawhy: building a distributed, credible and scalable research and data infrastructure for open science, scinlp: natural language processing and data mining for scientific text the comprehensive r archive network covid-19 data repository by the center for systems science and engineering covid-19: status of cases in toronto database resources of the national center for biotechnology information ape 5.0: an environment for modern phylogenetics and evolutionary analyses in r rentrez: an r package for the ncbi eutils api a contribution to the mathematical theory of epidemics the sir model for spread of disease: the differential equation model, loci.(originally convergence exact analytical solutions of the susceptible-infected-recovered (sir) epidemic model and of the sir model with equal death and birth rates devtools: tools to make developing r packages easier shiny: web application framework for r shinydashboard: create dashboards with 'shiny', r package version 0 shinycssloaders: add css loading animations to 'shiny' outputs interactive web-based data visualization with r, plotly, and shiny, chapman and hall/crc, 2020 dt: a wrapper of the javascript library 'datatables', r package version 0 dplyr: a grammar of data manipulation estimated personal protective equipment (ppe) needed for healthcare facilities personal protective equipment (ppe) burn rate calculator high-profile coronavirus retractions raise concerns about data oversight covid-19 pandemic reveals the peril of ignoring metadata standards artificial intelligence cooperation to support the global response to covid-19 the challenges of deploying artificial intelligence models in a rapidly evolving pandemic the r script containing the shiny app to be run should be placed in /etc/shiny-server and confiurations details about the shiny interface are adjusted in the /etc/shiny-server/shiny-server.conf file.permissions for the application file has to match the identity of the user launching the server, in this case the shiny user.at this point if the installation was sucessful and all the pieces were placed properly, when the shiny-server command is executed, a shiny hosted app will be accessible from localhost:3838.since the shiny server listens on port 3838 in plain http, it is necessary to setup an apache web server to act as a reverse proxy to receive the connection requests from the internet on ports 80 and 443, the regular http and https ports, and redirect them to port 3838 on the same host (localhost). mp wants to thank all his colleagues at scinet, especially daniel gruner for his continous and unconditional support, and marco saldarriaga who helped us setting up the vm for installing the shiny server. key: cord-011745-dbdtpojs authors: thompson, mark g.; sokolow, leslie z.; almendares, olivia; openo, kyle; farley, monica m.; meek, james; ray, julie; kirley, pamala daily; reingold, arthur; aragon, deborah; hancock, emily; baumbach, joan; schaffner, william; lynfield, ruth; ryan, pat; monroe, maya; cheng, po-yung; fry, alicia m.; shay, david k. title: effectiveness of nonadjuvanted monovalent influenza a(h1n1)pdm09 vaccines for preventing reverse transcription polymerase chain reaction–confirmed pandemic influenza hospitalizations: case-control study of children and adults at 10 us influenza surveillance network sites date: 2013-12-01 journal: clin infect dis doi: 10.1093/cid/cit551 sha: doc_id: 11745 cord_uid: dbdtpojs during 2009–2010, we examined 217 patients hospitalized with laboratory-confirmed pandemic influenza in 9 influenza hospitalization surveillance network sites and 413 ageand community-matched controls and found that a single dose of monovalent nonadjuvanted influenza a(h1n1)pdm09 vaccine was 50% (95% confidence interval, 13%–71%) effective in preventing hospitalization associated with a(h1n1)pdm09 virus infection. during 2009-2010, we examined 217 patients hospitalized with laboratory-confirmed pandemic influenza in 9 influenza hospitalization surveillance network sites and 413 ageand community-matched controls and found that a single dose of monovalent nonadjuvanted influenza a(h1n1)pdm09 vaccine was 50% (95% confidence interval, 13%-71%) effective in preventing hospitalization associated with a(h1n1) pdm09 virus infection. keywords. influenza; influenza vaccines; vaccine effectiveness; hospitalization; pandemic/prevention & control. the 2009 influenza a(h1n1) pandemic, which began in 2009 and continued into 2010, resulted in increased hospitalizations in the united states and globally [1, 2] . a recent review reported that monovalent a(h1n1)pdm09 vaccines were 69% effective in preventing medically attended influenza during the pandemic [3] . studies focused specifically on hospitalization have reported estimates of vaccine effectiveness (ve) that ranged from 49% to 90% [3] [4] [5] [6] ; however, to date, no estimate of ve against these outcomes in the united states has been published. we identified patients with community-acquired, laboratoryconfirmed a(h1n1)pdm09 infections through the influenza hospitalization surveillance network (flusurv-net) within the centers for disease control and prevention's (cdc) emerging infections program. flusurv-net conducts populationbased surveillance of influenza-associated hospitalizations among children and adults from select counties in 9 states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, oregon, tennessee) that represent approximately 7.3% of the us population [1, 7] . although a(h1n1) pdm09 vaccine was initially only available to priority groups [8] , enrollment for this study started after vaccine became available to the general population and at least 10% of the local population was estimated to be vaccinated (november-december 2009; supplementary figure) . cases were patients aged >6 months residing in a flusurv-net catchment area who tested positive for a(h1n1)pdm09 influenza by reverse transcription polymerase chain reaction (rt-pcr) assay from respiratory specimens collected shortly before (1%) or upon (99%) admission to the hospital for an acute respiratory illness; 86% of specimens were collected within 7 days of illness onset. patients in longterm care facilities and those with nosocomial infections were not enrolled. for each rt-pcr-confirmed hospitalized influenza case, 2 community controls matched by age group and county of residence were enrolled. persons who had not been hospitalized for a respiratory illness from 1 october 2009 until the hospital admission date of a corresponding case were eligible for enrollment. control children were matched on differing age bands depending on age (children aged 7-23 months were matched ±2 weeks of the case's birth; those aged 24-59 months were matched ±2 months of the case's birth; children aged 5-13 years were matched in 3-or 4-year age bands, ie, 5-7 years, 8-10 years, 11-13 years, and 14-17 years; and adults were matched in 5-year age bands, with the exception of a single 18-24 years age band). controls were recruited from individuals identified through birth certificate registries (for children aged <5 years) or lists of random landline telephone numbers (for those aged ≥5 years). structured telephone interviews (forms available upon request) were conducted with cases after hospital discharge and their matched controls; guardians were interviewed for participants aged <18 years. participant characteristics, vaccination status and date, and the presence of medical conditions associated with increased risk of influenza complications [9] were collected from telephone interviews, from reviews of medical records at primary care providers, and through review of hospital records for hospitalized cases. receipt of at least 1 dose of vaccine was documented by medical record or by self-report (if date and location of vaccination could be provided). high-risk conditions and vaccination status were documented by self-report only for the 35% of cases and 34% of controls for whom medical records were unavailable. immunization was defined as receipt of any monovalent a (h1n1)pdm09 vaccine ≥14 days before illness onset for the case; for matched controls, the illness onset date of their corresponding case was used. ve was estimated as 100% × (1odds ratio [ratio of odds of being immunized among the cases to the odds of being immunized among the controls]) using conditional logistic regression models. the following covariates in our adjusted models were similar to those used in other recent pandemic ve studies [5, 6, 8] : age, race, ethnicity, region, highrisk medical condition, and month of index-case illness onset. other covariates were considered for inclusion in the model as confounders if they were related to both vaccination and influenza status or if they affected the ve point estimate by >5%. we estimated that 78 cases were needed to achieve 80% power (α = .05) to detect a ve of 60% with 2 controls per case and a vaccination rate of 30% among controls. each participating site submitted a protocol for evaluation by their state institutional review board (irb), and irb approval was obtained if required. april 2010, a total of 329 hospitalized patients with rt-pcr-confirmed a(h1n1)pdm09 infection were identified as potential study cases; 24 pediatric and 88 adult patients were excluded either because they did not give consent (n = 107) or because vaccination status could not be confirmed (n = 5). enrolled cases were similar to all eligible cases with respect to age and onset month (data not shown). only 1 matched control was enrolled for 10% of the cases (21 of 217). compared with community controls, hospitalized influenza cases were more likely to be non-white, hispanic, and unmarried; have less education and lower incomes; lack private healthcare insurance; be obese; and have high-risk medical conditions (table 1) . among cases, the proportion immunized differed by age; among controls, hispanics and those with a high-risk medical condition were more likely to be immunized; no other significant association between participant characteristics and immunization status was observed. among 217 hospitalized influenza cases, 14% were immunized compared to 22% immunized among 413 community controls. the crude ve against a(h1n1)pdm09 for prevention of hospitalization was 38% (95% confidence interval [ci], 4%-61%) for all ages. the ve adjusted for age, race, ethnicity, region, high-risk respiratory condition, and month of index case illness onset was 46% (11%-67%). potential confounding was observed for education, insurance status, and presence of a nonpulmonary high-risk medical condition. adding these to the multivariate model resulted in a fully adjusted ve of 50% (95% ci, 13%-71%). hispanic ethnicity, lower education, lack of healthcare insurance, and pulmonary and nonpulmonary high-risk medical conditions were all statistically significant contributors in the final multivariate model (data not shown). in secondary stratified analyses, the adjusted ve for those with 1 or more high-risk medical condition was 54% (95% ci, 16%-75%), and the ve point estimate for those without a high-risk condition was similar at 46% (95% ci, −89% to 84%), although confidence intervals were wide given the small number of cases without a high-risk condition. in sensitivity analyses, ve estimates were unchanged when those vaccinated 7-13 days prior to the illness onset of the index case were considered immunized. we estimated that during the winter wave of the 2009 influenza a(h1n1)pdm09 pandemic in the united states, a single dose of monovalent nonadjuvanted a(h1n1)pdm09 vaccine was 50% (95% ci, 13%-71%) effective in preventing hospitalization associated with a(h1n1)pdm09 virus infection. this finding is similar to the adjusted ve of 56% (23%-75%) against medically attended a(h1n1)pdm09 influenza reported by a us study with the same vaccine options in which 9% of the cases were hospitalized [8] . our ve point estimate of 50% was consistent with ve estimates in an australian study of hospitalization and unadjuvanted pandemic vaccine that used a test-negative control design (ve = 49%; 95% ci, 13%-70%) [6] , but was substantially lower than the inpatient ve reported by a european study of adjuvanted pandemic vaccine that used a test-negative control design (ve = 90%; 95% ci, 48%-100%) [5] . the strengths of this study include case ascertainment through population-based surveillance and confirmation of abbreviations: bmi, body mass index; ca, california; co, colorado; ct, connecticut; ga, georgia; md, maryland; mn, minnesota; nm, new mexico; ns, not statistically significant (p > .05); or, oregon; tn, tennessee. a two controls who had not been hospitalized for a respiratory illness since 1 october 2009 were recruited matched by age and community. for cases aged 7-23 months, controls were within plus or minus (±) 2 weeks of the case's age at illness onset; for cases aged 24-59 months, controls were ±2 months of the case's age. potential controls for cases aged <5 years were selected at random from birth certificate registries and matched to the case's zip code. for cases aged >5 years, controls were within ±7 years of the case's age, resided in the case's county, and were contacted from a telephone sampling list generated by survey sampling international (shelton, connecticut). b pearson χ 2 test was used to assess differences between cases and controls in the distribution of participant characteristics. c pearson χ 2 test was used to assess differences between participant characteristic groups (rows) in the percentage vaccinated; differences in vaccination by participant characteristics were tested separately with cases and then controls. d characteristic describes interviewed parent/caregiver when participant is aged <18 years. e high-risk respiratory conditions included lung disease and asthma as indicated by self-report of physician diagnosis and/or a medical encounter with international classification of disease (icd-9) codes available upon request. other high-risk medical conditions were indicated by self-report of physician diagnosis and/or 1 or more medical visits for a condition associated with increased risk of influenza complications, including cancer, diabetes, and neurological disorders as well as heart, immune, and kidney disease (icd codes available upon request). medical conditions were determined by self-report for 35% of cases and 34% of controls with missing or unavailable medical records. f vaccination is defined as receipt of at least 1 dose of vaccine documented by medical record or self-report (if date and location were also provided); immunization is defined as receipt of any a(h1n1)pdm09 vaccine ≥14 days before illness onset of the case or index case if a matched control. the difference between the number vaccinated and the number immunized reflects the fact that 15 cases and 24 controls were vaccinated after the illness onset date (of the index case) and 8 cases and 11 controls were vaccinated 1-13 days before the illness onset date. vaccination status and dates were determined by self-report for 35% of cases and 34% of controls with missing or unavailable medical records. influenza by rt-pcr. the study addresses a gap in knowledge regarding ve against serious influenza complications during the pandemic and the ve of nonadjuvanted vaccines, which may have fewer local and systemic reactions but can be less immunogenic in some populations [10] . our study also has limitations. first, cases and matched controls differed on multiple characteristics. although adjusting for potential confounders increased the ve point estimate from 38% to 50%, residual or unmeasured confounding may have biased our results in unknown ways. second, our limited sample size resulted in ve estimates with wide confidence intervals and precluded stratification by age or consideration of site differences in a mixed-effects model. third, our findings may have been influenced by information and selection biases, as only residents with telephone landlines could be enrolled as controls (for those aged ≥5 years) and medical history was incomplete for one-third of cases and controls. because the proportion with missing information was similar for cases and controls, we do not expect this was a significant source of bias, but bias could have been introduced if the likelihood of recalling vaccination differed for cases vs controls with only selfreport data. fourth, information on vaccine type (inactivated vs live attenuated) and on receipt of a second recommended dose (among children aged <10 years) was not available for every subject, which likely resulted in underestimation of ve, as other studies found that ve improved after accounting for these differences [8] . finally, similar to other ve estimates [5, 6, 8] , we lacked information on medical utilization or possible infections during earlier waves of the pandemic. natural immunity acquired from infection prior to the availability of vaccine would lead to over-or underestimating ve if those immune were more or less likely to be vaccinated, respectively. in conclusion, our results suggest that a single dose of monovalent nonadjuvanted a(h1n1)pdm09 vaccine prevented onehalf of the potential hospitalizations associated with a(h1n1) pdm09 virus infection. this finding from the flusurv-net, which serves geographically and economically diverse communities across the united states, confirms previous reports of the preventive benefit of the vaccine [8, 11] and adds to the evidence indicating that influenza vaccines have the potential to prevent a substantial proportion of influenza-associated hospitalizations [12] . increase in rates of hospitalization due to laboratory-confirmed influenza among children and adults during the 2009-10 influenza pandemic epidemiology of 2009 pandemic influenza a (h1n1) in the united states efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis estimating the effect of influenza vaccines and pandemic vaccines, to prevent influenza hospitalizations during the autumn 2009 influenza pandemic wave in castellon, spain. a test-negative, hospital-based, case-control study effectiveness of h1n1/09 monovalent and trivalent influenza vaccines against hospitalization with laboratory-confirmed h1n1/09 influenza in australia: a test-negative case control study adult hospitalizations for laboratory-positive influenza during the effectiveness of non-adjuvanted pandemic influenza a vaccines for preventing pandemic influenza acute respiratory illness visits in 4 u.s. communities prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) immunogenicity and safety of as03-adjuvanted 2009 influenza a h1n1 vaccine in children 6-35 months effectiveness of 1 dose of influenza a (h1n1) 2009 monovalent vaccines in preventing reverse-transcription polymerase chain reaction-confirmed h1n1 infection among school-aged children in maine effectiveness of seasonal vaccine in preventing confirmed influenza-associated hospitalizations in community dwelling older adults disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the cdc.potential conflicts of interest. w. s. serves as a consultant for pfizer, inc, and dynavax technologies; has received an honorarium from sanofi pasteur; and is a member of the advisory board for the data safety monitoring board for merck & co, inc. r. l. has received royalties from a book chapter published by blackwell-wiley and travel expenses partially paid by the global pertussis initiative (parexel international). all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-256995-itiz6mqv authors: christoffersen, s. title: the importance of microbiological testing for establishing cause of death in 42 forensic autopsies date: 2015-05-31 journal: forensic science international doi: 10.1016/j.forsciint.2015.02.020 sha: doc_id: 256995 cord_uid: itiz6mqv abstract microorganisms have always been one of the great challenges of humankind, being responsible for both high morbidity and mortality throughout history. in a forensic setting microbiological information will always be difficult to interpret due to lack of antemortem information and changes in flora postmortem. with this study we aim to review the use of microbiological procedures at our forensic institute. in a retrospective study including 42 autopsies performed at our institute, where microbiological test had been applied, analyses were made with regard to: type of microbiological tests performed, microorganisms found, histological findings, antemortem information, c-reactive protein measurement and cause of death. fiftyone different microorganisms were found distributed among 37 cases, bacteria being the most abundant. nineteen of the cases were classified as having a microbiological related cause of death. c-reactive protein levels were raised in 14 cases of the 19 cases, histological findings either supported or were a decisive factor for the classification of microbiologically related cause of death in 14 cases. as a multitude of abundant microorganisms are able to cause infection under the right circumstances, all findings should be compared to anamnestic antemortem information, before conclusions are drawn. a definite list of true pathogens is nearly impossible to compile. microorganisms have always been one of the great challenges of humankind, being responsible for both high morbidity and mortality throughout history. in a forensic setting microbiological information will always be difficult to interpret due to lack of antemortem information and changes in flora postmortem. with this study we aim to review the use of microbiological procedures at our forensic institute. in a retrospective study including 42 autopsies performed at our institute, where microbiological test had been applied, analyses were made with regard to: type of microbiological tests performed, microorganisms found, histological findings, antemortem information, c-reactive protein measurement and cause of death. fiftyone different microorganisms were found distributed among 37 cases, bacteria being the most abundant. nineteen of the cases were classified as having a microbiological related cause of death. creactive protein levels were raised in 14 cases of the 19 cases, histological findings either supported or were a decisive factor for the classification of microbiologically related cause of death in 14 cases. as a multitude of abundant microorganisms are able to cause infection under the right circumstances, all findings should be compared to anamnestic antemortem information, before conclusions are drawn. a definite list of true pathogens is nearly impossible to compile. ß 2015 elsevier ireland ltd. all rights reserved. and/or swaps are collected once suspicion is raised by gross pathology findings during the autopsy. tissue samples were analyzed at the department of clinical microbiology, university hospital of odense. samples are routinely analyzed for bacteria and fungi, using agar culturing. pcr analysis for viruses is performed per request. crp was measured in full blood using ''quickread crp'' produced by orion diagnostica. the ''quickread crp'' supplies results in the range of 8-160 mg/l, results below or above this interval will be noted as <8 and >160 respectively. though an exact cut off value for crp on postmortem material is not easy established [6] , values above 10 mg/l are considered raised. the age ranged from 2½ months to 84 years, 8 cases were children under the age of 2, one was between 2 and 15 years old, 23 were between 16 and 60, and 10 were older than 60 years. twenty-eight were male and 14 were female. in five cases, no microorganisms were found. bacteria were found in 36 (85.7%) of the cases, amounting to 247 of 354 (69.7%) samples taken. viruses were found in 3 of 22 cases (1.4%). fungi were found in eight cases (19.0%), amounting to 21 of 354 (5.9%) samples taken. no other named fungi than candida albicans was found. in one case c. albicans was the only microbiological finding. bacteria were found in 16 cases where they were not related to cod. virus was only found in one case which was not classified as an infectious cod. in the 42 autopsies cod was classified as infectious in 18 cases (42.8%), distributed among: three cases of viral pneumonia (16.7%), three cases of bacterial pneumonia (16.7%), four cases of sepsis (22.2%), two cases of meningitis (11.1%), two cases of peritonitis, one due to accidental perforation of the intestine during surgery and one due to perforated appendicitis (11.1%), one case of pericarditis (5.6%) and three cases of combined opiate poisoning and infection (16.7%), table 1 . the cod of the remaining 24 cases, which were not related to microbiological findings, are listed in table 2 . in all, 52 different microorganisms were found including one report of ''normal flora'', table 3 shows the distribution. testing in one case, classified as viral pneumonia cod, did not yield any positive viral findings. however the histological findings were consistent with viral pneumonia (interstitial pneumonitis), and the person had been treated with antiviral antibiotics before death which could cause the negative viral findings [7] . crp was measured in 33 cases. in nine cases crp was below 8 mg/l. regarding the 18 cases classified as infectious cod, crp values were below 8 mg/l in two cases, 13 cases had elevated crp values (p < 0.05), and in three cases no measurement was performed. of the 18 cases with an infectious cod, histological examination was of importance in 14 cases (77.8%). in two of the 14 cases no microorganisms were found but the histological changes, crp values and other information lead to the conclusion (one case of viral pneumonia and one case of neutrophilic pneumonia in an opiate poisoning case). in four cases, histological findings did not show inflammation to support the microbiological findings, however crp levels above 160 mg/l and the bacteriological results were sufficient to verify the microbiologically related cod. culturing bacteria on agar plates is an old invention whereas polymerase chain reaction (pcr) used for viral detection is a fairly new technique and is still costly compared to agar cultures. hence bacteria and their colonization of the body is the most thoroughly investigated group of pathogens. a single study has used pcr for bacterial detection with excellent results [8] . though this might be the way to proceed in the future, the skill and equipment required for pcr, compared to using agar plates, could mean that it will not be the investigation of choice in the near future on a worldwide basis. the key point in interpreting postmortem microbiological results is to determine whether detected microorganisms arise from postmortem spread or are the result of true antemortem infection. larsen et al. suggest that to be sure a bacteria was present in the bloodstream before death, it must be cultured from at least two different sites [9] . however authors do not agree on the most reliable sites for sampling, blood, lung and spleen are generally mentioned, as well as cerebrospinal fluid [10, 11] . tuomisto et al. [8] suggest liver and pericardial sampling as the most reliable within the first five days after death. preferably, cultures should be verified by gross pathologic or histologic findings [7, 9] . when bacteria are cultured postmortem, one must consider several mechanisms which could lead to bacteria in the samples. four lines of spread have been suggested [7, 12] . a microorganism has genuinely invaded the bloodstream or target organ during life leading to bacteriaemia/ infection/sepsis, the group of cases which could be classified with a microbiological cod. 2. agonal spread: according to this theory the ischemia in the agonal period or during resuscitation lead to mucosal damage allowing bacteria to invade through the surface in question. 3. postmortem translocation: the movement of naturally present bacteria across the mucosal barriers, into the surrounding tissue, bloodstream and body. one should expect a mixed growth and not a single isolate bacteria [13] . 4. contamination: microorganisms are introduced into the tissue at time of sampling, and were not present in life. the theory of agonal spread is according to a review by morris et al., considered unlikely [13] . several papers find that elapsed time from death to sampling point does not have a significant influence on false positive bacterial results provided that bodies are kept refrigerated [11, [13] [14] [15] . however animal experiments performed by heimesat et al. [16] do show a rather large increase in bacteria in extra-intestinal tissue especially up to 72 h after death. perhaps even more interesting, they observe a rise in extraintestinal bacteria already five minutes after death. maujean et al. report an interesting case in which neisseria meningitidis was detected in a putrefied body after 10 days using molecular analyses [17] . pryce et al. compared antemortem vs. postmortem sampling and concluded that postmortem sampling is as valuable as antemortem, when related to an infectious cod [18] . the risk of contamination from the autopsy room seems to be small [19] . table 3 shows the vast diversity of microorganisms found in our cases, and illustrates the large number of possible pathogens. the majority of bacteria could be considered postmortem translocation, however as many of the microorganisms contributing to our normal flora can become pathogens under the right circumstances, all findings must be carefully considered. in our study bacteria were found in 36 cases, 16 of these cases were not classified as having infectious cod, adding to the presumption of a significant postmortem spread. the vast majority of the microorganisms found in these cases are part of our natural flora, and are to be considered opportunistic pathogens, several being able to cause both pneumonia and bacteremia. in six of these 15 cases no clear cod was found, four were sids and two unexplained under the age of two (table 4 ). histology did not show inflammation in any of the samples, and none of the cases had elevated crp values, though in two cases crp measurement was not performed. not surprisingly, these problematic cases are all infants. as several of the bacteria are possible pathogens, infection cannot be completely ruled out due to the fact that a not fully developed immune system could be responsible for the lack of both inflammatory response and rise in crp values. several more or less commonly encountered microorganisms, such as n. meningitidis, neisseria gonorrhoeae, haemophilus influenzae, salmonella species, staphylococcus aureus, streptococcus pneumoniae, b-hemolytic streptococci, klebsiella species, escherichia coli, mycobacterium tuberculosis, members of the enterobacteriaceae and c. albicans should, according to some authors, always be considered pathogens [10, 11, 13] . several of the above are however part of our natural flora, and may be considered opportunistic pathogens [20] , only causing disease in persons with an underlying illness, immune deficiency, or are perhaps only known to cause disease in children etc. [21] . generally all cases should be correlated to anamnestic information, if any present, both medical and socio-economic [22] before a microorganism is classified as a true infection. hence a definite list of true pathogens is impossible to comprise. considering that microbiological testing amounts to only 6.3% of the autopsies in the investigative period versus the yield of cases which were classified as microbiologically related cod (42.8%), this inexpensive testing seems to be rather well founded. perhaps microbiological testing should be applied in even more cases. in denmark around 5% of autopsies do not yield a viable cause of death, and though microbiological testing would be performed on most, the possibility remains that a few of these deaths could be due to infections/sepsis. in this study 13 of 18 cases with a microbiologically related cod had elevated crp values (p < 0.05). considering that result and the literature on the subject [6, 23] , one would suggest that crp measurement is carried out at the beginning of the autopsy. this would give an early indication of a possible infection in cases where antemortem information does not suggest such, allowing for microbiological sampling before significant handling of the body and a hence reduced risk of mechanical contamination, both by direct transfer of bacteria and by displacement of bowel-near blood in arteries and veins. besides the possibility of improved sampling methods we also need to consider other methods to improve the microbiological testing in relation to autopsy. pcr analyses have been proved useful [24] , and although it seems to be excellent with regards to virus investigations, it does not bypass the biggest pitfall in bacterial analysis, the postmortem spread. since it is even more sensitive than culturing [8] it might possibly yield an even higher number of positive results. pcr analysis targeting specific bacterial toxins or the toxin-genes might be of some use [25] , investigations into pm bacterial toxin production could be an interesting area for future work. microbiological sampling remains an important part of the autopsy yielding the cause of death in 42.8% of the cases in which it was performed. optimized storage of the body and precautions to avoid contamination greatly enhance the value of the results. the decision to classify a cod as microbiological rests on a combination of factors, including crp levels, histology and an analysis of the microbiological agents. a raised crp level is correlated with microbiologically related cod and if performed at the beginning of the autopsy could be used as a guideline to initiate a microbiological autopsy, before mechanical manipulation leads to contamination. histology is an important part of the microbiological investigation in almost 2/3 of the cases with a microbiologically related cod, verifying inflammation in the cultured material, and in a few cases being the decisive factor, though supported by other information, in the diagnosis where the microbiological results were negative. therefor all sites sampled for microbiological testing should also be histologically examined. in very few of our cases only one single microbiological agent was found or an agent which is without doubt the sole cause of death. several of the microorganisms contributing to our natural flora, colonizing human skin, respiratory tract or other inner surfaces or which are found in abundance in our surroundings can lead to life-threatening infections under the right circumstances, i.e. persons with underlying illness, immune suppression etc. conversely widely found microorganisms known to cause sepsis could be the result of postmortem spread, considering their established virulence, target age group and point of infection. investigators should always take into account all findings and anamnestic information in each case compared to the detected microorganisms before making any conclusions. hence a definite list of ''true pathogens'' is nearly impossible to compile, and we suggest consulting microbiological literature on each microorganism, once it has been deemed of interest due to the above factors. accuracy of cause of death determination without forensic autopsy examination postmortem diagnosis of sepsis the importance of the autopsy in emerging and reemerging infectious diseases determinants for autopsy after unexplained deaths possibly resulting from infectious causes, united states a study of pneumococci and allied organisms in human mouths and lungs after death the routine use of c-reactive protein in forensic investigations particularities regarding the etiology of sepsis in forensic services evaluation of postmortem bacterial migration using culturing and real-time quantitative pcr ii: comparison of a group of cancer patients with a control group procurement, interpretation, and value of postmortem cultures bacteriological investigationsignificance of time lapse after death postmortem bacteriology: a re-evaluation practical and theoretical aspects of postmortem bacteriology sterile site infection at autopsy in sudden unexpected deaths in infancy postmortem interval and bacteriological culture yield in sudden unexpected death in infancy (sudi) comprehensive postmortem analyses of intestinal microbiota changes and bacterial translocation in human flora associated mice the interest of postmortem bacteriology in putrefied bodies microbiological findings in sudden unexpected death in infancy: comparison of immediate postmortem sampling in casualty departments and at autopsy bacteriological sampling of postmortem rooms principles and practice of infectious diseases sudden death caused by fulminant bacterial infection: background and pathogenesis of japanese adult cases postmortem bacteriology in forensic pathology: diagnostic value and interpretation autopsy cases of fulminant bacterial infection in adults: clinical onset depends on the virulence of bacteria and patient immune status cerebrospinal fluid pcr analysis and biochemistry in bodies with severe decomposition staphylococcal toxins in sudden unexpected death in infancy: experience from a single specialist centre key: cord-255814-k203h9jq authors: abou-al-shaar, hussam; sabbagh, abdulrahman j. title: the undetermined destiny of case reports in the era of sophisticated medicine date: 2017-05-31 journal: world neurosurgery doi: 10.1016/j.wneu.2017.02.109 sha: doc_id: 255814 cord_uid: k203h9jq nan the undetermined destiny of case reports in the era of sophisticated medicine letter: w e read with great interest the editorial by dr. benzel. 1 the importance of case reports is indeed declining in recent years. the continuous development of medicine, the use of sophisticated methodologic tools, the increasing need for cutting-edge evidence-based medicine (which cannot be achieved with case reports), and the continuous restrictions from journals on case reports all have contributed to the "diminishing value" of case reports. however, a few questions remain to be answered here. should authors stop reporting their unique cases? should journals stop accepting such unusual cases? are we in an era where there is no more scientific value in case reports? looking back at history, we find various diseases and discoveries that were described initially as case reports. the perfect example of this is cushing disease. in 1910, dr. harvey cushing first encountered the disease in a 23-year-old woman, who is historically known as "minnie g." the patient had an unusual and complex clinical presentation in the form of obesity, hypertrichosis, amenorrhea, overdevelopment of secondary sexual characteristics, low-grade hydrocephalus, and increased cerebral tension. 2,3 such an unusual clinical combination was not yet described by any medical disorder at the time, which stimulated cushing to begin his lifelong interest and extensive research and investigations. cushing was confident that minnie's unusual presentation was due to a problem of the pituitary gland. because of minnie's case and similar unusual cases reported in the literature, cushing's efforts eventually culminated in 1932 in his pivotal work, "the basophil adenomas of the pituitary gland and their clinical manifestations (pituitary basophilism)." 4 in his article, cushing described 2 cases from his own experience (minnie g. was the first case) along with 10 other cases reported in the literature. 4 because of such single case reports, the clinical symptoms of the disease were described and were named after cushing. 4 not only did minnie g. aid in discovering a new syndrome, but her very unusual long-term survival intrigued carney 5 to speculate that she might have had the carney complex of primary pigmented nodular adrenocortical disease. the value of case reports in our opinion goes beyond just mere reporting of unique cases. it expands our understanding of the nature of a disease and helps us, although by a small step, advance our field. this is clearly evident in the case of some recent diseases, such as the middle east respiratory syndrome coronavirus. 6 the disease was initially described as a single case, and extensive reporting of more cases expanded our understanding of the nature of such an infection, its origin, route of transmission, pathophysiologic mechanisms, and clinical implications. 7 the value of case reports is also evident by their role in delineating hidden unusual disease associations, 8, 9 novel genetic discoveries, 10 new surgical techniques and technical nuances, 11 unique thought-provoking disease pathogenesis, 12 decision-making challenges and conundrums, 13 cutting-edge management innovations, 14 and unusual complications. 15 such unique cases carry new information that can be of huge educational value and are of great interest to the readers. therefore, the answers to the aforementioned questions are not easy. the continuous advancements in the medical field should not diminish the value of case reports. on the contrary, these advancements should aid case reports to be the initial building blocks and cornerstone that employ technical and methodological advances for better understanding of such rare cases and form an infrastructure for future cutting-edge evidence-based medicine. we hope that world neurosurgery continues to provide the platform that can accommodate such rare and innovative case reports and remains the strong infrastructure that authors can rely on to report such cases. the diminishing value of "the case report the pituitary body and its disorders: clinical states produced by disorders of the hypophysis cerebra cushing's case xlv: minnie g the basophil adenomas of the pituitary body and their clinical manifestations (pituitary basophilism) the search for harvey cushing's patient, minnie g., and the cause of her hypercortisolism isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for camel-to-human transmission of mers coronavirus frontal dermoid cyst coexisting with suprasellar craniopharyngioma: a spectrum of ectodermally derived epithelial-lined cystic lesions? hypothalamic-optochiasmatic pilocytic astrocytoma associated with occipital and sacral spinal cavernomas: a mere coincidence or a true association a novel missense mutation in the tbx5 gene in a saudi infant with holt-oram syndrome bolstering the nasoseptal flap using sphenoid sinus fat packing: a technical case report supratentorial neurenteric cysts-a fascinating entity of uncertain embryopathogenesis hemoglobin drop after anesthesia in craniosynstosis: dilemma of operate or not to operate brachytherapy in the treatment of recurrent aggressive falcine meningiomas free esophageal perforation following hybrid visceral debranching and distal endograft extension to repair a ruptured thoracoabdominal aortic aneurysm key: cord-264037-43yr6qon authors: kang, yun-jung title: lessons learned from cases of covid-19 infection in south korea date: 2020-05-07 journal: disaster medicine and public health preparedness doi: 10.1017/dmp.2020.141 sha: doc_id: 264037 cord_uid: 43yr6qon on december 31, 2019, the chinese government officially announced that the country had a single pneumonia case with an unknown cause. in the weeks after, south korea had 24 confirmed cases by february 8, and the number has increased steadily since then. the highly contagious virus known as coronavirus disease 2019 (covid-19) infected case no. 31 in daegu; she was the first patient related to sincheonji church. later, the number of cases involved with sincheonji skyrocketed. on march 6, 2020, the number of confirmed cases was 6284, with 42 dead. this study, through collecting epidemiological data about various covid-19 infection cases, discovered that getting together in large groups leads to mass infection, and that paying close attention to personal hygiene by means of wearing masks, sanitary gloves, etc., can prevent the spread of covid-19. additional epidemiological data and related studies on covid-19 infections in south korea are likely to support or slightly modify this conclusion. however, this study is significant in that it emphasizes the precautionary principle in preventing and managing infectious diseases, and has a suggestion for public health policies, which are currently in high demand. o n december 31, 2019, the chinese government announced officially that the country had a pneumonia case with an unknown cause. it is not clear when this case occurred initially. according to reports containing 41 confirmed cases from the medical teams in wuhan, china (december 1, 2019 to january 2, 2020), the first patient developed symptoms on december 1, but had never visited the huanan fish market. 1 later, on january 7, 2020, the chinese government reported a new kind of coronavirus as the cause of this pneumonia case and provided information on the virus to researchers around the world. 2 the world health organization (who) named the virus coronavirus disease 2019 (covid-19) temporarily, but as the english name is rather long in korean, the south korean government decided to call it "corona 19 (corona il-gu)," following the opinion of the korean centers for disease control and prevention. 3 coronavirus (cov) is a virus that can infect humans and various other animals. it is an rna virus with gene sizes from 27 to 32 kb, with four genera (alpha, beta, gamma, and delta). alpha and beta genera can infect humans and animals, while gamma and delta genera infect only animals other than human beings. as implied by its name, the shape of the virus observed through an electron microscope is a crown shape with characteristic protein spikes attached to a ball. the pathogen is covid-19, and the source of infection is estimated to be an animal, although the investigation is ongoing. the infection route channel is estimated to be animal → human → human. spread between humans is assumed to be due to droplet infection. secondary infection cases in households and hospitals have been confirmed. clinical manifestations are fever, respiratory symptoms (coughs and dyspnea), and pneumonia. 4 case related to the sincheonji church in daegu (case no. 31), the number of covid-19 cases by sincheonji members in south korea increased sharply. the south korean government, using the church member registry of 244,743 members it acquired from sincheonji headquarters, analyzed the connection between church members and 4212 covid-19 cases confirmed until march 2. according to the analysis, 93% of the confirmed cases were related to sincheonji. 7 the possibility of mass infection in church services was suggested. as more than 6000 covid-19 cases were confirmed, the cases of infection became more diverse. this study collected and studied various cases of covid-19 infections to the time of this writing to determine measures of prevention against the spread of the disease, while suggesting a managerial direction for public health, which is in urgent demand now. reports were collected from south korea based on data from the korea centers for disease control and prevention, along with data and presentation examples from the korea centers for disease control and prevention from february 8 to march 6, 2020. respiratory syndrome caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. a person who has been confirmed to be infected with an infectious pathogen according to the examination criteria for diagnosis. test criteria for diagnosis: virus isolation from samples, detection of specific genes in samples. the korea centers for disease control and prevention is an organization belonging to the ministry of health and welfare of korea and is located in cheongju city, north chungcheong province. this institution is responsible for the prevention, investigation, quarantine, testing, research, and long-term transmission management of infectious diseases, chronic diseases, rare intractable diseases, and injured diseases to improve the national health of south korea. the term large-scale group infection refers to a condition in which a large number of people are exposed to each other in a short period of time, because some infectious diseases are clustered among schools, companies, dormitories, factories, or people in a limited physical space at once, and the conditions for causing infectious diseases are established as a group. some disorders occur sporadically in time or place. it is not so important in public sanitation, but it is sometimes necessary to monitor the situation as it happens as an example of group outbreak. nationally, 71.7% of the confirmed covid-19 cases in korea are verified to be related to mass infections. the other 28.3% cases are due to sporadic infections or the causes are still being investigated. investigating the mass infection cases by regions, confirmed that cases related to sincheonji church in daegu accounted for 72.4% of the overall 3397 confirmed cases in the city. more cases were confirmed in public and medical facilities while investigating people who had had contact with the sincheonji followers. 8 in busan and gyeongnam, the number of covid-19 confirmed cases related to a retreat to oncheon church is 35. people from busan account for most of the cases, with the number of 33; the other two people were from gyeongnam. among the 35 confirmed cases, 28 were geochang church bore the most covid-19 confirmed cases as a single group in a single region of gyeongnam province. therefore, gyeongnam province government sent an epidemiological investigation team to geochang and started an in-depth investigation in cooperation with the city government of geochang. the number of the members of geochang church congregation is 60, with 39 people living in geochang and 21 coming from other towns. among the 39 members from geochang, 10 people were confirmed to be infected, while the other 29 were confirmed negative. sixty-two people who had contacted the confirmed cases are all confined in their own homes. 10 among the 39 pilgrims of andong, gyeongbuk parish of the catholic church, who had participated in the pilgrimage to israel, 30 people were confirmed to be infected by covid-19. nineteen other people who had contacted with the confirmed cases were also tested to be positive, making 49 the total number of the confirmed cases. as some of the pilgrims are reported to have visited restaurants and eateries after returning to korea, there is a possibility of regional spread. 11 the mass infection among the saengmyeongsam church congregation, which bore 10 confirmed cases of covid-19, is reported to have been caused by worshipping and eating together in a small and crowded indoor environment. a sincheonji believer and lecturer from sincheonji church, daegu, who attended the service, is indicated to be the index case. 12 person a, a confirmed case who is currently being treated in a management ward, reported that he was infected after visiting a wedding hall in daegu. he spent only 1 h and 10 min in the wedding hall. on the date of visiting, his wife and son wore masks, unlike himself. 25 elevator person a (53, male), a curate of myeongseong church in gangdong-gu, and person b (41, female), a resident of the same apartment building he lived in, took the same elevator, and both were confirmed positive. person b did not wear a mask in the elevator and became infected with covd-19 after the short time spent in an elevator. 26 in busan, a middle school student (teenager) was confirmed to be infected with covid-19 on february 28, 2020, after visiting the same internet café where a member of the oncheon church had visited. 27 person a, a tour guide, confined himself in his own home when he first developed symptoms (a sore throat) on january 31, 2020. since then, he recorded his covid-19 symptoms meticulously in a daily journal. when going to hospitals, he avoided using public transportation and walked, taking only uncrowded routes. he wore masks and gloves even in his home, which led to the negative confirmation of 23 people who had come in contact with him, including his mother living with him under the same roof. 28 patient number 63 from bukgu, busan, who is in his 30s, confined himself to his home when he heard that he was confirmed positive of covid-19 and drove by himself even when he visited the selective care center. he always wore masks in his own home and separated his living area, saving his parents; they were both confirmed negative. 28 a covid-19 confirmed case c (58, male) wore masks and sanitary gloves even in his homes when he developed symptoms of the virus. he sterilized the dishes he used in hot water right after using. even though he was confirmed negative once, he recorded his symptoms and the places he went just in case. after he was confirmed, he provided this information to the prevention authorities and contributed to their quick reaction. 29 pyeongtaek city government in gyeonggi-do announced that all the family members of the second covid-19 confirmed case were confirmed negative of the virus on february 24, 2020. cctv footages showed that the patient always wore the mask. 30 cases of covidfigure 1 ). locations of mass infections are churches, hospitals, sanitariums, private educational institutes, and dance studios, where many people gather in a closed and crowded environment and tend to contact each other at a close distance. the chinese national hygiene and health committee admitted the possibility of infection through aerosols in its 6th edition of covid-19 treatment guide, providing "long-time exposures to dense aerosol in relatively closed environment." 31 aerosol means solid or liquid small particles floating in the air, ranging from 0.001 μm to 100 μm. 32 according to scientific definitions, small particles that can travel far distances for a long time are classified as airborne, while bigger aerosols are classified as droplets. 33 this means that the spread through aerosols can manifest in two types, one being droplet infection by direct contact and one being airborne spread. generally, droplet infection occurs when the infected person sneezes, coughs, talks, or exhales; this is called the first aerosolization. 34 in contrast, airborne spread is due to the spread of droplet nuclei with the size of <5 μm, left behind when the water in droplets evaporates. as the droplet nuclei are light and can float in the air for a long time, they can be especially dangerous. 35 although religious services conducted in crowded spaces like churches and temples are vulnerable to infections, there are no specific prevention guides from the government. most churches hosted services as usual, despite the possibility of regional infection with more and more cases of covid-19 cases being confirmed. contact between hundreds and thousands of the members in the same space was inevitable, making people vulnerable to covid-19 with its strong infectivity. furthermore, the korea centers for disease control and prevention suggests the possibility of limited but constant spread in groups through holiday services and small meetings. 36 private educational institutes and physical training facilities bear similar dangers. even though the government postponed the start of semesters for kindergartens, elementary schools, middle schools, high schools, and universities for fear of mass infections, many private educational institutes and physical training centers are insisting on operation, threatening covid-19 prevention. according to a report on there are even cases of infection that go against the explanation provided in the chinese national hygiene and health committee's 6th edition covid-19 treatment guide, which says that the condition for spread through aerosol is "long-time exposures to dense aerosol in relatively closed environment." 32 the cases of infection in the elevator and the wedding hall, where the patients spent only a short amount of time together, prove this false. the patients of these two cases are similar in that they did not wear masks. from the perspective of prevention and management, blocking the route of airborne infection is critical. there were also many desirable cases where the infection spread was prevented by using masks and sanitary gloves (table 3) . [28] [29] [30] patients, after feeling their symptoms or finding out that they had contact with confirmed cases, confined themselves to their homes and shunned crowded places to prevent infecting others. they always wore masks and sanitary gloves, and even though their uninfected family members were under the same roof, these measures allowed them to be confirmed negative. the mask used at this time was a disposable non-woven-material mask. in contrast, when the confirmed cases did not wear masks or confined themselves in distribution of covid-19 occurrence types. cases of covid-19 infection in south korea disaster medicine and public health preparedness separate rooms in their homes, all their family members were diagnosed to be positive. 24 these are cases where wearing masks and using proper individual hygiene decided the positive outcome of prevention. the temporary conclusion of this study, based on limited epidemiological data and information on confirmed cases currently available, is that group meetings lead to mass infections of covid-19, and that caring for individual hygiene by wearing masks and sanitary gloves can prevent its spread. however, in the current epidemic, the group that needs the most masks are the medical staff working on the front lines. therefore, an individual's wearing of a mask should be limited to patients suspected of having an infection. this conclusion can be supported or modified according to additional epidemiological data and research results on covid-19 infections in south korea. however, this study is meaningful in that it emphasizes the precautionary principle in preventing and managing infectious diseases, and has a suggestion for public health, which is currently in urgent demand. clinical features of patients infected with 2019 novel coronavirus in wuhan china novel wuhan (2019-ncov) coronavirus the outbreak cases with the novel coronavirus suggest upgraded quarantine and isolation in korea corona corresponds to korea, being caught by sinchon and conservatives the government "93% of confirmed people are infected with sincheonji" president moon "practical actions 66% of korean corona19 confirmed 'cold infection'… the largest is shincheon daegu church corona 19 group occurrence related to busan hot spring church… "33 busan, 2 gyeongnam". newsis epidemiological survey of geochang churches in korea. hankyoreh zumba · pilgrimage · hospital community outbreaks "another primer ?news_id=n1005680958&plink. (korean) suwon city, 199 members of life sam church. 10 confirmed. sport news subsequent hospital infections occur… concerns about analysis of local health care system mary's hospital confirmed by 14… "the biggest seoul outbreak arcid=0014290920&code= 61121111&cp=nv. (korean) confirmation of 3 medical staffs, 14 days of quarantine medical staff at bundang jesaeng hospital and 8 patients corona 19 'group infection'. financial news chilgok severe disability facility 'millal house', corona 19 confirmed by 22 gyeongsangbuk-do, more than half of new patients second south hospital situation ona… bonghwa blue sanitarium 49 infections worried that the dance instructor confirmed by corona19 in cheonan will become a local super evangelist 114 quarantines' 13 confirmed people related to the apartment in seongdong-gu we are frustrated with school infection spread… busan office of education appeals to parents the sixth person who confirmed coin karaoke… small scale infection is a reality. hankyoreh → no. 6 → 2 family' 3 cases of infection in korea ?volumeno=27398212&member no=15305315. (korean) confirmation "don't be attentive in 1 hour of daegu wedding corona 19' confirmed. ohmy news where do youth acting school?… 'pc · karaoke' in various places never go to harm"… 'corona journal' with few copper wires and symptoms wearing masks and hygiene gloves at home… carefully record the symptoms and movement pyeongtaek's 2nd corona 19 confirmed family 'negative'. yunhap news chinese authorities add coronal 19 disturbance path aerosol technology: properties, behavior, and measurement of airborne particles preventing airborne disease transmission: review of methods for ventilation design in health care facilities viral infections acquired indoors through airborne, droplet or contact transmission aerobiology and its role in the transmission of infectious diseases coronavirus infection-19 domestic outbreaks postponement of school opening, 'combination', and teaching instructors… outbreak 'emergency'. nocut news the authors declare that they have no competing interests. the author (y.j. kang) wrote the entire manuscript and holds final responsibility for the decision to submit the manuscript for publication. the author read and approved the final manuscript. key: cord-253542-twn07poq authors: nikolay, birgit; salje, henrik; sturm-ramirez, katharine; azziz-baumgartner, eduardo; homaira, nusrat; ahmed, makhdum; iuliano, a. danielle; paul, repon c.; rahman, mahmudur; hossain, m. jahangir; luby, stephen p.; cauchemez, simon; gurley, emily s. title: evaluating hospital-based surveillance for outbreak detection in bangladesh: analysis of healthcare utilization data date: 2017-01-17 journal: plos med doi: 10.1371/journal.pmed.1002218 sha: doc_id: 253542 cord_uid: twn07poq background: the international health regulations outline core requirements to ensure the detection of public health threats of international concern. assessing the capacity of surveillance systems to detect these threats is crucial for evaluating a country’s ability to meet these requirements. methods and findings: we propose a framework to evaluate the sensitivity and representativeness of hospital-based surveillance and apply it to severe neurological infectious diseases and fatal respiratory infectious diseases in bangladesh. we identified cases in selected communities within surveillance hospital catchment areas using key informant and house-to-house surveys and ascertained where cases had sought care. we estimated the probability of surveillance detecting different sized outbreaks by distance from the surveillance hospital and compared characteristics of cases identified in the community and cases attending surveillance hospitals. we estimated that surveillance detected 26% (95% ci 18%–33%) of severe neurological disease cases and 18% (95% ci 16%–21%) of fatal respiratory disease cases residing at 10 km distance from a surveillance hospital. detection probabilities decreased markedly with distance. the probability of detecting small outbreaks (three cases) dropped below 50% at distances greater than 26 km for severe neurological disease and at distances greater than 7 km for fatal respiratory disease. characteristics of cases attending surveillance hospitals were largely representative of all cases; however, neurological disease cases aged <5 y or from the lowest socioeconomic group and fatal respiratory disease cases aged ≥60 y were underrepresented. our estimates of outbreak detection rely on suspected cases that attend a surveillance hospital receiving laboratory confirmation of disease and being reported to the surveillance system. the extent to which this occurs will depend on disease characteristics (e.g., severity and symptom specificity) and surveillance resources. conclusion: we present a new approach to evaluating the sensitivity and representativeness of hospital-based surveillance, making it possible to predict its ability to detect emerging threats. we propose a framework to evaluate the sensitivity and representativeness of hospital-based surveillance and apply it to severe neurological infectious diseases and fatal respiratory infectious diseases in bangladesh. we identified cases in selected communities within surveillance hospital catchment areas using key informant and house-to-house surveys and ascertained where cases had sought care. we estimated the probability of surveillance detecting different sized outbreaks by distance from the surveillance hospital and compared characteristics of cases identified in the community and cases attending surveillance hospitals. we estimated that surveillance detected 26% (95% ci 18%-33%) of severe neurological disease cases and 18% (95% ci 16%-21%) of fatal respiratory disease cases residing at 10 km distance from a surveillance hospital. detection probabilities decreased markedly with a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 distance. the probability of detecting small outbreaks (three cases) dropped below 50% at distances greater than 26 km for severe neurological disease and at distances greater than 7 km for fatal respiratory disease. characteristics of cases attending surveillance hospitals were largely representative of all cases; however, neurological disease cases aged <5 y or from the lowest socioeconomic group and fatal respiratory disease cases aged !60 y were underrepresented. our estimates of outbreak detection rely on suspected cases that attend a surveillance hospital receiving laboratory confirmation of disease and being reported to the surveillance system. the extent to which this occurs will depend on disease characteristics (e.g., severity and symptom specificity) and surveillance resources. we present a new approach to evaluating the sensitivity and representativeness of hospitalbased surveillance, making it possible to predict its ability to detect emerging threats. • many countries rely on hospital-based surveillance for the detection of infectious diseases of national and global public health relevance. • it is often difficult to access suitable external reference data to assess the capacity of a surveillance system to detect cases and outbreaks or to characterize cases. • we demonstrate a novel approach using healthcare utilization data to evaluate the sensitivity and representativeness of severe infectious disease surveillance in bangladesh. • the capacity to detect cases and outbreaks decreased with distance from surveillance hospitals. • cases captured by surveillance differed from cases in communities by age and socioeconomic status. • geographic coverage of surveillance could be improved by including other hospitals in the surveillance system. • the presented approach is applicable for a wide range of infectious diseases in different settings, taking some practical considerations into account. • hospital-based surveillance may have low sensitivity in rural areas at greater distances from surveillance hospitals, suggesting a risk of unrecognized transmission of emerging infectious diseases. a well-functioning disease surveillance system is crucial for the identification and control of outbreaks, and hence the prevention of national and global health emergencies [1] . the world health organization (who) highlighted the value of national surveillance systems in the international health regulations (2005) , an agreement among all member states to develop and maintain sufficient capacity for the detection, reporting, and control of public health threats of international concern [2] . infectious disease surveillance should enable (i) the timely detection of outbreaks, (ii) the quantification of health problems, (iii) the identification of subpopulations at risk, and (iv) the assessment of temporal trends including the impact of control strategies [3, 4] . national surveillance systems typically collect data from patients seeking care at sentinel hospitals or other healthcare facilities and can provide useful information for public health purposes. however, hospital-based surveillance generally underestimates disease burden since only a proportion of cases visit a hospital for care [5] . low case detection may also undermine the value of hospital-based surveillance for outbreak detection. moreover, if patients captured by the surveillance system are not representative of all cases in the community, surveillance statistics could lead to erroneous interpretations of disease patterns and misallocation of prevention resources. in particular, sex, socioeconomic status, or distance can affect healthcare seeking at hospitals, especially where access to care is limited [6] [7] [8] [9] . surveillance evaluation guidelines, such as those established by the us centers of disease control and prevention, list sensitivity and representativeness among the attributes that a public health surveillance system should possess and that require assessment [10, 11] . in order to follow these guidelines, we need external reference data that are often unavailable in resource-poor settings [12] . here, we present a new approach to evaluating the capacity of a surveillance system to detect and characterize disease cases, with emphasis on outbreaks of emerging infections that often occur as small case clusters in remote areas. we apply our methodology to assess hospital-based surveillance of severe neurological infectious disease and fatal respiratory infectious disease in bangladesh. the field teams obtained written informed consent from participants or their guardians (if <18 y of age) during community surveys. healthcare utilization survey protocols were reviewed and approved by the ethical review committee of the international centre for diarrhoeal disease research, bangladesh. how epidemiological studies can be used to identify cases with severe symptoms in communities and capture their personal and healthcare utilization characteristics (data collection stage) (fig 1) . in addition to detailing how we collected the data in this study, we provide information about how the approach could be varied in other settings. we subsequently demonstrate how such data can be used to evaluate the sensitivity and representativeness of surveillance systems (evaluation stage). we then apply our approach to the detection of severe neurological infectious diseases and fatal respiratory infectious diseases in bangladesh as a case study. selecting study locations. the first step was to randomly select communities at differing distances from the surveillance hospitals. we specified catchment areas of selected hospitals based on hospital records and subsequently randomly selected small administrative units from which all communities were surveyed. selection of communities could also be done through census data or using detailed population maps of the area. identifying people with diseases in the selected community. study teams visited the selected communities and identified cases that had had the disease of interest. the retrospective identification of severe disease cases in the community was based on syndromic criteria, used as a proxy for clinical case definitions that would be applied in healthcare facilities. the in the bangladesh example, the catchment areas of surveillance hospitals were first defined based on hospital records (e.g., areas where >50% or >75% of cases reside) [13, 14] . subsequently, small administrative units were chosen at random from within the catchment area, and all communities in the selected areas were surveyed. cases in the community were identified based on lists of deaths in addition to community networking strategies (rural settings) or house-to-house surveys (urban settings). information on symptoms (to establish case definitions), healthcare seeking behavior, and characteristics of cases was collected. in other settings, the exact survey procedures may vary according to the context. identification of such cases in the community is often the most problematic step, and the optimum strategy will depend on the local context, the severity of the disease, and the specificity of disease symptoms. collecting information on healthcare seeking and personal case characteristics. to estimate case detection probabilities, identify biases in case statistics, and characterize the healthcare utilization behavior in the population, we needed information about the healthcare seeking and personal characteristics of cases. in particular, we needed to identify whether the cases attended a surveillance hospital. such information was obtained during household visits of identified cases. to understand the impact of distance from the hospital, we approximated the locations of households by the central positions of the small administrative units. alternatively, household locations could be recorded precisely using gps devices. quantifying the probability of detecting a case. we estimated the case detection probability as the proportion of cases who reportedly attended a surveillance hospital among all cases identified in the community. we further assessed how this probability changed with distance from the surveillance hospital. quantifying the probability of detecting outbreaks. we subsequently used the estimated case detection probabilities to quantify the capacity of the surveillance system to identify disease outbreaks. we estimated outbreak detection probabilities for varying outbreak sizes and for outbreaks occurring at different distances from surveillance hospitals. assessing the representativeness of detected cases. we evaluated the representativeness of detected cases by estimating the difference between case statistics (proportions of specific case characteristics) based on all cases in the community and based on identified cases who attended the surveillance hospital. the investigated characteristics included sex, age, and socioeconomic status. assessing alternative surveillance strategies. to investigate how sensitivity and representativeness of the surveillance system could be improved by integrating other healthcare providers, we applied the evaluation procedures as described above to other healthcare provider types. we demonstrate the application of the proposed evaluation strategy by using it to assess the capacity of hospital-based surveillance for severe infectious diseases in bangladesh, which is based on tertiary care hospitals located throughout the country. we used data from two surveys carried out in catchment areas of some of these hospitals that investigated the healthcare utilization behavior of individuals with severe neurological infectious disease or fatal respiratory infectious disease (fig 2a) [14, 15] . these disease types are of great public health relevance in bangladesh (e.g., japanese encephalitis and influenza) but also represent symptoms typical of other emerging infectious diseases (e.g., nipah and severe acute respiratory syndrome). a first survey collected data between 10 june 2008 and 30 march 2009 about cases with symptoms of severe neurological infection that occurred within the previous 12 mo in 60 small administrative units (mean population size of 28,000 people) in the catchment areas of three surveillance hospitals [14] . a second survey collected data between 3 april 2012 and 22 february 2013 about acute respiratory infection (ari)-related deaths that occurred within the previous 24 mo in 22 administrative units in the catchment areas of 11 surveillance hospitals [15] . we considered ari-related deaths as a proxy for respiratory disease of sufficient severity to require medical attention. the surveillance hospital in dhaka city was excluded from the original studies because of the difficulty of defining the catchment area (a step necessary for the original study purpose), as people nationwide seek medical care in dhaka. the surveys followed procedures as previously described and summarized below [13, 14] . characteristics of the study population are described in fig. a in s1 text. [16] . sixty-eight percent of the population in bangladesh lives >30 km from a surveillance hospital (including the dhaka surveillance hospital), a distance at which case and outbreak detection probabilities are low. (c) probability of surveillance case detection by distance. the observed probability was calculated as a moving average over a 25 km distance window. case detection probabilities were estimated using logbinomial regression models including distance as an explanatory variable. evaluating hospital-based surveillance the catchment areas of selected hospitals were first specified based on hospital records (s1 text). small administrative units (mean population of 28,000 people) were subsequently selected randomly within the catchment areas, and all communities in the selected areas surveyed. the identification of cases in selected communities was based on social structures, i.e., cases were identified by visiting public meeting points, such as mosques, markets, or tea-stalls, where health problems in the community are often publicly discussed. cases were subsequently confirmed by household visits. in urban areas, house-to-house surveys were conducted to compensate for less pronounced community structures. additional fatal respiratory infectious disease cases were identified through lists of deaths provided by administrative officers. for both disease types, the identification of cases was based on syndromic criteria. we defined severe neurological infectious disease as fever with altered mental status for >6 h or with unconsciousness for !1 h, or fever with altered mental status, unconsciousness, or a new onset seizure that resulted in death. fatal respiratory infectious disease (ari-related death) was defined as having any two of the following symptoms in the 30 d prior to death: sudden onset of fever, cough, breathing difficulty, feeding difficulty, or runny nose. deaths in children aged <5 y were also classified as ari-related deaths if there was a sudden onset of breathing difficulty in the 30 d prior to death. during surveys, information was collected on healthcare utilization behavior and personal characteristics of identified severe neurological and fatal respiratory disease cases. cases or their household members were asked whether the case visited the surveillance hospital or any other healthcare provider, including other nonlocal hospitals, during his/her illness. further, information on sex, age, socioeconomic status, and geographic location of households of cases was collected. we defined "community cases" as all severe neurological or fatal respiratory disease cases identified during community surveys (whether they attended a surveillance hospital or not) and "surveillance cases" as the subset of community cases who reportedly attended a surveillance hospital. for each case identified in community surveys, we identified whether they attended their nearest surveillance hospital. we then estimated the distance to that surveillance hospital as the distance between the residence administrative unit centroid and that specific surveillance hospital using qgis [17] . age was categorized as <5, 5-14, 15-59, and !60 y. a socioeconomic status index was generated by principal component analysis based on household assets (electricity, working television, bicycle, motorcycle, sewing machine, mobile phone) and categorized into tertiles (lowest, middle, and highest) [18] . in sensitivity analyses, we explored the use of continuous age and socioeconomic status classified into quintiles (s1 text). socioeconomic status was missing for 45 of 1,633 fatal respiratory disease cases, who were excluded from the analysis where this information was required. three fatal respiratory disease cases were excluded from all analyses due to missing healthcare seeking information. we estimated the disease-specific case detection probability as the proportion of cases who reportedly sought care at a surveillance hospital among all cases identified during community surveys (number of surveillance cases/number of community cases) and computed 95% confidence intervals (95% cis) based on the clopper-pearson exact method [19] . we quantified case detection probabilities by distance from a surveillance hospital using log-binomial regression analysis separately for severe neurological and fatal respiratory disease cases. we further investigated more complex functional forms of distance in log-binomial regression models. we fitted models with polynomial terms up to the fifth degree and models with basic splines with knots at various positions (between 20 and 50 km distance). model fit was compared based on the akaike information criterion (aic), and the models with lowest aic were selected. the fit of selected models was compared to the observed proportion of cases who attended surveillance hospitals at different distances (moving average over a distance window of 25 km). we estimated the proportion of the population living >30 km and >50 km from a surveillance hospital using gridded population density estimates of 100 × 100 m resolution [16] . to quantify the capacity of the surveillance system to detect outbreaks of varying sizes, we calculated the probability that at least one case was detected: pr outbreak1 is the outbreak detection probability based on a one-case threshold, pr is the case detection probability, and s is the outbreak size. this calculation assumes that the probability of detecting a sentinel case is independent of other cases. we used distance-specific case detection probabilities estimated by log-binomial regression and obtained confidence intervals of outbreak detection probabilities based on the 95% ci limits of case detection probabilities. we further estimated the size of the smallest outbreak that would be detected with !90% probability by distance from the surveillance hospital. for emerging infectious diseases of global health importance, such as nipah, severe acute respiratory syndrome, or avian influenza, a single detected case may be considered an outbreak. for other disease systems (e.g., endemic diseases or diseases for which differential diagnosis is difficult), an outbreak may be declared only after more than a single case is detected over a specified period of time and within specified geographic boundaries [20] . we can extend the framework to estimate the probability of identifying an outbreak with different outbreak thresholds applied, and we provide examples for outbreaks defined as detection of at least two cases or at least five cases. we calculated the probability of detecting at least two cases (pr outbreak2 ) as one minus the probability of detecting no cases (pr 0 ) and exactly one case (pr 1 ): likewise, we estimated the probability of detecting at least five cases (pr outbreak5 ) as one minus the probability of detecting no cases (pr 0 ) and exactly one (pr 1 ), two (pr 2 ), three (pr 3 ), and four cases (pr 4 ): we investigated the representativeness of surveillance cases (sex, age, and socioeconomic group) by comparing the proportion of cases with a specific characteristic (and exact binomial confidence intervals) among community cases to the proportion of cases with that characteristic among surveillance cases. we quantified the absolute difference in proportions (proportion of cases with characteristic among surveillance cases minus proportion among community cases) with 95% cis and p-values using bootstrapping (2,000 bootstrap iterations) [21] . based on the collected healthcare utilization data, we evaluated how the sensitivity and representativeness of a surveillance system may be improved by integrating other healthcare providers. we classified healthcare providers as (i) surveillance hospitals, (ii) other hospitals (government and private clinics), (iii) qualified private practitioners, and (iv) the informal sector (unqualified practitioners such as traditional healers, village doctors, homeopaths, and pharmacies). we estimated the proportion of cases attending each healthcare provider class, with exact binomial confidence intervals, and estimated outbreak detection probabilities based on proportions attending the surveillance hospital plus (i) other hospitals, (ii) qualified private practitioners, or (iii) informal healthcare providers. furthermore, we compared the proportion of cases with each characteristic (sex, age, and socioeconomic group) among community cases to the proportion among those attending each healthcare provider class and quantified absolute differences in proportions with 95% cis and p-values using bootstrapping (2,000 bootstrap iterations). all statistical analyses and graphics were implemented in the r computing environment; maps were created using qgis software [17, 22] . the studied communities were located within 95 km (severe neurological infectious disease) and 62 km (fatal respiratory infectious disease) of a surveillance hospital. in these communities, 76 of 426 severe neurological disease cases (18%, 95% ci 14%-22%) and 234 of 1,630 fatal respiratory disease cases (14%, 95% ci 13%-16%) attended a surveillance hospital. adjusting for distance, the case detection probability was nearly twice as high among severe neurological disease cases than among fatal respiratory disease cases (risk ratio 1.8, 95% ci 1.4-2.3; p < 0.001). at 10 km distance, an estimated 26% (95% ci 18%-33%) of severe neurological disease cases and 18% (95% ci 16%-21%) of fatal respiratory disease cases were detected by the hospital-based surveillance. the detection probability decreased with distance from the surveillance hospital, and the decline was faster for fatal respiratory disease than for severe neurological disease. a 10 km distance increase resulted in a 12% (95% ci 4%-19%; p = 0.003) relative reduction in case detection probability for severe neurological disease but a 36% (95% ci 29%-43%; p < 0.001) relative reduction for fatal respiratory disease (fig 2c) . including more complex functional forms of distance in the log-binomial regression models did not improve model fit based on aic (table a and figs . b and c in s1 text). the probability of detecting an outbreak of exactly three cases (if a single detected case was considered an outbreak) dropped below 50% at distances greater than 26 km for severe neurological disease and at distances greater than 7 km for fatal respiratory disease (fig 3a) . fig 3b and 3c show the minimum number of cases required for surveillance to detect outbreaks with a probability of !90% if different outbreak thresholds are applied. for outbreaks defined as detection of at least one case, we found that an outbreak of fatal respiratory disease required 12 cases (95% ci 11-13) to be detected with 90% probability at 10 km from a surveillance hospital, but 30 cases (95% ci 24-39) to be detected at 30 km. in contrast, the impact of distance on the outbreak size requirement was much more limited for severe neurological disease: eight cases (95% ci 6-12) at 10 km and 11 cases (95% ci 9-14) at 30 km. for outbreaks defined as detection of at least two cases, 14 severe neurological disease cases (95% ci 11-20) and 20 fatal respiratory disease cases (95% ci 18-23) would be necessary for an outbreak to be detected at 10 km distance, and 19 severe neurological disease cases (95% ci 15-24) and 51 fatal respiratory disease cases (95% ci 41-66) at 30 km. the necessary outbreak sizes increased further when a five-case threshold was applied, so that 28 severe neurological disease cases (95% ci [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] and 39 fatal respiratory disease cases (95% ci 35-44) would need to occur for an outbreak to be detected at 10 km distance, and 36 (95% ci 30-46) and 97 (95% ci 79-128), respectively, cases at 30 km. surveillance hospital attendance among community cases varied by case characteristics, leading sometimes to biased disease statistics among surveillance cases (table b in s1 text). for severe neurological disease, individuals aged <5 y represented 48% of community cases but only 29% of surveillance cases (p < 0.001). additionally, the proportion of cases in the lowest socioeconomic group was lower among surveillance cases than among community cases (43% versus 57%; p = 0.012), while the proportion of individuals aged 15-59 y was higher (43% versus 29%; p = 0.005) (fig 4a) . for fatal respiratory disease, the proportion of individuals aged !60 y (47% versus 62%; p < 0.001) was lower among surveillance cases than among community cases, while the proportion of individuals aged <5 y (24% versus 18%; p = 0.020), individuals aged 15-59 y (27% versus 18%; p < 0.001), and cases in the highest socioeconomic group (43% versus 37%; p = 0.022) was higher (fig 4b) . we observed a slight difference in the proportion of females for fatal respiratory disease (34% among surveillance cases versus 38% among community cases; p = 0.108), but not for severe neurological disease (39% versus 40%; p = 0.861). results were consistent in sensitivity analyses with age as a continuous variable and socioeconomic status classified into quintiles (figs. d and e in s1 text). a substantial proportion of cases (severe neurological disease 42% [95% ci 38%-47%]; fatal respiratory disease 26% [95% ci 24%-28%]) visited multiple healthcare providers during their illness. forty-eight percent (95% ci 44%-53%) of severe neurological disease cases and 31% (95% ci 29%-34%) of fatal respiratory disease cases attended any hospital, including surveillance hospitals (fig 5) . including other hospitals that were attended by cases in the surveillance system could have increased the overall case detection probability by 31% (absolute increase) for severe neurological disease cases and 17% for fatal respiratory disease cases. the capacity to detect outbreaks would have increased, so that outbreaks containing four severe neurological or eight fatal respiratory disease cases would have been detected with !90% probability for any distance in the range 0-40 km from the original surveillance hospital, compared to 13 and 47 cases, respectively, with the current system ( fig. f in s1 text) . however, since individuals who attended any hospital had similar characteristics in terms of sex, age, and socioeconomic status as those attending surveillance hospitals (fig. g in s1 text), this expansion would not have increased disease detection in key groups such as the lowest socioeconomic group. only with the informal sector incorporated in the surveillance system would cases in such groups be detected. we described an analytic approach for evaluating the sensitivity and representativeness of hospital-based surveillance systems and applied it to surveillance for severe neurological diseases and fatal respiratory infectious diseases in bangladesh. we quantified the proportion of cases detected and the probability that the surveillance system would detect different sized outbreaks by distance from the surveillance hospital. finally, we characterized biases in surveillance statistics and identified potential improvements to the surveillance platform. we estimated that approximately one-quarter of severe neurological disease cases and onefifth of fatal respiratory disease cases occurring 10 km from a surveillance hospital would be detected with current surveillance. the proportion of cases attending a surveillance hospital declined significantly with increasing distance between individuals' residence and the surveillance hospital, substantially faster for fatal respiratory disease than for severe neurological disease. these low detection probabilities mean that hospital-based surveillance in bangladesh (like in most other resource-poor countries presumably) would likely miss a high proportion of single-case public health events. of greater relevance is that surveillance system capacity to detect outbreaks and detection probabilities increased substantially with the number of cases. the required number of cases to detect outbreaks with high probability varied with disease type and distance from the surveillance hospital. it could be as low as about ten cases if the outbreak occurred <10 km from the surveillance hospital but increased quickly with distance for evaluating hospital-based surveillance fatal respiratory disease. for outbreaks defined as a single detected case, we found that more than half of outbreaks with ten cases of fatal respiratory disease would be missed if the outbreak occurred >32 km from the hospital. such detailed quantification of outbreak detection probability is essential to ascertain the likelihood that an emerging threat can be detected early enough to be contained [23] . in some circumstances, authorities may have to wait until more than a single case is detected to recognize that an outbreak is occurring. in particular, difficult differential diagnoses and lack of appropriate diagnostic tests mean that only when a number of cases are detected from the same area and over a short time frame will an outbreak be identified and further investigations conducted. in addition, where a low background level of transmission is expected (such as with endemic diseases), public health authorities may wait until a particular threshold is exceeded before declaring an outbreak. in both of these scenarios, where multiple cases need to be detected by the hospital before an outbreak is recognized, the optimal number of detected cases and their spatial and temporal separation will depend on the disease system. we can incorporate this information into our flexible framework and provide examples where we calculate the size an outbreak needs to be for scenarios where at least two or five cases need to be detected (fig 3b and 3c ). in particular, this demonstrates that if an outbreak is identified only once five cases are detected at the surveillance hospital, the size of the outbreak would have to be substantially larger (e.g., nearly 100 total cases of a fatal respiratory disease at 30 km from a surveillance hospital) for there to be a 90% chance of an outbreak being identified. this highlights the possibility that, by the time an outbreak reaches sufficient size to be detected by the system, outbreak control measures may be much less effective at controlling spread. thresholds for case counts that trigger an outbreak response should be crafted taking this possibility into account. low detection probabilities for outbreaks that occur far from surveillance hospitals are an important concern because pathogens with high case fatality such as japanese encephalitis and nipah virus are nearly exclusively found in rural communities in bangladesh [24, 25] , and evaluating hospital-based surveillance these communities are usually located far from surveillance hospitals. rural environments are also considered to be at highest risk for the emergence of novel pathogens [26, 27] . population distribution maps suggest that 68% of the population in bangladesh live in communities >30 km from a surveillance hospital (representing 108 million individuals) and 40% live >50 km from a surveillance hospital (representing 63 million people) (fig 2b) . strengthening healthcare-based surveillance in these areas should be a priority, and cost-effective approaches to achieving surveillance targets need to be identified. there is increasing recognition of the value of novel data sources to improve the sensitivity of infectious disease surveillance, some of which can provide crucial information in remote areas [20] . novel approaches include surveillance for media reports of disease clusters, as used for various infectious diseases in bangladesh [12, 28] , and training of local drug sellers to recognize and report disease symptoms, as rolled out nationally to enhance tuberculosis surveillance in ghana [29] . other surveillance data streams, such as monitoring over-the-counter medication sales, telephone triage, and webbased queries, have been successfully integrated in surveillance systems in resource-rich settings [30] . we found that cases attending surveillance hospitals were not necessarily representative of all cases in the community. in particular, the youngest severe neurological disease cases and the oldest fatal respiratory disease cases were less likely to attend surveillance hospitals, and attendance was also lower among cases in the lowest socioeconomic group. similar variation in hospital attendance has been reported in other resource-poor settings [6, 8, 9] , indicating that hospital-based surveillance in these countries may have comparable limitations. disease statistics obtained through hospital-based surveillance have to be interpreted in the light of detected biases, and correction factors may need to be applied. for example, underestimating severe neurological disease among young children may mislead any future japanese encephalitis vaccination strategy [31, 32] . differential surveillance hospital attendance may also influence the capacity to detect emerging infections, such as the avian influenza a (h7n9) virus that emerged in 2013 in china with observed cases mainly among elderly men [33] . overall, access to appropriate care was poor-over 30% of community cases with severe disease or who died in our study never saw a qualified provider. such low access is a common problem in low-income settings and means that a large proportion of the population, and particularly subgroups that are potentially at highest need, do not receive the required medical attention [6] [7] [8] [9] . for example, difficulties accessing qualified healthcare providers for elderly people, who are often at greatest risk of respiratory disease, can have severe consequences for the outcome of disease. previous studies have demonstrated that accessibility to healthcare is a significant predictor of morbidity and mortality among elderly individuals with respiratory disease [34] . the study showed that healthcare utilization behavior varied by disease type, which may be due to different characteristics of cases such as their age, socioeconomic status, and geographic location (fig. a in s1 text) . the majority of fatal respiratory disease cases were !60 y old and may have faced limitations in mobility; moreover, rapid progression of disease to death may have prevented cases in this age group from seeking appropriate care. cases and their family members in general may have also perceived neurological symptoms as more severe, resulting in higher motivation to attend a qualified healthcare provider [7] . we evaluated potential improvements of surveillance by analyzing healthcare seeking behavior among cases identified in communities. while the majority of individuals did seek care, much of this was in the informal sector, which cannot easily be incorporated into surveillance activity. nevertheless, including other hospitals attended by cases in the surveillance system (the exact location and number of these hospitals was unfortunately not identified during surveys) would double case detection probabilities and allow detection of medium-sized outbreaks (<10 cases) in a wider geographic area. however, in the case of bangladesh, such extension is likely to be prohibitively expensive. mapping other hospitals in bangladesh that may serve as surveillance sites would allow testing of various surveillance scenarios to identify the optimal location of surveillance sites while keeping the same total number or to quantify the number of sites needed to achieve a target surveillance coverage [35] . many emerging infectious diseases originate as spillover infections of zoonotic diseases into the human population [36] . therefore, mapping the occurrence of relevant zoonotic diseases (e.g., avian influenza) and combining such maps with the estimated outbreak detection probabilities would allow highlighting of surveillance gaps for particular types of emerging infectious diseases. the capacity of surveillance systems to detect outbreaks will depend not only on the probability that a case attends a surveillance hospital, but also on whether the case undergoes confirmatory laboratory testing and is subsequently reported through the surveillance system by the hospital. here we assumed a "best-case scenario" with a fully functional surveillance system at the hospital level, where each case who attends the surveillance hospital is ultimately recognized and confirmed as a case. case detection at the hospital may however be incomplete, since case definitions at hospitals may differ from syndromic definitions, a surveillance sampling frame may be applied, or resources and trained personnel for the diagnosis and reporting of cases may be limited [37] . the calculation of case and outbreak detection probabilities may be adjusted for misdiagnosis and underreporting at hospitals if such information is available. we further assumed complete detection of cases in communities during surveys. although a few cases may have been missed, this assumption is justified as we investigated severe disease conditions that are easily remembered by family and community members. moreover, survey procedures combining interviews of key informants and house-to-house visits were specifically designed to capture near-complete case information. further, any missed cases are unlikely to impact our estimates, as such impacts would occur only if there was differential healthcare seeking between those detected and those missed. we investigated spatial differences in hospital attendance based on the straight-line distance of communities from the surveillance hospital. if available, other distance measures such as travel distance or travel time may provide a more accurate indicator of distance from the surveillance hospital. in some cases, these measures may strongly vary with the season, and it would be interesting to explore how that may impact the probability of detecting an outbreak. we assumed that cases did not visit other surveillance hospitals than the catchment hospital. given the poor road infrastructure in the country, it would be very unusual to travel to a tertiary care hospital that was not the closest one. it is possible that some individuals traveled to dhaka; however, these are likely to be wealthier individuals who would visit small private healthcare facilities that are not part of the surveillance network. the surveillance hospital in dhaka was not included in our study. this is unlikely to have biased our assessment of the performance of the surveillance system outside the capital. indeed, this would introduce a bias only under the unlikely scenario that many cases in our study who did not attend the nearest surveillance hospital (and were therefore not captured there) instead attended the surveillance hospital in dhaka (and were captured there). surveillance system performance in the capital city may however differ from elsewhere, and a comprehensive assessment of the national surveillance system would therefore have to include dhaka. moreover, hospital-based surveillance is only one surveillance type in bangladesh, and other data sources need to be considered to assess the country's overall capacity to detect public health events. the described methodology is applicable to assessing surveillance for other severe diseases in resource-poor settings, keeping in mind practical constraints. conducting community surveys may be labor intensive, time consuming, and expensive depending on the setting and may be particularly challenging in densely populated areas such as dhaka. nonetheless, such surveys are valuable tools for obtaining external reference data and simultaneously assess heterogeneities in healthcare access. the effectiveness of community networking may depend on the social structures in the study area; where social links are weaker (e.g., in urban areas), house-to-house surveys, even though more labor intensive, may be more suitable for the identification of cases in the community. the proposed strategy is valid for diseases of sufficient severity to require medical attention and to be remembered by cases and family members. the approach is syndromic (i.e., disease types are classified based on a set of symptoms), and the classification specificity may vary by disease. in conclusion, this study allowed us to quantify the sensitivity and representativeness of hospital-based surveillance and to identify weaknesses, particularly in detecting small-to medium-sized outbreaks in remote areas. these findings highlight difficulties that low-middle-income countries may have in meeting international health regulations requirements, despite considerable investment in hospital-based surveillance platforms. international infectious disease law: revision of the world health organization's international health regulations geneva: world health organization public health surveillance toolkit. a guide for busy task managers. washington (district of columbia): world bank guide to monitoring and evaluating. geneva: world health organization world health organization. who report on global surveillance of epidemic-prone infectious diseases. geneva: world health organization neonatal morbidity and care-seeking behaviour in rural bangladesh health care utilization and symptom severity in ghanaian children-a cross-sectional study inequalities in public health care delivery in zambia inequities among the very poor: health care for children in rural southern tanzania updated guidelines for evaluating public health surveillance systems framework for evaluating public health surveillance systems for early detection of outbreaks low-cost national mediabased surveillance system for public health events influenza-associated mortality in 2009 in four sentinel sites in bangladesh a novel low-cost approach to estimate the incidence of japanese encephalitis in the catchment area of three hospitals in bangladesh incidence of influenza-associated mortality in bangladesh disaggregating census data for population mapping using random forests with remotely-sensed and ancillary data qgis development team. qgis geographic information system. version 2.12. open source geospatial foundation project constructing socio-economic status indices: how to use principal components analysis the use of confidence or fiducial limits illustrated in the case of the binomial innovation in observation: a vision for early outbreak detection an introduction to the bootstrap r: a language and environment for statistical computing. vienna: r foundation for statistical computing strategies for containing an emerging influenza pandemic in southeast asia recurrent zoonotic transmission of nipah virus into humans hospital-based surveillance for japanese encephalitis at four sites in bangladesh emerging infectious diseases of wildlife-threats to biodiversity and human health investigating the zoonotic origin of the west african ebola epidemic early detection of pandemic (h1n1) intensifying tb case detection: update 2012-ghana. geneva: world health organization syndromic surveillance for local outbreak detection and awareness: evaluating outbreak signals of acute gastroenteritis in telephone triage, web-based queries and over-the-counter pharmacy sales targeting vaccination against novel infections: risk, age and spatial structure for pandemic influenza in great britain optimizing infectious disease interventions during an emerging epidemic an overview of the characteristics of the novel avian influenza a h7n9 virus in humans pneumonia and influenza hospitalizations in elderly people with dementia optimizing provider recruitment for influenza surveillance networks global trends in emerging infectious diseases hospitalization records as a tool for evaluating performance of food-and water-borne disease surveillance systems: a massachusetts case study the international centre for diarrhoeal disease research, bangladesh (icddr,b) acknowledges with gratitude the commitment of the us centers for disease control and prevention to its research efforts. icddr,b is also grateful to the governments of bangladesh, canada, sweden, and the uk for providing core/unrestricted support. conceptualization: bn hs sc esg. key: cord-264266-6xvj9zey authors: chakrabarti, sankha shubhra; kaur, upinder; banerjee, anindita; ganguly, upasana; banerjee, tuhina; saha, sarama; parashar, gaurav; prasad, suvarna; chakrabarti, suddhachitta; mittal, amit; agrawal, bimal kumar; rawal, ravindra kumar; zhao, robert chunhua; gambhir, indrajeet singh; khanna, rahul; shetty, ashok k; jin, kunlin; chakrabarti, sasanka title: covid-19 in india: are biological and environmental factors helping to stem the incidence and severity? date: 2020-05-09 journal: aging dis doi: 10.14336/ad.2020.0402 sha: doc_id: 264266 cord_uid: 6xvj9zey the ongoing corona virus (covid-19) pandemic has witnessed global political responses of unimaginable proportions. many nations have implemented lockdowns that involve mandating citizens not to leave their residences for non-essential work. the indian government has taken appropriate and commendable steps to curtail the community spread of covid-19. while this may be extremely beneficial, this perspective discusses the other reasons why covid-19 may have a lesser impact on india. we analyze the current pattern of sars-cov-2 transmission, testing, and mortality in india with an emphasis on the importance of mortality as a marker of the clinical relevance of covid-19 disease. we also analyze the environmental and biological factors which may lessen the impact of covid-19 in india. the importance of cross-immunity, innate immune responses, ace polymorphism, and viral genetic mutations are discussed. could also be infected by sars-cov-2. older individuals with pre-existing chronic health condition shave a higher risk of developing severe complications after sars-cov-2 infection. covid-19 has emerged as a pandemic of respiratory illness ever since the first cases appeared in wuhan, china, in december 2019 [1] . currently, it has spread to 203 countries/territories worldwide, with >920,000 infected cases and >46,000 casualties (www.worldometers.info/coronavirus/). this ongoing pandemic has witnessed global political responses of unimaginable proportions. many nations have implemented lockdowns that involve mandating citizens not to leave their residences for nonessential work. the rules of lockdown vary from country to country. the indian prime minister announced a nation-wide lockdown from the midnight of 24 th march 2020. only essential services would be functioning and government services apart from health, law and order, banking, power and a few others have been suspended altogether. while the response by the indian government both at the central and state levels has been commendable and timely given the global covid-19 crisis, we deliberate some of the other reasons why the authors believe covid-19 may have a lesser impact on india. the world's second-most populous country lies around the 40 th rank among nations for the number of diagnosed covid-19 cases (www.worldometers.info/coronavirus/). the first case of covid-19 was detected in india in the southern state of kerala on 30 th january 2020 and was a medical student who had returned from wuhan. two other cases, also medical students back from wuhan soon surfaced from kerala (https://weather.com/en-in/india/news/news/2020-02-14-kerala-defeats-corona virus-indias-three-covid-19-patients-successfully). the indian government implemented thermal screening of inbound international air-travelers. such measures were implemented for china around17 th january and then, in a stepwise fashion, expanded to include all international passengers (https://pib.gov.in/pressreleaseiframepage. aspx?prid=1599901). interestingly, thermal screening is far from a fool-proof way to detect sars-cov-2 carriers, many of whom may be in the middle of the incubation period or mild cases and hence asymptomatic. further, the quarantine of international travelers on arrival was initiated only for those from china and its immediate neighbors at the beginning and then expanded for travelers from other countries, that too in a phased manner, while observing global trends of covid-19 spread. domestic travel in india continued unabated without incorporating any screening. further, the initial testing criteria with confirmatory qrt-pcr (quantitative reverse transcription polymerase chain reaction)-based tests for suspected patients, released by the indian council of medical research (icmr), emphasized testing only those with laboratory-proven contacts or recent foreign travel (www.icmr.nic.in/content/covid -19) . with such restrictions, the movement of several sars-cov-2 positive patients, whether asymptomatic or symptomatic, must have occurred all over india. however, 9 weeks after the first case was diagnosed, india has a covid-19 count of 2300 (www.mohfw.gov.in/). in contrast, the first two italian cases were chinese tourists who arrived in milan on 23 rd january, and tested positive on 30 th january, the same time when the first indian case was recorded [2] . the case positivity in italy exploded after the fourth week since march 1 st when daily more than 250 new cases (more than 500 new cases daily since 4 th march) were diagnosed (https://ourworldindata.org/coronavirus). it is curious that among the two countries which reported their first case at almost the same time, the progression has been so disparate. one may argue that testing in india has been sluggish in contrast to aggressive testing by some developed nations. however, india had tested samples (nasal/throat swabs for qrt-pcr) from 26798 patients suspected to have covid-19, as of 27 th march which still gave a testing positivity rate of 2.6%, a figure which has not increased significantly over time or with changes in testing criteria (https://icmr.nic.in/sites/default/files/ whats_new/icmr_website_update_27march_9am_ist .pdf). a further update by the icmr pegged the figure at 42788 samples by 30 th march (2.5% test positivity). in comparison, italy at the top of the spectrum had carried out nearly 206886 tests as of 20 th march with 41035 positives, which translates into a positivity rate of tested individuals of 19.8%. south korea, on the other hand, adopted a comprehensive strategy of community testing. such an approach translated into 8652 positives in 316664 tests (2.7%) (www.statista.com/statistics/1028731/ covid19-tests-select-countries-worldwide/). a few other countries at different ends of the spectrum are represented in the graph in figure 1 . one instantly notices the surprisingly low rates of covid-19 positivity in india, and the south korean example implies that higher testing may not necessarily alter the incidence rates of covid-19. this low rate of covid-19 positivity remaining constant over a couple of weeks has led most experts to believe that community spread of the disease has not taken place in india as yet. such inference is, however, intriguing and needs careful analysis. the reports of hospitalization of covid-19 patients appeared in china from mid-december 2019 and given the average incubation period of 14 days for the manifestation of the disease, the viral spread likely started in china in early december 2019 [1] . the geographical proximity of india to china and the regular unrestricted movement of people between two countries through multiple daily direct and indirect flights throughout december 2019 until mid-january 2020 should have made a densely populated nation like india rapidly invaded by covid-19. considering these, we propose several possibilities for the low incidence of covid-19 in india, which include both environmental and biological factors. the population density of india is 414 persons/km 2 , which is higher than the six countries with maximum covid-19 cases. such a finding is a paradox as countries with higher densities and people staying closer to each other are theoretically at a higher risk of contracting communicable diseases transmitted by fomites or aerosols. similarly, the examples of south korea (517 persons/km 2 ) and japan (333 persons/km 2 ), which have a relatively lower incidence of covid-19 and a higher population density, support this curious case. trying to explain this paradox would require further research. in the köppen classification of climatic zones of the world, both wuhan, the apparent epicenter of the pandemic and most of italy are categorized as type c (mild temperate) whereas much of india has either a dry (type b) or tropical climate (type a). however, this again raises queries on why iran having a dry desert climate (type b) bore the brunt of covid-19 cases [3] . it has been suggested that high temperatures and high relative humidity levels significantly reduce sars-cov-2 transmission [4] . it may be fortuitous for india that as it enters the weeks deemed to mark its entry into phase 3 of the epidemic, the climate over large portions of the country is taking a turn for this same high temperature, high humidity state. like other animal and human corona viruses, sars-cov-2 is also a positive-strand rna (approximately 30kilobases) virus which codes for a replicase enzyme associated with other enzymatic activities and several structural proteins of which the spike protein (s) is essential for viral entry into host cells [5] . sars-cov, hcov-nl63 and sars-cov-2 enter the host cells through the protein angiotensin 1 converting enzyme 2 (ace2) on the cell membrane and the viral entry requires the cleavage of s protein by a serine protease (tmprss2) [5, 6] . ace2 protein is expressed in many different types of cells, and the virus can invade multiple organs [1] . since pulmonary alveolar cells and mucosa of the gastrointestinal tract have high expressions of ace2, sars-cov-2 can easily invade these organs [1, 7] . the invasion of lungs leads to the most critical pathology of pneumonia with severe respiratory distress and hypoxemia that accounts for the significant mortality in this disorder. enhanced production of pro-inflammatory cytokines and chemokines by immune effector cells leading to a cytokine storm is thought to be the cause of multiple systemic and respiratory symptoms. the genomic sequences of sars-cov-2 from multiple isolates show more than 99% identity which is sufficiently different from other human corona viruses and its origin has been traced to a bat corona virus [1] . apart from sars-cov and mers-cov which caused severe respiratory diseases following outbreaks in 2003 and 2012, there are four endemic human corona viruses, hcov-229e, hcov nl-63, hcov-oc4, hcov-hku1 in populations that are responsible for various types of respiratory illness which are generally self-limiting in young and immunecompetent persons [8] . the defense against any viral infection involves both innate immunity and adaptive immunity. while adaptive immunity relies on antibodies and t-cells which can recognize viral antigens with high degrees of specificity, the innate immunity utilizes receptors (pattern recognition receptors of several types) which recognize broad structural motifs present in bacteria or viruses but generally absent in host cells. there are currently no established data on the specific role of either humoral or cellular immunity or innate immunity in patients recovering from covid-19, but immune response associated with sars-cov and mers-cov has been studied in some detail [9] . it can be assumed that some degrees of sequence homology or conformational similarities among the structural proteins, especially the s protein, of sars-cov-2 and the endemic corona viruses (hcov-229e, hcov nl-63, hcov-oc4, hcov-hku1) may result in cross-reactive immunity (circulating antibodies or primed t-cells) in persons with prior exposure to the latter viruses, and this may modulate the course and outcome of covid-19. this kind of crossreactive immunity modulating the host-response to viral infection is well-known and widely studied in infections with flaviviruses (between different subtypes of dengue viruses or between the dengue virus and zika virus) [10, 11] . although the cross-reactive immunity may be protective in some cases, it may lead to augmented harmful reactions in other cases which has been termed antibody-dependent enhancement (ade), and these processes have been established from epidemiological studies as well as in animal models [10, 12] . thus, the possibility of a protective cross-immunity in the indian population against covid-19 cannot be ignored in explaining a rather mild effect of the current coronavirus pandemic in india in comparison to that in europe and the usa. it is important to note that the presence of crossreactive antibodies in the sera of patients infected with different types of human corona viruses have not been studied extensively. however, one study reported that sera of subjects who had sars-cov infection developed cross-reactive antibodies to hcov-229e and hcov-oc43 [13] . similarly, sera of convalescent patients of sars-cov have been shown to contain neutralizing antibodies against mers-cov [14] . likewise, sera of convalescent patients of sars-cov contain neutralizing antibodies that inhibit the entry of sars-cov-2 in vero cells [6] . therefore, cross-reactive antibodies generated as a result of infections from other human corona viruses may have a protective role in a population affected by covid-19. of note, antibodies against mers-cov have been detected in a significant fraction of persons exposed to camels and dromedaries without any clinical evidence of prior mers suggesting that mers-cov can infect individuals without any symptoms, and yet induce signs of protective immune response [15] . it is noteworthy that many corona viruses are widely present in cattle, pigs and chickens producing a variety of diseases affecting the respiratory and gastro-intestinal systems in these species [5] . the animal corona viruses do not infect human beings unless a mutation breaks the species-barrier. nonetheless, a large number of people working in dairy farms and livestock sectors come in close contact with animals and extensively handle the raw meat of these species in markets and houses with bare hands, which may expose the circulating immune cells to viral proteins through minor injuries in the skin. such exposures may result in the development of crossimmunity because of possible antigenic similarities among human and animal corona viruses. another interesting observation is the apparent exclusion of malaria-endemic zones by the covid-19 epidemic. india, large parts of africa, and parts of south america that report ongoing malaria transmission have had a low incidence of covid-19. the reasons are not known, but an explanation may lie in the ensuing cytokine storm, that may be involved in the severe respiratory manifestations of covid-19 [16] . it is now widely accepted that not only malaria but many other acute infective conditions such as sepsis have deleterious effects mediated through the human body's response to the infective agent, in the form of a cytokine storm [17] . it is plausible that persons in malaria-endemic zones, which may also be endemic for several tropical pathogens including viruses, have recurrent cytokine fluctuations in response to minor and subclinical infections. these recurrent fluctuations may have a de-sensitizing effect on the body's immune system, which prevents an uncontrolled and detrimental immune response and thus severe clinical disease in sars-cov-2 infection. hence, many infected patients, may not manifest any symptoms and, as a corollary, may not meet testing criteria, resulting in a low positivity rate. it may be mentioned here that a novel therapeutic approach has been tried recently in covid-19 utilizing the immunomodulatory role of mesenchymal stem cells to attenuate the cytokine storm [18] . apart from cross-immunity, an essential mechanism of the variable outcome of covid-19 may lie in the polymorphism of ace2 protein, which is coded by a gene on the x-chromosome. several single nucleotide polymorphisms of this protein have been described which may have implications in altered expression levels of ace2 or its interaction with s protein of sars-cov-2. however, a detailed analysis of such polymorphisms in different populations vis-à-vis the outcome of covid-19 will require more time [19] . further, in viral infections, host antiviral mirnas play a crucial role in the regulation of immune response, depending upon the viral agent. many known human mirnas appear to be able to target viral genes and their functions by interfering with replication, translation, and expression. in this context, an interesting, but yet to be peer-reviewed research article, performing a sequence-based analysis has reported a unique mutation in the indian sars-cov-2 strain affecting the s protein of the virus that allows it to be a target for a mirna (hsa-mir-27b) [20] . while not directly relevant to issues discussed in this article, a possibility also exists that many cases may be missed due to the sensitivity of the tests being performed. mostly pharyngeal and nasal swabs are used for qrt-pcr based diagnostics. a recent article mentions these samples as having only 32% and 63% positivity while testing for covid-19 [21] . another vital aspect of covid-19 is mortality in diagnosed cases. mortality is the statistic that is most relevant for developing nations such as india. acute viral illnesses as well as acute infections due to any pathogenic agent, are prevalent in india, owing to dismal standards of hygiene, especially among a large section of its population which lives below the poverty line. an exact number cannot be ascribed to the incidence of viral infections, because of the sheer diversity of such infections ranging from respiratory viral infections (influenza, rhinovirus, adenovirus, coronavirus, etc.) to several arthropod-borne viral fever syndromes. the diagnostic facilities are nonexistent in the periphery, and the majority of these infections may be subclinical or may present with atypical manifestations [22] . an idea can be obtained from the data of the integrated disease surveillance programme (idsp) of the ministry of health and family welfare (mohfw) of the government of india. during 2017, the idsp reported 1683 disease outbreaks in india, of which 71% were due to viral pathogens [23] . the authors, in their clinical practice, also treat patients regularly who present with classic symptoms of viral respiratory disease. sometimes, the same patient with comorbidities such as copd visiting multiple times in a year. whereas such patients in affluent nations would be subjected to additional testing and probably annual influenza vaccination, such measures are mostly non-existent in the indian scenario, apart from a few of the apex centers. the majority of such patients with fever, upper and lower respiratory tract symptoms, bodyache and headache may either not turn up at clinics, dismissing their symptoms as another bout of viral fever and resorting to home remedies, or even in case they present to a trained practitioner, supportive management is at best done. even drugs such as oseltamivir are scarcely used in routine practice. even with community transmission of the virus (phase 3 of the epidemic), most cases may turn out to be subclinical and mild. in the end, mortality and not morbidity is the statistic that matters. such a scenario brings us to the critical question; what is the actual attributable mortality of covid-19? a peek into the italian scenario may be helpful. nearly 85% of covid-19-related deaths in italy have been individuals in the 70+ years age group. among the deceased patients, chart details were available for 481. out of these only 6 patients did not have any comorbidities. among the 9 patients of age < 40 years who died (m=8, f=1), clinical information was available for 7, and each had pre-existing serious comorbidity(www.epicentro.iss.it/coronavirus/bollettino /report-covid-2019_20_marzo_eng.pdf). even in india, more than half of the deaths that have ensued have affected patients more than 60 years of age, the accepted geriatric age cutoff in the country. although the chart details of the indian patients are unavailable, the available data collected from reputed news sources is presented in table 1 . most cases with fatality, whether young or old evidently had comorbidities. it is common sense that in a patient with a fatality, in the presence of severe renal or pulmonary or cardiac disease, the mere presence of covid-19 positivity does not confirm the role of coronavirus in causing death. detailed case histories may be revealed in the coming days, which may further clarify the matters. there may be a faulty statistical angle to covid-19 mortality in india too. italy (23%) stands second in the world after japan (27%) in having a population of > 65 years age (www.worldatlas.com/articles/countries-withthe-largest-aging-population-in-the-world.html). in lifeexpectancy it also ranks second after japan among all countries with a population more than 10 million. italian healthcare provides for universal coverage and is largely free. what this means is a sizeable section of the population in italy has survived to an age that would be impossible for them to do up to, in other countries. contrast this to the situation in india where healthcare services do not have this much peripheral penetration. further, transport and awareness issues as well as societal preferences for the younger earning population, results in neglect of the elderly and failure of free healthcare to translate into health gains for the older population. india's life expectancy stands at 69 years, interestingly less than the mean age of covid-19 fatalities in italy. so, india may not have the most vulnerable age group for covid-19 to exert its effect to the fullest. overall, if one analyzes the mortality statistics of india in comparison to other countries (fig. 2) , it is clear that the curve has been flat for quite long. a plot between total confirmed cases and total confirmed deaths clarifies the situation in india further in comparison to other major countries (fig. 3 ). the different restrictive measures adopted by the central and the state governments in india like visa and travel restrictions, home isolation and quarantine, and finally nation-wide lockdown are classical methods of prevention of the community spread of sars-cov-2. these methods must have played a role in limiting the effect of this pandemic on the indian population. however, we strongly feel that various biological and environmental factors have also contributed significantly to this process, which may result in covid-19 in india behaving more mildly in contrast to its global effects. the careful analysis of such biological factors will certainly open new avenues to combat outbreaks of novel viruses, through research on cross-immunity, immuno-modulation and perhaps lifestyle management and dietary manipulations. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak the first two cases of 2019-ncov in italy: where they come from present and future köppen-geiger climate classification maps at 1-km resolution high temperature and high humidity reduce the transmission of covid-19 coronaviruses: an overview of their replication and pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor. cell covid-19: faecal-oral transmission? human coronaviruses: what do they cause? coronavirus infections and immune responses cross-reactive immunity among flaviviruses. front immunol cross-reaction, enhancement, and neutralization activity of dengue virus antibodies against zika virus: a study in the mexican population human polyand cross-reactive anti-viral antibodies and their impact on protection and pathology antigenic cross-reactivity between severe acute respiratory syndromeassociated coronavirus and human coronaviruses 229e and oc43 cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study mesenchymal stem cell infusion shows promise for combating coronavirus (covid-19)-induced pneumonia understanding the role of inflammatory cytokines in malaria and related diseases transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations comparative analyses of sar-cov2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis detection of sars-cov-2 in different types of clinical specimens morvan's syndrome-is a pathogen behind the curtain? emerging/reemerging viral diseases & new viruses on the indian horizon prof chakrabarti thanks the maharishi markandeshwar (deemed to be) university, mullana for research support. key: cord-259368-k8t8brjy authors: ren, xiang; li, yu; yang, xiaokun; li, zhili; cui, jinzhao; zhu, aiqin; zhao, hongting; yu, jianxing; nie, taoran; ren, minrui; dong, shuaibing; cheng, ying; chen, qiulan; chang, zhaorui; sun, junling; wang, liping; feng, luzhao; gao, george f.; feng, zijian; li, zhongjie title: evidence for pre‐symptomatic transmission of coronavirus disease 2019 (covid‐19) in china date: 2020-08-07 journal: influenza other respir viruses doi: 10.1111/irv.12787 sha: doc_id: 259368 cord_uid: k8t8brjy background: between mid‐january and early february, provinces of mainland china outside the epicentre in hubei province were on high alert for importations and transmission of covid‐19. many properties of covid‐19 infection and transmission were still not yet established. methods: we collated and analysed data on 449 of the earliest covid‐19 cases detected outside hubei province to make inferences about transmission dynamics and severity of infection. we analysed 64 clusters to make inferences on serial interval and potential role of pre‐symptomatic transmission. results: we estimated an epidemic doubling time of 5.3 days (95% confidence interval (ci): 4.3, 6.7) and a median incubation period of 4.6 days (95% ci: 4.0, 5.2). we estimated a serial interval distribution with mean 5.7 days (95% ci: 4.7, 6.8) and standard deviation 3.5 days, and effective reproductive number was 1.98 (95% ci: 1.68, 2.35). we estimated that 32/80 (40%) of transmission events were likely to have occurred prior to symptoms onset in primary cases. secondary cases in clusters had less severe illness on average than cluster primary cases. conclusions: the majority of transmissions are occurring around illness onset in an infected person, and pre‐symptomatic transmission does play a role. detection of milder infections among the secondary cases may be more reflective of true disease severity. a novel coronavirus named "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) was first identified in january 2020 as the pathogen responsible for a cluster of cases of atypical pneumonia in wuhan, a large city located in hubei province in central china. 1, 2 genetic analysis of the virus indicates that it originated from a bat coronavirus. 2 sars-cov-2 is considered distinct from sars-cov or mers-cov, and coronavirus disease 2019 caused by it has rapidly become a global health concern. 3 incidence of infections slowly increased through january 2020 with a reproductive number estimated to be in the range 2.2-3.6. [4] [5] [6] [7] starting in mid-january 2020, covid-19 cases began to be identified in other cities in china and also in other countries. 8, 9 a number of studies suggested the probable phenomenon of covid-19 transmissions during incubation period, [10] [11] [12] these studies used cluster cases in limited number of families for analysis, and the relative frequency of pre-symptomatic transmission was not quantified. moreover, there was an analysis on publicly available data indicating the existence of negative serial intervals, also implying pre-symptomatic transmission. 13 here, we retrospectively analyse data on cases identified outside of hubei province through the chinese public health event surveillance system at the early stage of transmission in china, in order to provide insights on the transmission dynamics of covid-19. data were extracted from the epidemiological reports of the chinese public health event surveillance system, through which the first confirmed case or potential outbreaks leading to clusters of suspected cases for each county were required to be investigated and reported. the extracted variables included demographic data, possible exposure and travel history, and clinical data using a structured form. the events reported by 29 january 2020 were included for data extraction. for events by 23 january 2020, which was the date of "locking down" wuhan city, all events (168 cases) in the system were extracted, and we used these data to construct an epidemic curve and estimate the incubation period distribution. of the events (281 cases) reported between 24 january and 29 january 2020, we focused on those events which included probable human-to-human transmission or epidemiologically linked cases, so that we could capture a larger number of these clusters for analyses of serial intervals, transmission events and comparative severity between primary cases and secondary cases. all cases included in our analyses were laboratory-confirmed cases, and the case definitions followed we drew the epidemic curve by illness onset for cases who reported that they had been in wuhan in the 14 days of preceding onset, and a separate epidemic curve for the other cases. cases with onset dates closed to the end of epidemic curve may not be reported due to delays in seeking medical attention and consequent delays in laboratory testing. to allow for onset-to-reporting delays, we used the nowcasting approach described by van de kassteele et al 15 to jointly estimate the augmented case number on the most recent dates and the onset-to-reporting distribution. based on 1000 augmented epidemic curves, then we fitted exponential growth models to obtain estimates of the growth rate and doubling time. we examine the characteristics of confirmed cases and compared the demographics, onset symptoms and results of some clinical tests between confirmed sporadic and cluster primary cases and cluster secondary cases. we also obtained information on secondary cases that had not been to wuhan but had close contact with another case that had been to wuhan and were presumed to be infected by that particular case. in these pairs of primary and secondary cases, we fitted a normal distribution to the serial intervals between illness onset dates, allowing for negative and zero serial intervals, and correcting for growth rates in the early stage of an epidemic. specifically, the parameters of the fitted normal distribution were corrected to normal (µ′,σ) where µ′ = µ + σ 2 ρ for the epidemic growth rate r estimated below (personal communication, neil ferguson). because information was available on the periods during which each secondary case had been exposed to their presumptive infector, we were able to identify cases where exposure is likely to have occurred before or after illness onset in the primary case infector. when the exposure window overlapped the onset date, we used our fitted incubation period distribution to resample 1000 infection times at random, excluding any that fell outside the exposure window, and counted the proportion of resampled times that fell before symptom onset in the infector. we collected detailed information on 168 confirmed cases, including 145 and 23 reporting that they had or had not, respectively, been in wuhan in the past 14 days. the demographics of the 145 and 23 cases were very similar (data not shown). the median age was 48 f i g u r e 1 panel a: occurrence by date of illness onset of cases identified outside of hubei province in persons with a history of travel from wuhan in the 14 d prior to onset. panel b: occurrence by date of illness onset of cases identified outside of hubei province in persons without a history of travel from wuhan in the 14 d prior to onset. panel c: augmented occurrence (yellow bars) by date of illness onset of cases identified outside of hubei province in persons with a history of travel from wuhan in the 14 d prior to onset with back filled cases considering delays between illness onset and seeking care and being tested (range 10-81) and 91 (54%) were male. only three (2%) of the cases who had visited wuhan also reported visiting the huanan seafood wholesale market. figure 1 shows the epidemic curve by illness onset of the cases in persons that had been to wuhan compared to cases in other persons that were presumed to represent onwards transmission. we used data augmentation to correct for reporting delays in cases with recent onset ( figure 1c ) and estimated that the growth rate was 0.14 per day (95% ci: 0.11, 0.17), and the doubling time was 5.3 days (95% ci: 4.3, 6.7). in this analysis, the mean onset to reporting delay was estimated to be 5.7 days (95% ci: 5.3, 6.1). we obtained data on exposure windows for 98 cases in individuals that had been in wuhan, by assuming that they had been infected while they were in wuhan. we fitted a lognormal distribution to the data on exposure periods and onset dates, correcting for epidemic growth, and estimated that the incubation period had mean 5. (14%) occurred in those who had a meal together, 2 (3%) were colleagues working together, 2 (3%) were those who took the same vehicle, and 1 (1%) occurred among neighbours. the identified 80 observations of transmission events were used for estimation of the serial interval ( figure 2b ). we fitted a normal distribution to these data, with allowing 4 negative serial intervals and correcting for epidemic growth, and estimated that the serial interval distribution had mean 5.7 days (95% ci: 4.7, 6.8) and standard deviation 3.5 days ( figure 2b ). in detailed investigations of contact patterns, we obtained information on the period when the secondary infection could have occurred, and related this to the illness onset date of the infector. figure 3 shows that of 80 pairs identified, pre-symptomatic transmission occurred in 9 pairs, post-symptomatic transmission occurred in 16 pairs, and in the remaining 55 pairs, transmission could have occurred either before or after the corresponding infector's illness onset date. in these 55 pairs, we used monte carlo simulations based on the incubation period distribution described above to estimate that 23/55 of transmission events had greater than 50% chance to be pre-symptomatic transmissions. thus, in total we infer that 32/80 (40%) of the observed transmission events were likely or very likely to have occurred prior to the onset of symptoms in the infector. we examined demographic and clinical characteristics of all the confirmed sporadic and clusters cases ( table 1) . age distributions were similar between the two groups of patients, while less male cases were identified in cluster secondary cases (43% vs 60%). cluster primary and sporadic cases were generally more severe, with 26% and 7% in severe and critical conditions, respectively, compared with 12% and 1% in cluster secondary cases. nearly all the primary and sporadic cases (96%) had a radiologic indication of pneumonia, compared with 74% in the secondary cases. among 80 cluster secondary cases, 87% had been classified as mild cases, while 66% (110/166) of the cluster primary and sporadic cases were mild. the primary and sporadic cases showed largely similar onset symptoms as the secondary cases. fever is the most common symptom, followed by headache, fatigue, dry cough and myalgia. generally higher proportions of the cluster primary cases and sporadic cases reported an onset of the symptoms than cluster secondary cases (table 1 ). in addition to systemic and respiratory presentations, a small proportion of the cases also reported gastrointestinal symptoms, including vomiting and diarrhoea. in this study, we report estimates of the transmission dynamics of covid-19 based on cases identified outside of hubei province. importantly, we examine quantitative evidence for pre-symptomatic infectiousness, a feature which complicates control strategies. we transmission events used to infer the occurrence of pre-symptomatic transmission. dots indicate the dates of onset of primary and secondary cases, and the shaded area in each row indicates the period of exposure of the secondary case to the primary case during which the secondary infection is thought to have occurred. brackets indicate the exposure window when the primary case was thought to have been infected. data were resolved to the nearest day, and so transmission windows are plotted from the start of the first date to the end of the last date, onset dates are plotted in the middle of the corresponding day, and if the secondary onset date is the same as the primary onset date, then the former is offset slightly so that both can be seen note: for patients having clinical test results of both lymphocyte count and proportion, a normal lymphocyte count refers to both the lymphocyte count within the range of 1-4 × 10 9 /l and the lymphocyte proportion to be 20%-40%. patients with either lymphoycyte count or lymphocyte proportion lower or higher than the normal range will be classified as "decreased" or "increased," respectively. a category of lymphocyte count is determined by both the count and proportion of lymphocyte (the proportion is derived as lymphocyte count divided by white blood cell count) in the blood test. demonstrate that the phenomenon of pre-symptomatic transmission is not uncommon, having occurred in a minimum of 9/80 transmission events ( figure 3 ). using statistical inference based on our estimated incubation period distribution, we estimated that pre-symptomatic transmission was likely to have occurred in up to 40% of the 80 transmission events in our data set. this observation should be interpreted in the context of isolation of some cases after illness onset, reducing the amount of post-symptomatic transmission that might otherwise have occurred. this also implies that social distancing measures may be some of the most important strategies to reduce transmissibility, for example closing schools and encouraging, or facilitating working at home, in addition to control of onwards transmission by sequestering symptomatic persons at home or in isolation facilities. our finding was consistent with other reports that the serial interval distribution was similar to, or much lower than the incubation period distribution, 13,18-21 consistent with the occurrence of pre-symptomatic transmission in case reports. [22] [23] [24] [25] [26] pre-symptomatic infectiousness is generally not thought to occur for most respiratory viruses, but measles is a well-known example of a respiratory infection that can be spread before symptom onset, 27 and viral shedding during the incubation period has also been reported for influenza. 28 viral shedding has been reported during the incubation period for covid-19, 29 and there were also asymptomatic cases for covid-19 across all age groups, in varying proportions depending on the intensity of surveillance and testing. 30, 31 in conducting investigations of clusters of cases, it is often found that secondary cases have on average milder illnesses than primary cases; for example, this was noted for middle east respiratory syndrome coronavirus infections. 32 here, we found that secondary cases were more likely to have milder disease (table 1) , which would be consistent with the existence of some milder covid-19 infections, which were not laboratory confirmed. the cluster investigations that we report here are not likely to represent the full spectrum of mild infections, and better information would be provided by prospective studies of close contacts with repeated collection of respiratory swabs and sera. our estimates of the growth rate of infections in wuhan in early january 2020 of around 0.14 per day (95% ci: 0.11, 0.17) are very consistent with previous reports. 4, 6 our estimate of the effective reproductive number in wuhan is now slightly lower than the previous estimate of r 0 4 and some other estimates of r 0 5-7 but that is because of the shorter serial interval. we included a limited number of cases in other cities in china that were detected in persons that had not been to wuhan ( figure 1b ), but the sample size was insufficient at the time of analysis to determine whether there has been sustained transmission in any other cities. the incubation period averaged 4.6 days and up to 11.1 days in 95% of infections (figure 2a) , which is important for specifying quarantine periods and also for understanding transmission dynamics. the city of wuhan was put under lockdown on 23 january 2020, and other nearby cities were also locked down on the following days. this stopped exportation of infections to other chinese cities, and the effect of this intervention has become more apparent in early february, given the delays that occur between infection, illness onset, admission to hospital and then laboratory testing. we restricted the study participants to covid19 analysis were also subject to ascertainment of symptom onset date, which was self-reported by the patient and could be less reliable especially at the early stage of symptom presentation. in conclusion, our analysis showed evidence indicative of pre-symptomatic transmission of covid-19. quarantine of exposed persons having close contact with infected cases before symptom onset could reduce the risk of further transmission. we gratefully thank benjamin j. cowling, peng wu, jessica y. wong, yiu chung lau, tim k. tsang from school of public health, university of hong kong for their guidance and support on study design, data analysis and interpretation. we appreciate the staff members of the county-, city-and province-level centers for disease control and prevention of china for their fieldwork on case investigation, data collection and validation. clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china a novel coronavirus outbreak of global health concern early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia pattern of early human-to-human transmission of wuhan nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov in china, from 2019 to 2020: a data-driven 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coronavirus disease (covid-19) based on symptom onset data quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing presymptomatic transmission of sars-cov-2 -singapore delivery of infection from asymptomatic carriers of covid-19 in a familial cluster asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility a covid-19 transmission within a family cluster by presymptomatic infectors in china transmission of covid-19 in the terminal stages of the incubation period: a familial cluster evidence base of incubation periods, periods of infectiousness and exclusion policies for the control of communicable diseases in schools and preschools the dynamic relationship between clinical symptomatology and viral shedding in naturally acquired seasonal and pandemic influenza virus infections viral load of sars-cov-2 in clinical samples estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship a well infant with coronavirus disease 2019 (covid-19) with high viral load middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility evidence for pre-symptomatic transmission of coronavirus disease 2019 (covid-19) in china key: cord-259126-5d4p8woi authors: pinotti, francesco; di domenico, laura; ortega, ernesto; mancastroppa, marco; pullano, giulia; valdano, eugenio; boëlle, pierre-yves; poletto, chiara; colizza, vittoria title: tracing and analysis of 288 early sars-cov-2 infections outside china: a modeling study date: 2020-07-17 journal: plos med doi: 10.1371/journal.pmed.1003193 sha: doc_id: 259126 cord_uid: 5d4p8woi background: in the early months of 2020, a novel coronavirus disease (covid-19) spread rapidly from china across multiple countries worldwide. as of march 17, 2020, covid-19 was officially declared a pandemic by the world health organization. we collected data on covid-19 cases outside china during the early phase of the pandemic and used them to predict trends in importations and quantify the proportion of undetected imported cases. methods and findings: two hundred and eighty-eight cases have been confirmed out of china from january 3 to february 13, 2020. we collected and synthesized all available information on these cases from official sources and media. we analyzed importations that were successfully isolated and those leading to onward transmission. we modeled their number over time, in relation to the origin of travel (hubei province, other chinese provinces, other countries) and interventions. we characterized the importation timeline to assess the rapidity of isolation and epidemiologically linked clusters to estimate the rate of detection. we found a rapid exponential growth of importations from hubei, corresponding to a doubling time of 2.8 days, combined with a slower growth from the other areas. we predicted a rebound of importations from south east asia in the successive weeks. time from travel to detection has considerably decreased since first importation, from 14.5 ± 5.5 days on january 5, 2020, to 6 ± 3.5 days on february 1, 2020. however, we estimated 36% of detection of imported cases. this study is restricted to the early phase of the pandemic, when china was the only large epicenter and foreign countries had not discovered extensive local transmission yet. missing information in case history was accounted for through modeling and imputation. conclusions: our findings indicate that travel bans and containment strategies adopted in china were effective in reducing the exportation growth rate. however, the risk of importation was estimated to increase again from other sources in south east asia. surveillance and management of traveling cases represented a priority in the early phase of the epidemic. with the majority of imported cases going undetected (6 out of 10), countries experienced several undetected clusters of chains of local transmissions, fueling silent epidemics in the community. these findings become again critical to prevent second waves, now that countries have reduced their epidemic activity and progressively phase out lockdown. two hundred and eighty-eight cases have been confirmed out of china from january 3 to february 13, 2020. we collected and synthesized all available information on these cases from official sources and media. we analyzed importations that were successfully isolated and those leading to onward transmission. we modeled their number over time, in relation to the origin of travel (hubei province, other chinese provinces, other countries) and interventions. we characterized the importation timeline to assess the rapidity of isolation and epidemiologically linked clusters to estimate the rate of detection. we found a rapid exponential growth of importations from hubei, corresponding to a doubling time of 2.8 days, combined with a slower growth from the other areas. we predicted a rebound of importations from south east asia in the successive weeks. time from travel to detection has considerably decreased since first importation, from 14.5 ± 5.5 days on january 5, 2020, to 6 ± 3.5 days on february 1, 2020. however, we estimated 36% of detection of imported cases. this study is restricted to the early phase of the pandemic, when china was the only large epicenter and foreign countries had not discovered extensive local transmission yet. missing information in case history was accounted for through modeling and imputation. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 our findings indicate that travel bans and containment strategies adopted in china were effective in reducing the exportation growth rate. however, the risk of importation was estimated to increase again from other sources in south east asia. surveillance and management of traveling cases represented a priority in the early phase of the epidemic. with the majority of imported cases going undetected (6 out of 10), countries experienced several undetected clusters of chains of local transmissions, fueling silent epidemics in the community. these findings become again critical to prevent second waves, now that countries have reduced their epidemic activity and progressively phase out lockdown. why was this study done? after being first identified in china in january 2020, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reached all countries worldwide within a few months [1] . massive intervention measures [2] were implemented by chinese authorities in late january to control the epidemic. countries outside china promptly reinforced border controls and intensified active surveillance to rapidly detect and isolate importations, trace contacts, and isolate suspect cases [3, 4] . such a massive response did not prevent countries to face importations of cases and experience extended independent chains of local transmission [5] [6] [7] , ultimately resulting in sustained local spread. the effectiveness of measures aiming at preventing importations and local transmissions critically depends on disease epidemiology and natural history of the infection [8, 9] , as well as the volume of importations [3] . for the case of coronavirus disease 2019 , the presence of an incubation period, during which infected individuals carry on their usual activities (including travel), was a major challenge for screening controls at airports [8] . moreover, mild non-specific symptoms and transmission before the onset of clinical symptoms [10] [11] [12] compromised infection control measures for importations and onward transmissions [9] . imported cases went undetected and contributed to the global spread of the disease [13] [14] [15] [16] [17] [18] . here we systematically collected and analyzed data on the first 288 covid-19 confirmed cases outside china, to characterize the international spread of covid-19 pandemic during its early phase. we show that the detailed information carried by case histories provided early evidence of the covid-19 specific features that enabled the virus to escape containment efforts and reach pandemic proportion. we analyzed importations that were successfully isolated and those leading to onward transmission, characterizing their case timeline. we developed a statistical model to describe trends in importations up to mid-february and quantified the proportion of undetected imported cases. we collected all international cases confirmed by official public health sources in the period from january 3 to february 13, 2020. case history was reconstructed by searching the scientific literature, official public health sources, and news . case history included dates of travel and symptom onset, date of covid-19 confirmation, date of hospital admission, date of case isolation, travel history, epidemiological link with other cases, and hospitalization history. international cases included imported cases, secondary cases out of china, and repatriations. cases from cruises were not considered here. information was extracted by ldd and eo and checked by mm. additional cases in the period from february 14 to february 27 were collected and used to validate the results. the full database, along with the database describing clusters, was made publicly available [40] . this study is reported as per the strengthening the reporting of observational studies in epidemiology (strobe) guideline. strobe checklist can be found in the supporting information (s1 strobe checklist). the study did not have a protocol. for imported cases with full information on the timeline of events, we computed the average duration from travel to onset, from travel to hospitalization, and from hospitalization to reporting. we used analysis of variance to compare groups of imported cases that generated or did not generate local transmissions. we extended the analysis to all imported cases combining cases with full and partial information on the timeline. we used the analysis of variance and multiple imputation for missing data. results were combined using rubin's approach [41] . we modeled the total number of imported cases out of china over time accounting for date of travel, delay in reporting, and source areas. we distinguished between 3 different sources: hubei province (h), the rest of china (c), other countries (o). we modeled imported cases over time as a piecewise exponential function depending on the source and on travel restrictions in place. we assumed a different situation in hubei province and the rest of the world because of the level of awareness in the different phases of the outbreak. the exponential functions are defined as follows: where r pre h is the growth rate of cases coming from hubei, and r pre c ; r o , with r pre c ¼ r o , the growth rates of cases coming from the rest of china and other countries, respectively. travel restrictions were modeled by assuming a discontinuity in the growth rate. for hubei, we assumed the growth rate to change from r pre h to r post h after the travel ban of january 23, 2020 (indicated with t h ); for the rest of china, we assumed an analogous change from r pre c to r post c after january 29, 2020 (t c ), date of first flight cancellations [42] . no change was considered for the other countries (r o constant over time), as no restrictions of travel were established toward these countries. the scale parameters of the exponential functions (i pre h ; i post h ; i pre c ; i post c ; i o ) were assumed to be different among the 3 sources, to account for different traveling volumes and dates of beginning of importations. we modeled the time τ from importation to detection of a case with a gamma distribution, g t (τ), conditioned to the date of case importation, t. the distribution g t (τ) was assumed to have constant coefficient of variation (sd/mean) achieved by a constant shape parameter and a rate parameter varying smoothly in time to capture change in surveillance efficiency. we used a bayesian framework to fit the model to imported cases by origin, travel date, and confirmation date. cases with partial information (e.g., missing date and/or origin of travel) were included by defining latent variables marginalized out during inference. the model was then used to predict imported cases 2 weeks in the future. all details of the analysis are reported in the supporting information (s1 text). we analyzed clusters of transmission generated by imported cases (index case(s) in each cluster) to estimate undetected importations. a cluster can be seeded by more than 1 index case, e.g., by infected family members who traveled together. the number of clusters of local transmission was modeled with a multinomial distribution according to the number of index cases and whether these were imported cases. as detailed in the supporting information, the likelihood function was a function of 4 observable quantities: the number of observed clusters with 1 imported index case (x 1 ); the number of observed clusters with more than 1 imported index case (x 2 ); the number of known imported cases causing no onward transmission (ỹ); the number of clusters for which an index case was not identified (z); and a fifth and unobserved quantity, the number of undetected imported cases who did not start onward transmission (w). maximization of the likelihood allowed to estimate w from the records of x 1 , x 2 ,ỹ, z and to compute the expected number of unobserved imported cases. additional details are described in the supporting information (s1 text). we collected 288 cases, including 163 imported cases, 109 cases involved in local transmissions, 30 repatriations, and 1 case of unknown origin. fifteen cases were classified as both imported and local transmissions, since they contracted the infection outside china and traveled to a different country once infected (es01, es02, gb03, gb04, gb05, gb06, gb07, gb08, kr12, kr16, kr17, kr19, my09, th20, and th21 in our database [40] ). fig 1 summarizes the timeline of imported cases. symptom onset occurred after the travel to the destination country for almost all cases for which date of travel and of onset are available (68 out of 73, 93%). complete information was available for 51 (31%) imported cases, with quality of information decreasing over time (s2 text, fig a) . among imported cases with full information, the delay from travel to hospitalization was longer in cases that generated secondary transmissions (mean of 10 ± 0.97 days compared with 5.5 ± 0.67 days, p = 0.003). overall, the duration from travel to first event (whether symptom onset, or hospitalization for asymptomatic) was also longer, although the difference was not statistically significant (5.0 ± 0.9 days versus 3.7 ± 0.5 days, p = 0.08). durations of hospitalization were instead comparable among the 2 groups of cases (1.5 ± 0.7 days versus 2.6 ± 0.4 days for cases that generated or did not generate secondary transmissions, respectively). including imported cases with missing information through imputation, we found the same trend though smaller in magnitude and not statistically significant (delay from travel to hospitalization 9.8 ± 1.2 versus 8.3 ± 0.5 days p = 0.3; delay from travel to onset 5.8 ± 1.1 versus 4.2 ± 0.5 p = 0.16, for cases that generated or did not generate secondary transmissions, respectively). this suggests that importations with missing information may be closer in characteristics to index cases leading to onward transmission. the statistical model predicted a decrease in the average time from travel to detection from 14.5 ± 5.5 days on january 5, 2020, to 6 ± 3.5 days on february 1, 2020 (fig 2) . the model predicted a rapid exponential growth of importations from hubei, with a growth rate r pre h ¼ 0:26 (95% ci 0.21-0.31), corresponding to a doubling time of 2.8 days. in comparison, the exponential growth from other territories (rest of china and countries other than china) was slow, r pre c ¼ r o ¼ 0:04 (95% ci 0.00-0.08). after the implementation of travel restrictions, a negative growth rate was estimated, signaling a decline in imported cases. table b ). the predicted trend of all imported cases over time is shown in fig 3, compared with the observed data. reported importations were predicted to remain stationary in the second and third week of february and to rise again because of the effect of transmission clusters outside china. imported cases after february 13, 2020, were in agreement with model predictions (fig 3) up to february 24. data in the successive 3 days diverged from our predictions as they were linked with early detection of multiple imported cases in the middle east and europe from sustained local transmission in iran and italy. forty-two transmission clusters were identified out of china in the timeframe under study. table 1 summarizes the size and country of each cluster. clusters were grouped according to whether the index case (1) was a traveling case identified prior to cluster detection, (2) a traveling case not identified or identified retrospectively once the cluster was observed, or (3) completely unknown. assuming that clusters of unknown origin were linked to one of the already observed imported cases-or, in other words, not linked to an undetected imported case-led to an estimate of 76 (95% ci 49-118) undetected imported cases. in this scenario, detected cases would amount to 65% of all imported cases. assuming instead that all clusters of unknown origin were due to undetected imported cases increased the number of undetected cases to 225 (95% ci 186-369), i.e., detected cases would correspond to only 36% of the total. we reviewed here all confirmed cases out of china from january 3 to february 13, 2020, and gathered detailed information on case history and epidemiological links to (1) identify salient epidemiological features, (2) assess the impact of travel restrictions in china on the importations of cases worldwide, and (3) evaluate the effectiveness of control measures against importations. we found a rapid exponential growth of importations from hubei, up to the closure of wuhan airport preventing further travel of cases, combined with a slower growth from other countries in south east asia. the estimated growth of importation before interventions is compatible with a doubling time of 2.8 days. time from travel to detection considerably decreased across time, from 14.5 ± 5.5 days on january 5, 2020, to 6 ± 3.5 days on february 1, 2020. however, our estimates indicate that only 36% of imported cases were detected. this study is restricted to the early phase of the pandemic, when china was the only large epicenter and extensive silent transmission in other countries, such as iran and italy, was not discovered yet. the substantially larger growth of importations from china compared with other territories is related to a stronger epidemic activity in the hubei province, origin of the outbreak, with respect to other affected areas [5] . this difference is likely an outcome of the containment measures in china [2, 43, 44] and of the increased awareness following their implementation [45] [46] [47] [48] [49] , leading to more efficient control. identification, rapid management of cases, and contact tracing were indeed proposed by the world health organization as key to contain the epidemic globally. we found that the travel ban in wuhan produced a sharp decline in importations from the region. combined with local containment measures in the rest of china and other countries in southeast asia, this resulted in a substantial overall reduction of exported cases worldwide. indeed, after peaking at the end of january, registered traveling cases declined to plateau at very low levels. on one side, this shows that strict travel bans may be beneficial by reducing importations to manageable levels and by giving countries the time to prepare and strengthen their surveillance systems in the short term, as signaled by a reduction of the interval from travel date to detection over time. on the other hand, however, the decline likely occurred too late when local spreading was already established in many countries outside the epicenter of traveler(s) identified prior to cluster detection a 15 cde01 (16), cfr02 (12) , cvn02 (7), ckr01 (5) (2), cjp05 (2), cjp07 (2), csg03 (2), csg05 (2), csg06 (2), csg07 (2), csg08 (2), csg10 (2), csg12 (2), cth01 (2), cth02 (2), cth03 (2), cae01 e a the index case was identified independently from secondary transmissions. b cluster associated with 2 traveling cases. c the index case was either identified retrospectively following case investigation prompted by the detection of secondary cases or the identity was not identified; however, the cluster was linked to a specific location/circumstance visited by chinese travelers (shop, conference, bus tour). the epidemic [50, 51] . at the end of february, sustained community transmission in iran and italy led to exportations to several other countries in the world in a timeframe of few days [52, 53] . this was facilitated by the fact that no local transmission was reported in these countries, preventing the alert to other countries for them to deploy targeted surveillance and control. at that time, indeed, case definition for the importation of a covid-19 suspect case was based exclusively on china as the origin of exposure or travel [54, 55] , with few exceptions including east asian countries [56, 57] . monitoring imported cases is critical in territories with sporadic epidemic activity where the attention is concentrated on preventing the introduction of cases. at the time of writing, countries such as china and australia show limited local transmission and are concentrating strong efforts in border controls to avoid a second epidemic wave, that could be fueled by the large proportion of the population still susceptible to sars-cov-2. strict measures, such as massive testing and quarantine of incoming travelers, are being implemented to block silent introductions of cases as it occurred at the beginning of the pandemic. we found that during the first half of february, countries outside china witnessed an increasing reporting of clusters with no known epidemiological link [5, 16] . our estimates indicate a detection ability of 36% to ascertain imported cases in countries outside china. this means that approximately 6 imported cases out of 10 have gone undetected. previous detection rates estimates range from 27% [15] to 38% [15, 17] , with variations across countries [13, 15] . ascertainment was estimated to be even lower (approximately 10%) when assessed on repatriations [58] . here, we excluded from this analysis all repatriation events and cruises with outbreaks, as conditions for detection and identification may be different. underdetection may have been due to several different factors including asymptomatic infections, infections with mild clinical symptoms, health-seeking behavior and declaration of travel history, case definition, and underdiagnosis. a relative long prodrome phase preceding symptom onset (approximately 2 days [59] ) may have limited the tracing and isolation of contacts. we found, indeed, that traveling cases generating transmissions following importation were, in general, spending more time in the community prior to hospitalization-although this result was diluted upon imputation of missing values-signaling that the period of mild or no symptoms during which the individual carries on a normal life is important for the generation of secondary cases. the epidemic emergency starting with one single critical case on february 21, 2020, in italy and accumulating hundreds of cases in few days [16] showed that clusters had gone undetected and epidemiological links with the index case were not found. subsequent developments of the pandemic showed that underdetection of importations was a clear pattern in fueling large-scale epidemics that spread unseen in several countries before the first critical cases were finally detected [52, 60] . our study is affected by limitations. underdetection of imported cases during the early phase of the pandemic might have been higher than what estimated here, as our analysis is conditional to the identification of clusters of cases. underdetection may also proceed from the imperfect characteristics of rt-pcr (reverse transcription-polymerase chain reaction) tests used to identify infected cases. some cases tested for sars-cov-2 could have been falsely negative, and this would affect both analyses presented in the manuscript. this is in line with our conclusion that a large part of imported cases may have been undetected. as the number of detected imported cases grew considerably, the quality of the information on case history degraded over time. we used modeling and imputation to account for the missing information regarding the most recent imported cases. extension of this study beyond the early phase of the pandemic is limited by the strong spatiotemporal heterogeneities currently characterizing the pandemic. our assumptions are based on a context of localized epicenter and border controls targeted against importations from china and countries in south east asia who experienced local transmission during the early phase. as the pandemic regime shifted toward a multiple delocalized epicenter in late february, additional travel bans were implemented worldwide to prevent importations from europe and north america. this led to heavy disruptions of the flight network and a change of focus toward within-country transmission. our findings provide critical epidemiological understanding on the rate of importation and detection of cases during the early phase of the pandemic. though effective in reducing international spread, the travel ban in wuhan did not prevent the seeding of the pandemic in other countries, later becoming new epicenters of the pandemic. the epidemiological features of covid-19 facilitated the silent spread of the epidemic in seeded countries. the lessons learnt during the early phase of the pandemic become critical again to prevent second waves now that countries have reduced their epidemic activity and progressively phase out lockdown. supporting information s1 text. statistical methods. modeling traveling cases, delay from arrival to detection and index case detection probability. (pdf) s2 text. additional results. dataset of international cases, results of likelihood estimation, sensitivity analyses, and analysis of imported clusters. (pdf) s1 european centre for disease prevention and control. covid-19 situation update worldwide an investigation of transmission control measures during the first 50 days of the covid-19 epidemic in china world health organisation. coronavirus disease (covid-19) technical guidance covid-19: what is next for public health? the lancet coronavirus disease (covid-2019) situation report 37 we are calling on all countries to invest urgently in preparedness we are concerned about the number of cases with no clear epidemiological link estimated effectiveness of symptom and risk screening to prevent the spread of covid-19. elife factors that make an infectious disease outbreak controllable chinese center for disease control and prevention. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china. zhonghua liu xing bing xue za zhi transmission of 2019-ncov infection from an asymptomatic contact in germany epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study estimates of the severity of coronavirus disease 2019: a model-based analysis early dynamics of transmission and control of covid-19: a mathematical modelling study report 6: relative sensitivity of international surveillance. imperial college london covid-19 response team covid-19-situazione nel mondo using observational data to quantify bias of travellerderived covid-19 prevalence estimates in wuhan, china. lancet infect dis feasibility of controlling covid-19 outbreaks by isolation of cases and contacts world health organization. coronavirus disease (covid-19) situation reports european centre for disease prevention and control. covid-19 situation update worldwide queensland covid-19 statistics covid-19 current health situation emirates news agency bayerisches staatsministerium fü r gesundheit und pflege ministère des solidaités et de la santé uk government public health. news and communication italian ministry of health covid-19 india about coronavirus disease 2019 (covid-19) malaysia ministry of health. press releases updates on novel coronavirus disease (covd-19) the russian government public health agency of sweden. covid-19 cdc [internet updates on covid-19 local situation ministry of health thailand covid-19 international cases as of multiple imputation for nonresponse in surveys airlines around the world are suspending flights to china as the coronavirus spreads the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak. science assessing spread risk of wuhan novel coronavirus within and beyond china early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study. lancet digit health risk of mers importation and onward transmission: a systematic review and analysis of cases reported to who public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey survey on the likely behavioural changes of the general public in four european countries during the ebola outbreak: media events track changes in observed reproductive number potential short-term outcome of an uncontrolled covid-19 epidemic in cryptic transmission of sars-cov-2 in washington state. medrxiv preliminary estimation of the novel coronavirus disease (covid-19) cases in iran: a modelling analysis based on overseas cases and air travel data covid-19: italy confirms 11 deaths as cases spread from north european centre for disease prevention and control. case definition and european surveillance for human infection with noel coronavirus (sars-cov-2) global surveillance for human infection with coronavirus disease (covid-19) covid-19: guidance for staff in the transport sector israel bans foreigners coming from east asian countries over virus fears the rate of underascertainment of novel coronavirus (2019-ncov) infection: estimation using japanese passengers data on evacuation flights temporal dynamics in viral shedding and transmissibility of covid-19 european centre for disease prevention and control. threat assessment brief: outbreak of novel coronavirus disease 2019 (covid-19): situation in italy key: cord-262104-oig3qrr7 authors: brüssow, harald title: covid‐19: test, trace and isolate‐new epidemiological data date: 2020-06-08 journal: environ microbiol doi: 10.1111/1462-2920.15118 sha: doc_id: 262104 cord_uid: oig3qrr7 in the absence of an efficient drug treatment or a vaccine, the control of the covid‐19 pandemic relies on classic infection control measures. since these means are socially disruptive and come with substantial economic loss for societies, a better knowledge of the epidemiology of the new coronavirus epidemic is crucial to achieve control at a sustainable cost, and within tolerable restrictions of civil rights. this article is protected by copyright. all rights reserved. comprising 10'800 participants (gudbjartsson et al., 2020) . very similar information was reported in data describing household transmission in wuhan, where children showed a 4% infection rate compared with 17% in adults. . likewise, in a study from hunan children had a threefold lower infection risk than adults . in contrast, the infection risk in children from shenzhen, china was similar to that in adults (bi et al., 2020) . however, all studies concur that disease in children is generally mild, if not asymptomatic. asymptomatic cases raise problems for contact tracing and containment, but it is currently not clear to what extent infected children transmit the disease. three asymptomatic sars-cov-2-positive adolescents showed transmission to family members (liao et al., 2020) while data for transmission from children are still absent. one special problem should still be mentioned in this context: in italy, pediatric hospitalization decreased substantially during the covid-19 epidemic. when the general population avoids hospitals for fear of infection, it can have a negative health impact. an increased number of deaths occurred in italy due to delayed arrival of children in hospitals, while no child died from covid-19 in italy (lazzerini et al., 2020) . the fear of hospitals has also been responsible for a deficit in the treatment against stroke in adults in the us (kansagra et al., 2020) . the statistics for the covid-19 epidemic have shown that the older population has suffered the greatest loss of life, and that nursing homes have been hotspots for transmission. two detailed studies from the us document these given facts. at the end of february 2020, a cluster of 167 epidemiologically linked covid-19 cases were reported in several long-term care facilities in washington state. of those, 144 cases were residents (median age 83 y), 55% of them were hospitalized, and 33% died. in comparison only 16 cases had been visitors, 50% of whom were hospitalized, but none died. further 50 cases were among the health care personnel, of whom only 6% were hospitalized, but again none died. factors favoring the outbreak were: health care personnel who showed up to work with symptoms; some of the personnel worked at more than one facility; and some residents were transferred between facilities. in the early stages of the epidemic, contributing factors to this outbreak were: an unawareness of the risk; a lack of diagnostic tests; and inadequate personal protection equipment (mcmichael et al., 2020) . currently, 1.3 million us americans reside in nursing homes. one in ten of >1'300 accredited nursing facilities reported covid-19 cases. an epidemiological survey in such a facility demonstrated the extent of the problem: the first infection in this nursing home was introduced by a symptomatic nurse, and then a week later the first resident tested positive. a further 5 days later, half of the residents from this unit tested positive for viral rna. during the following 2 weeks, 30 to70% of the residents in other units of this home became infected, along with 19% of the staff. notably, more than half of the residents were asymptomatic when they tested positive for viral rna. four days later, 89% of them had developed symptoms. the rest remained asymptomatic. mortality was high at 26%. importantly, asymptomatic, pre-symptomatic, and symptomatic residents did not differ with respect to viral load and infectious virus release. all viruses showed identical genome sequences guangdong cases. in guangdong province of china, all return visitors from wuhan/hubei province, their contacts, and all of the local hospitalized patients were tested for viral rna. 1.6 million tests were used to identify 1'400 sars-cov-2-positive cases; 1000 patients had had exposure to infected people from hubei. half of the local transmissions occurred within households. by mid-february, the local spread was controlled, but in march new cases were imported from abroad. sputum samples showed the highest viral titers, followed by oropharynx, stool, and finally nasopharynx samples. critical and severe cases showed higher viral titers than moderate and mild cases, but the differences were small. viruses from 53 patients were sequenced and compared with 177 sars-cov-2 sequences deposited in the database. single nucleotide polymorphism (snps) were detected at 97 nucleotide positions scattered through the viral genome; 77 variant sites were only seen in a single virus isolate. on a phylogenetic tree, the guangdong sequences were interspersed between the viral sequences from wuhan and those isolated abroad, documenting a recent, single source outbreak of a virus showing a low mutation rate (lu et al., 2020a) . usa west-to-east spread. the first case of covid-19 in the us was reported on jan 19 at the northwest coast of washington state and was imported from china. from march 1 to 19 the number of cases in the us increased from 70 to 13'000. epidemiologists investigated the first nine covid-19 cases on the east coast (connecticut) that were observed in mid-march with genome sequencing. only one sequence exactly matched the viral sequences from china, but the patient had not traveled to china. the viral sequences from seven further patients, clustered with a large us clade known from washington state, documented a rapid west-to-east national spread of the novel coronavirus in the us. international air travel restrictions had no, or low, impact on the epidemic spread in the us. the viral genome was rooted in a single ancestor coronavirus in wuhan by fewer than 10 mutations, and it had accumulated about 2 nucleotide changes per month during its spread across the us, which is a low mutation rate for a 30'000 nucleotide long viral rna genome. with the portable oxford nanopore technologies minion platform, viral genomes were sequenced within 14 hours after having received the sample, theoretically allowing near real-time molecular epidemiology of the epidemic spread (fauver et al., 2020) . china. in wuhan, 105 index cases of patients suffering from moderate covid-19 symptoms (fever, cough, fatigue) were investigated for secondary transmission to 392 household contacts. the average household size was 4 persons. the index case persons remained at home for a documented number of days before seeking medical advice. in total, 64 contacts (16%) were infected, 9 without symptoms, and the rest experienced moderate disease. the time lapse between primary and secondary infections was 6 days. the transmission probability was age-dependent: it was 4% in children and 17% in adults. the highest transmission rate was seen in 50 to 60 year-olds, but not in >60 year-old household members. spouses experienced a 28% infection transmission rate. when the index case was quarantined directly after symptom onset, transmission rate was 0% . in shenzhen, 391 cases were recorded, 77% of which were detected through the surveillance of symptoms. most cases were mild (26%) or moderate (65%). only 9% of infections were associated with severe disease, which correlated with male sex and older age; 3 patients died. the researchers identified 1'281 close contacts for the index cases; 8% of these had become infected. an increase to 16% in the rate of infection was seen in those who lived or traveled with index case persons. interestingly, infection risk was comparable for all age groups ranging from 0 to 70 y old. however, half of the children showed no fever, and severe infections were rare in people under the age of 40. notably, 80% of secondary infections were traced to only 9% of the index case persons (bi et al., 2020) suggesting an important role of "super-spreaders" in infection transmission. it would be helpful for public health if characteristic traits of super-spreaders were known. adding viral-specific igg antibody tests to the toolbox of covid-19 have allowed the connection between three previously separated infections clusters in singapore. an infected traveler from wuhan attended a church meeting, thereby infecting a secondary person (case x), who transmitted the virus to another subject during a family gathering, who then transmitted the virus to a large number of people in a second church. case x tested negative for viral rna and represented the missing link between the events. case x showed a strong serological response to sars-cov-2, then connecting the links in the chain (yong et al., 2020) . iceland. from among 9000 high risk icelanders (persons who were symptomatic, or had contact with infected persons, or who traveled to a high risk country) 13 % tested positive for sars-cov-2 rna. children under 10 years from that high risk group showed a 7% rna detection rate compared with 14% in subjects older than 10 y. before mid-march, travel exposure to austria and italy was a common denominator in the positive subjects. after mid-march, travel to uk was the biggest risk factor. in mid-march, a representative sample of 11'000 subjects from the general population of iceland (360'000 inhabitants) was tested, and 0.8% tested positive for sars-cov-2 rna. in april, another sample of 2000 subjects from the general population showed a similar rate of viral excreters. in the general population, males were more frequently virus-positive than females. the highest prevalence was seen in 40 y-old subjects, while no children under 10 y were infected. five hundred viral genomes, isolated from infected icelanders, were sequenced. the genomes were clustered into 42 distinct clades. during the early epidemic phase in iceland, nearly all sars-cov-2 isolates belonged to the a2 clade which was also frequently found in central european populations. viral genome sequencing identified networks of up to 14 linked infections. transmission occurred in the early phase via international travel, but later via infection from family members (gudbjartsson et al., 2020) . airborne: cough, sneeze and speech. australian engineers evaluated the literature about the reach of pathogen transmission by coughing and sneezing. the 1 to 2 meter rule has been set since the 1940s by photography, physical calculations, and through simulations. distinct models (turbulent jets vs. puffs) have been used, but not all used appropriate parameters for humidity and temperature. it remains unclear which conditions apply to the human respiratory excretions when handling infected patients in a clinic (bahl et al., 2020) . newer high speed pictures of a coughing volunteer show a turbulent jet plume that extends over 0.5 meter (tang and settles, 2020) . high speed pictures of a sneezing volunteer revealed exhaled air, muco-salivary filaments and drops. the turbulent puff cloud disintegrated into droplets that settled within 1 to 2 m distance. some droplets evaporate and become suspended in the puff and travel a room within a few minutes to land 6 to 8 m away from the sneezing person (bourouiba, 2020) speech generated droplets in front of the mouth increased with the loudness of the voice. holding a cloth in front of the mouth suppressed the droplet detection (anfinrud et al., 2020) . a who communication led to a controversy saying that there is not enough evidence that sars-cov-2 transmission is airborne. who defines airborne transmission as being via aerosols as opposed to transmission by droplets. since most covid-19 transmission seems to occur through close contact, droplets have been considered to be the more likely vehicles. whatever the exact transmission route by air, avoiding crowds, standing next to a person for too long, and increasing the rate of ventilation in closed rooms without air recirculation are reasonable precautions (lewis, 2020) . physicians recommended face mask use, even when scientific evidence for its effectiveness is lacking, in comments to leading medical journals. recent reviews have concluded that: no randomized trials with masks have been conducted; that the benefit of masks over no masks (but not of respirator masks over paper masks) was shown in an influenza epidemic in australia; that some benefit of masks was seen when worn by symptomatic, but not by asymptomatic cases during an influenza epidemic; that no data exist which directly support the use of mask wearing by the public; that no significant effect was seen for household use of masks against influenza transmission. therefore, who initially recommended masks only for symptomatic cases. cdc first advised against mask use by the public but have now changed their policy by recommending even self-made cloth masks for wide use. harm (e.g. increase of co 2 level under the mask) is low if not used by small children or elderly people with disabilities (cheng et al., greenhalgh et al., 2020) . as assessed by viral rna detection, air and surfaces in a wuhan hospital were widely contaminated during the height of the epidemic in china. contamination levels were greater on the intensive care unit ward than on the general ward. the transmission of sars-cov-2 might reach up to 4 meters distance. however, no medical staff in that particular hospital was infected. the authors admit two limitations of the study which prevent firm conclusions from being made. first, viral rna detection does not mean infectious virus detection, and second, the minimum infectious dose of sars-cov-2 for humans is unknown . when reviewing the official recommendations, the consensus seems to be to use a respirator for high risk interventions which create aerosols in the icu, and to use surgical masks on the general ward with low risk activities (bahl et al., 2020) . singapore hospital. the environment of three covid-19 patients from a ventilated hospital infection ward in singapore was tested for viral rna presence by rt-pcr. after routine cleaning of high touch areas and of the floor, no viral rna was found in the air, on hospital room surfaces or on the personal protection equipment of the treating physician. before routine cleaning, however, 16 of 20 room sites (table, chair, floor, window, toilet) and the shoe protection of a physician tested positive. room air samples and hospital corridor floors were, however, negative for viral rna. for infection control, regular room cleaning and handwashing were judged to be essential (ong et al., 2020) . primit study. it is more problematic to establish a control when a family member with mild infection remains at home. there is only one behavioral intervention study that has proved to reduce respiratory viral transmission within households, the primit germ defense study. the key interventions are web-based instructions about handwashing given to 10'000 intervention subjects in uk, but not to 10'000 controls. infection transmission was reduced by 20% and infection severity was also reduced, albeit modestly (little et al., 2015) . the rationale behind the idea was to reduce the viral load by which contacts are particularly exposed, such as through hand-to-eye contact, since the conjunctiva supports sars-cov-2 replication (hui et al., 2020; little et al., 2020) . in italy, the epidemic started in lombardy and veneto. lombardy strengthened their hospital capacity and increased icu beds, while veneto opted for strict containment and mass testing in 4% of the population. lombardy experienced 47'000 more cases than veneto and also had a much higher crude case fatality rate (18% vs 6%). the higher death rate is probably explained by the delayed public health response (odone et al., 2020) as also seen in the us (anonymus, 2020) . the swedish government had recommended a number of trust based measures (social distancing from old people, handwashing, home office, travel reduction), but refrained from closing borders, schools, restaurants and bars, partly because the swedish law does not allow lockdowns. the case reduction of seasonal influenza and norwalk diarrhea provided documented effectivity of the measures taken (paterlini, 2020) . however, compared with neighboring finland and norway, sweden experienced a tenfold higher number of deaths for a 1.5 fold larger population, but the absolute numbers are still small (3700 vs. 300 for sweden and finland, respectively, may 18). in the us, the individual states and cdc have many legal options for quarantine (for the segregation of exposed people) and isolation (separation of infected people from the general population) in such cases as with sars. the establishment of broad sanitary cordons in which entire geographical areas are quarantined (as has happened in wuhan) will raise constitutional questions in the us. the us recommendations say that patients who show mild symptoms should stay home and notify their employer electronically. low-wage workers cannot afford to stay off work, but the us senate is in the process of establishing bills for paid sick leave and unemployment insurance (parmet and sinha, 2020) . kong. the control measures that stopped the epidemic locally have included: intense infection surveillance of incoming travelers; isolation of covid-19 cases in hospitals; contact tracing and quarantine in holiday camps; and school closure but no lock-down, thus preventing the crisis from having a negative economic impact. a total of 715 cases were confirmed, half imported, and the rest locally transmitted with a reproduction number that quickly decreased to values around 1; and 13% of virus-positive subjects were asymptomatic. the control measures also stopped an ongoing seasonal influenza epidemic. surveys showed that the population agreed to participate in the measures. they kept social distancing and made behavioral changes (99% wearing masks outside house, 93% increased hand hygiene, 83% staying at home as much as possible). the study could not differentiate the impact of each individual measure. unfortunately the full effect of school closure is still unknown because the susceptibility of children for covid-19 and their capacity to transmit the infection has not yet been established . china has contained the covid-19 epidemic through a combination of different measures including drastic ones. an international team of epidemiologists developed a computer model that described the dynamics of the epidemic and tested the impact of the different containment measures by using computer simulations. without any intervention, a 100-fold higher number of cases would have occurred in china, resulting in over 10 million cases. without travel restrictions, the epidemic would have expanded more widely over the western provinces. early detection and isolation of patients reduced the number of cases by 5fold, while social distancing and contact reduction led to a 2.6-fold reduction. however, without contact reduction, the epidemic would have, over time, increased exponentially across the regions. initiating the intervention one week earlier would have decreased the number of cases by 66% or, if done one week later the number of cases might have increased by 3-fold. a delay of 2 or 3 weeks would have increased cases by 7-and 18-fold, respectively. lifting travel restrictions will result in a new rise in case numbers, but even moderate levels of social distancing could keep this increase in check. partial maintenance of npi may prevent, or at least delay, the arrival of second wave infections (lai et al., 2020) . contact surveys were conducted in wuhan and shanghai during the height of the covid-19 epidemic in china. before the epidemic, people reported between 15-18 contacts (two-way conversations, physical contact) per day. this number was reduced to 2 during the containment period. contact reduction was most significant for school-age children who, before the intervention, reported the greatest numbers of contacts out of all of the age groups, followed by adults at the workplace. during containment, contacts were mainly within families (80-95%). the survey was consistent with data from inner city mobility. all contacts of patients in hunan province were placed under medical observation and tested for excretion of viral rna. from these contact data it was deduced that children (<14 y) had an infection rate that was only a third as high as adults (15-65y), while older individuals had a 50% higher infection rate than young adults. based on these data and on a mathematical infection model, the authors concluded that social distancing alone is sufficient to control covid-19 spread. proactive school closure alone cannot interrupt transmission but can reduce the peak incidence of the disease by half, and it can delay the epidemic ). these are model simulations based on assumptions on infection transmission by children for which only few data are currently available. the reopening of schools in several countries will hopefully settle some questions with observational data. psychologists argue that contact-seeking is a basic human response to danger. this inclination takes over when an invisible infection threat is perceived. this instinct is only opposed by disgust when infected persons show appalling clinical signs which is not the case for sars-cov-2 infected subjects in pre-symptomatic or asymptomatic state. it will be increasingly difficult for health authorities to impose social distancing, as proven by the street demonstrations against containment measures in the us and in european countries by differently motivated opposition groups. the authors argue that the increased use of the internet as a substitute for contact can become an important public health tool to achieve physical distancing without social distancing (dezecache et al., 2020) . by may 18, the johns hopkins university registered 4.7 mio cases worldwide. the lion's share is from the us with 1.4 mio cases, compared with 84'000 cases reported by china. one should interpret these data with caution. the definition for a confirmed case of covid-19 was changed five times in china, which accounts for the increase in knowledge about the epidemic. scientists from the who collaboration center in hong kong calculated that when the fifth version is applied, the total number of cases in china would increase from 55'000 to 230'000, but the transmission patterns in mainland china would not change . the case number also depends on the intensity of viral testing and the capacity of the public health system to report the number of cases. with nearly 90 '000 deaths, the us number greatly surpasses the 4'600 deaths reported in china. it is still difficult to assess the morbidity and mortality impact of the covid-19 pandemic on the population. even mortality rate is not a clear figure since it is reported differently in different countries. while death is a clear diagnosis, the cause of death isn't. it might not be evident whether somebody died with or from covid-19, particularly in nursing homes. some countries attribute death to covid-19 if the virus was present at death. others attribute each death in nursing homes to covid-19 during the height of the epidemic -as was done in belgium, which explains its high mortality data. many deaths occurred while people also had underlying health problems (comorbidities), therefore they were already at increased risk of death. an international consortium of demographers called for the publication of excess mortality data. by comparing mortality statistics for a given epidemic period with a corresponding time period during previous years without that epidemic, the absolute impact of an infection can be assessed. such data are still largely lacking in the literature. excess mortality rates should best be calculated for both sexes and for each 5-y age range separately (leon et al., 2020) . first data have just now been reported: in march/april the care sector of england and wales alone has seen 20'000 excess deaths over the figures of previous years. covid-19 deaths at care homes were three times as high as covid-19 deaths in hospitals (burki, 2020) . excess mortality calculations have been done globally for seasonal influenza, arriving at 300'000 to 600'000 influenza-associated deaths occurring annually (iuliano et al., 2018) . while the majority of influenza mortality applies to elderly people, the death rate is also substantial for children at an estimated 34'000 deaths in 2018 . in comparison, the global death toll of covid-19 is now 315'000 (status may 18). a direct comparison of these two figures is difficult for two reasons: death levels are affected by vaccination campaigns against seasonal influenza and by strict containment measures for covid-19. it seems plausible that without any containment measures covid-19 mortality would surpass greatly the number of deaths from seasonal influenza. the presumption that the covid-19 mortality is comparable to that of seasonal influenza deaths is fundamentally flawed because it compares numbers which are obtained by different methods. the death rate for covid-19, which has just crossed the 100'000 figure in the us, is an actual count of dead patients. in contrast, the 23'000 to 61'000 annual deaths from seasonal influenza quoted after influenza epidemics in the us are estimates by the cdc of influenza deaths based on calculations from models. the death counts actually reported to us health authorities ranged from 3'400 to 15'000 deaths per year during an influenza epidemic in the us. expressed as deaths per peak week, influenza claimed a maximum 1'616 deaths per week, while covid-19 took about 15'000 lives per week at its peak in the us (faust and del rio, 2020) . wuhan. an international consortium of epidemiologists has estimated that 66'000 people were infected in wuhan and that 2800 have died; 70% were infected through household contact, 25% through public contact, and 5% in hospitals. from a peak number of 2000 new infections, wuhan currently has 10 or fewer new infections per day. safe strategies are now needed for the exit from lockdown measures. when lifting the lockdown to a 100% pre-quarantine social contact level, a computer model showed that a 95% face mask wearing would be needed to ensure a complete elimination of infections. in contrast, with only 50 % face mask use and a lifting date of strict measures before april 25 to pre-quarantine level, the conditions in this model would lead to a major second wave of infection. maintaining a contact rate below pre-quarantine level combined with a high percentage of face mask wearing is essential while now the restrictions have been lifted in wuhan-at least until a vaccine becomes available. however, face mask provision for such large populations represents logistical challenges and must not cause a shortage of protective gear for health personnel (zhang et al., 2020a) . in mainland china, 13'400 confirmed cases and 120 deaths were reported by march 18. in beijing and shenzhen, most cases have been imported from wuhan and the reproduction number r remained below 1.0. in shanghai and wenzhou, local cases dominated but r rose to greater than 1 for only one january week. case fatality was 1% compared with 6% in hubei. relaxing the restrictions could lead to a second wave of exponential infection from imported cases in a nonimmune, susceptible population. maximizing economic productivity under the < 1 constraint can, according to this study, only be possible with a real time prevalence determination of new infections through extensive testing . other epidemiologists working on outbreak data from mainland china observed a sub-exponential increase of cases from the beginning, instead of an expected exponential growth for an unconstrained epidemic. model calculations showed that the containment measures (the quarantine of exposed, and the isolation of infected persons) which depleted the number of susceptible individuals for the virus, reproduced the actually observed case development. similar strategies are recommended in the event of a future outbreak (maier and brockmann, 2020) . harvard model. epidemiologists from harvard university derived projections from model calculations about the future dynamics of the covid-19 epidemic. when anticipating short term immunity (as observed for seasonal common cold coronaviruses), they predict annual winter epidemics for sars-cov-2. with intermediate levels of immunity persistence, epidemics would become biannual. long-term immunity (as in the case of sars-cov) would lead to the extinction of the virus, even in the absence of social distancing. they also calculated that it needed 20 weeks of social distancing to reduce the peak number of infected persons. if the reproduction of the virus is reduced by more than 60% through lockdowns, the infection peak is predicted to shift to the next winter season with high numbers because no herd immunity has been achieved (kissler et al., 2020) . a central concept of epidemiology is herd immunity; the percentage of persons with protective immunity needed in a population to stop the propagation of an infectious agent. when this threshold is crossed, the remaining susceptible persons are protected from infection. the threshold level depends on the "force" of the infectious agent which is expressed by the basic reproduction number r 0 ,which is defined as the number of secondary infections caused by an index case. in infection modeling, herd immunity threshold and r 0 are linked by a simple mathematical function. sars-cov-2 has a higher r 0 "infectious force" than influenza virus, but a much lower one than "flying infections" such as chickenpox or measles. it is anticipated that a population needs a herd immunity of 50 to 80 % protected people to stop the covid-19 epidemic. the initial strategy of the uk government was to let the epidemic roll over the country to achieve this herd immunity, in contrast to containment policies which prevent exposure, but which also prevent immunity development in the population. this strategy has theoretical advantages (fewer economic losses when a lockdown is avoided, and a protected population in the event that no vaccine becomes available). however, it comes at a cost. if you allow, let's say, 60% of the population to get infected, you can calculate the cost of this strategy with the help of the infection fatality rate (ifr). in contrast to the case fatality rate (cfr) which expresses the number of deaths per clinically ill patients (which varies from 1.4% to 15% for covid-19), ifr is the number of deaths per infected individual. the number is, of course, lower than crf. while we know, approximatively, the number of covid-19 deaths and covid-19 cases, we do not definitively know the number of infected persons, since this would require large and systematic seroprevalence studies, but which are lacking. current estimates suggest 0.6% as a realistic approximation for ifr. with that figure, one can calculate that achieving herd immunity through natural infection with sars-cov-2 would cost the lives of 150'000 uk citizen or more than 1'000'000 us citizens. this death toll was considered as too high by the uk government, which then changed strategy by declaring a late containment strategy (randolphe and barreiro, 2020) . delays in imposing containment measures were predicted to lead to 10-fold or higher number of cases and fatalities. this prediction might explain why the us and uk have such high case and death statistics in international comparison. mobile phone technology. epidemiologists from the uk and us have developed a real-time datacapture platform applicable for mobile phone use for the self-guided collection of population-level data (cope consortium). the app queries location, age, health risk factors and asks daily for new symptoms and diagnostic test results. a test run with 1.6 million users in uk showed that the most common symptoms were fatigue and cough. anosmia (loss of smell) appeared as a strong predictor of covid-19, while fever was not a diagnostic criterion unless combined with other symptoms. the reported symptoms predicted that there would be changes in the number of cases as indeed reported from health authorities 5 to 7 days later. the tool will be important for a controlled safe exit from confinement measures. also, long-term effects of the disease, and the impact of the covid-19 epidemic on social relations, mental health, and financial outcome can be evaluated with this tool. machine learning could also reveal new disease manifestations of the epidemic (drew et al., 2020) . anosmia is an interesting symptom. a preliminary evaluation of a covid-19 symptom tracker smartphone app from uk users showed that the loss of smell was reported by 59 % of people with respiratory infection who tested positive for sars-cov-2, compared with 18% of respiratory patients who tested negative. clinical criteria which allow a diagnosis of covid-19 without a viral rna test would be welcome for mass screening and telemedicine in an epidemic situation. french physicians have also reported that many covid-19 patients reported loss of smell and loss of taste, without nasal congestion. when these criteria were combined in a retrospective questionnaire this combination of signs had a sensitivity of 42% and a specificity of 95% for detecting covid-19 patients (bénézit et al., 2020) . these observations are not surprising since the highest expression level of the sars-cov-2 receptor ace-2 was shown in the respiratory tract, more specifically in the nasal epithelia. us physicians even suspected that sars-cov-2 might, in addition to the respiratory and alimentary tract, also infect cranial nerves (i.e. being neurotropic) which potentially explains the observation of neurological signs in 9 % of covid-19 patients (chu et al., 2020) . seroprevalence studies. antibody tests are an important tool in a staggered release of population groups out of lockdowns because such tests identify people who have been exposed to the infection and who are potentially immune to infection. so far, only preliminary data became available from 3'300 volunteers from santa barbara county in california. one out of 66 (1.5%) showed antibodies to sars-cov-2. this number is 50-fold higher than the number of the official case count was for this area in early april. in a preliminary survey in geneva, less than 5 % of a population sample showed viral-specific antibodies in preliminary surveys. in a town with 12'000 inhabitants in germany, 500 people were antibody tested following carnival parties and an infection rate of 15% was determined. these datasets cannot be extrapolated to the population at large. in addition, the antibody tests were only validated with a small set of positive and negative test samples, raising concerns about the reliability of the results (mallapaty, 2020; sood et al., 2020) . the next challenge will be the acquisition of reliable antibody data for representative samples from entire populations. the covid-19 epidemic continues to challenge our societies by its toll in deaths, by the disruption of social life; by its disastrous impact on the world economy; by increasing the debt of many nations; by endangering the survival of many industries; and by reversing the worldwide trend for poverty relief. for microbiologists, the covid-19 crisis has also revealed shortcomings in the public health sector, particularly in that of countries which were exemplary in this field during past decades. in the us, the covid-19 epidemic has taken more lives in one month than over 8 years during the vietnam war. with more than 1.7 million cases, and more than 100'000 deaths, the "america first" slogan has become sadly ironic in the context of covid-19. an article in the leading us medical journal, the new england journal of medicine, attributes this calamity to insufficient diagnostic testing caused by the delivery of faulty tests by cdc; non-approval through the fda of working tests by who resulting in a delayed start of viral detection activities; and then followed then by a shortage of test reagents. public health workers were therefore blind to the unfolding of the us epidemic and unable to design efficient containment measures, short of a lockdown. epidemiologists were left without population data for modelling the epidemic in the us at a moment when the country started reopening economic and public activity. in comparison with other countries the us has tragically "failed the test". in the words of this article "the us once a leader, seem oddly lost" (schneider, 2020) . an editorial in the leading british medical research journal, the lancet, comes to the same conclusion: the cdc, once a pillar and international reference for combating diseases worldwide, instrumental in eradicating smallpox and coping with aids or ebola, has lost its technical competence and public trust due to contradictory scientific messages and the undermining of trust in scientific evidence by the current us administration. according to the lancet editors, the "us administration is obsessed with magic bullets-vaccines, new drugs-while only basic public health principles, like test, trace, and isolate, will see the emergency brought to an end" (the lancet, 2020). the situation is not better in the uk, once also renowned for its excellent public health research, particularly in the field of respiratory infections (remember the common cold research unit). at the end of may, the uk directly follows the us in the international mortality ranking list with more than 38'000 covid-19 deaths. the late onset of large scale testing, the lack of personal protective equipment, and a delayed introduction of containment measures have certainly contributed to this high death toll. a correspondent to the lancet deplores that the situation in europe was no better with respect to the lack of a coordinated response to the pandemic. the european center for disease prevention and control (ecdc), which was established in 2004 to create a complement to the us cdc, failed to become a hub in europe of knowledge for covid-19 and a coordination center for europe-wide epidemic counter-strategies. ecdc is underfunded (cdc in 2020: 8 billion $, ecd 60 million $) and understaffed (cdc: 10'800, ecdc: 270 employees). an emergency structure for a pandemic was not set up, and ecdc played essentially no role in pandemic crisis management, which was done according to eu laws by national organizations without any european coordination (jordana and triviño-salazar, 2020) . even the city-state of singapore, where the early handling of the covid-19 was lauded as exemplary public health action, had "blind spots" on their screen in overlooking the miserable living conditions of migrant workers that became hotspots of covid-19 transmission. of special global health concern are refugee camps from bangladesh to europe. a refugee camp on lesvos/ greece has just one water tap per 1300 residents, making efficient handwashing an impossible mission (newland, 2020) . governments plan to spend billions on rescuing what they consider to be essential national industries. it will be important that they also find the money needed for covid-19 containment among migrant workers, refugees and populations at risk in developing countries . the beneficial epidemic effect of the lockdowns, obtained at enormous economic costs, would be canceled out if a second wave epidemic should start from these settings with relatively unrestrained viral transmission. at present it is not clear which institution, if not the united nations' suborganizations, will be able to implement such measures. when the leading nation of the western hemisphere leaves now the who, this is a disastrous signal for global public health at this crucial moment of the covid-19 pandemic. 2020) visualizing speech-generated oral fluid droplets with laser light scattering 100'000 and counting. the economist presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility airborne or droplet precautions for health workers treating covid-19? utility of hyposmia and hypogeusia for the diagnosis of covid-19 epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series a sneeze england and wales see 20 000 excess deaths in care homes wearing face masks in the community during the covid-19 pandemic: altruism and solidarity comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study 2020) pandemics and the great evolutionary mismatch rapid implementation of mobile technology for real-time epidemiology of covid-19 assessment of deaths from covid-19 and from seasonal influenza coast-to-coast spread of sars-cov-2 during the early epidemic in the united states asymptomatic transmission, the achilles' heel of current strategies to control covid-19 face masks for the public during the covid-19 crisis spread of sars-cov-2 in the icelandic population aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards temporal dynamics in viral shedding and transmissibility of covid-19 estimates of global seasonal influenza-associated respiratory mortality: a modelling study where are the ecdc and the eu-wide responses in the covid-19 pandemic? collateral effect of covid-19 on stroke evaluation in the united states tropism, replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures projecting the transmission dynamics of sars-cov-2 through the postpandemic period effect of nonpharmaceutical interventions to contain covid-19 in china delayed access or provision of care in italy resulting from fear of covid-19 covid-19: a need for real-time monitoring of weekly excess deaths first-wave covid-19 transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment is the coronavirus airborne? experts can't agree the characteristics of household transmission of covid-19 epidemiological and clinical characteristics of covid-19 in adolescents and young adults an internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (primit): a primary care randomised trial reducing risks from coronavirus transmission in the home-the role of viral load genomic epidemiology of sars-cov-2 in guangdong province sars-cov-2 infection in children effective containment explains subexponential growth in recent confirmed covid-19 cases in china antibody tests suggest that coronavirus infections vastly exceed official counts epidemiology of covid-19 in a long-term care facility lost in transition covid-19 deaths in lombardy, italy: data in context air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2(sars-cov-2) from a symptomatic patient children with covid-19 in pediatric emergency departments in italy covid-19 -the law and limits of quarantine closing borders is ridiculous': the epidemiologist behind sweden's controversial coronavirus strategy herd immunity: understanding covid-19 failing the test -the tragic data gap undermining the u.s. pandemic response seroprevalence of sars-cov-2-specific antibodies among adults coughing and aerosols reviving the us cdc covid-19: pcr screening of asymptomatic healthcare workers at london hospital effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study global burden of respiratory infections associated with seasonal influenza in children under 5 years in 2018: a systematic review and modelling study virological assessment of hospitalized patients with covid-2019 connecting clusters of covid-19: an epidemiological and serological investigation detection of covid-19 in children in early changes in contact patterns shape the dynamics of the covid-19 outbreak in china what is required to prevent a second major outbreak of sars-cov-2 upon lifting the quarantine of wuhan city, china. preprint to the lancet acknowledgements i thank jacqueline steinhauser and sophie zuber for critical reading of the manuscript. the author consults nestlé, his former employer, on the scientific aspects of the covid-19 epidemic, but he does not consider this as a conflict of interest. key: cord-255140-3dwqqgv1 authors: christian, michael d. title: biowarfare and bioterrorism date: 2013-07-04 journal: crit care clin doi: 10.1016/j.ccc.2013.03.015 sha: doc_id: 255140 cord_uid: 3dwqqgv1 bioterrorism is not only a reality of the times in which we live but bioweapons have been used for centuries. critical care physicians play a major role in the recognition of and response to a bioterrorism attack. critical care clinicians must be familiar with the diagnosis and management of the most likely bioterrorism agents, and also be adequately prepared to manage a mass casualty situation. this article reviews the epidemiology, diagnosis, and treatment of the most likely agents of biowarfare and bioterrorism. weapons and their potential use by states with the consideration of use by nonstate actors (eg, terrorists). 3 more recent definitions of bioterrorism include both a broader range of potential biological agents as well as considering more diverse groups of potential targets and impacts of biological weapons. spencer 4 defines bioterrorism as "the use of micro-organisms as weapons of catastrophic effect which can be described as: the category or method of use of a weapon system that results in a significant negative impact on a nation's physical, psychological or economic well-being, thereby causing a major modification of routine activity." this definition highlights several key points. first, it highlights that a wide range of microorganisms must be considered and that their impact is not merely physical but may also include psychological and economic factors. spencer further elaborates on his definition of bioterrorism by stating "bioterrorism is best described as the use of micro-organisms (pathogens) or the products of living organisms (toxins) to inflict harm on a wider population, including animals and crops." 4 the elaboration on his original definition highlights that not only are humans directly vulnerable to bioterrorism but we are vulnerable through indirect attacks on our livestock or crops, which has also been termed agroterrorism. 5, 6 other investigators broaden the definition further to not only include microorganisms and biotoxins but also larger organisms, specifically insects. 6 as with many other medical issues that intensivists face in their busy clinical and academic practices, pressured by ever-increasing time and budgetary restraints, there is a necessity to prioritize efforts and resources toward the most common and higherimpact concerns. it is difficult to provide a clear-cut answer as to where bioterrorism should be prioritized on this list. although some experts state that the risk of a largescale bioterrorist attack is low, 7 in a more recent analysis, us senators graham and talent quote their conclusion form the commission on the prevention of weapons of mass destruction proliferation and terrorism in 2010, which stated "unless the world community acts decisively and with great urgency, it is more likely than not that a [biologic] weapon of mass destruction will be used in a terrorist attack somewhere in the world by the end of 2013." 8 anthrax in particular remains such a concern, because of both the lethality of the agent and also the potential availability given the number of governments that produced weaponized anthrax in the past. 9 the later issue is of concern because of both the availability of expertise as well as the risk of residual caches of anthrax in failed states that are vulnerable for misappropriation. in addition, it may require less expertise to develop aerosolizable anthrax then previously believed. 8, 9 box 1 lists the capabilities required of any organization, whether state or nonstate, to conduct and deliver a bioterrorist attack. even a small or moderate bioterrorism event has the potential to overwhelm local medical resources and cause significant civil and economic disruption as a result of the psychological impact of such an attack. given the potential risk for a bioterrorism event and the major impact that could occur, these factors alone provide a strong argument as to why an understanding of bioterrorism is warranted for critical care physicians. however, if these arguments are not persuasive enough, the knowledge that critical care physicians will play a key role in a bioterrorist event, and that their effectiveness in responding to the event is dependent on their medical knowledge regarding bioterrorism agents, should compel one to take the time to read this review. agents used for bioterrorism typically cause critical illness and therefore are of clinical relevance to the intensivist. several of the category a and b organisms (see later discussion) also produce human infections in nature, and therefore, knowledge of their presentation and treatment can also be applied in nonbioterrorism settings. the us centers for disease control and prevention (cdc) has identified and categorized a list of potential bioterrorism agents ( table 1 ). the agents identified by the cdc have been accepted, by most authorities globally, as the highest priority for preparedness and research. those agents from category a form the primary focus of this article. however, relman 10 reminds us that it is important not to solely focus on the agents from the cdc list because they were largely driven by past military programs and do not include agents that were not of particular interest or relevance in the past but may be in the future given technological advances. in addition, military programs focused on weaponizing agents, whereas terrorists could seek to manipulate the natural spread of existing organisms or the development of novel strains. 10 agents this section provides a basic overview and description of the commonly considered bioterrorism threats. specific details about the epidemiology, diagnosis, treatment, and outcomes associated with each of the organisms follow in the subsequent sections. anthrax the bacteria causing anthrax is bacillus anthracis, an encapsulated, grampositive, spore-forming bacilli. 7, 9, 11, 12 when seen on a gram stain, it is often described as box cars given its appearance as a series of railway boxcars in a train viewed from above ( fig. 1) . bacillus anthracis is a soil-borne organism and can be found in the environment globally. the organism grows quickly on standard culture media (6-24 h) and its spores are highly resistant, potentially being viable for decades. in naturally occurring infections, the organism may infect humans by transcutaneous inoculation (cutaneous anthrax), ingestion (gastrointestinal [gi] anthrax, including oropharyngeal), or inhalation (thoracic anthrax). in terms of use as an organism of bioterrorism, it is most likely to be delivered in its spore form as an inhaled agent. once inhaled, bacillus anthracis spores enter alveolar macrophages by phagocytosis and are transported to regional lymph nodes, where they germinate, typically within 2 to 5 days but they may be delayed up to 60 days. 9, 13, 14 symptoms start after germination and bacterial replication begins to occur. what causes the variability in incubation period associated with the time from infection with spores to germination to the vegetative bacillus is unknown. bacillus anthracis produces 2 exotoxins: edema and lethal, comprised of 3 components: (1) edema factor (ef), which impairs neutrophil function and disrupts cell water hemostasis, resulting in massive edema; (2) lethal factor (lf), which causes release of tumor necrosis factor a and interleukin 1b, which are believed to mediate the severity of illness produced; and (3) protective antigen, found in combination with both ef and lf, which allows binding and transportation of the other 2 toxins to and across cell membranes. in addition to the toxins, there are several other virulence factors, including an antiphagocytic capsule. anthrax is not transmitted person to person, but patients should be isolated with droplet precautions as part of standard febrile respiratory infection precautions until the cause of their illness is confirmed. patients with cutaneous anthrax require contact precautions. decontamination should include removing clothing and jewelry and washing skin with soap and water. caution must be exercised not to generate secondary aerosols when handling contaminated clothing items. environmental contamination from patients exposed to an aerosol of bacillus anthracis spore can be performed with a 0.5% hypochlorite bleach solution. this procedure is not sufficient for site decontamination after the release of bacillus anthracis spores, because much more extensive decontamination is required. [15] [16] [17] plague plague is caused by yersinia pestis, a nonmotile, gram-negative, bipolar coccobacillus that can be found worldwide. 7, 18, 19 human infections occur in nature regularly, and plague is endemic in regions such as the southwest united states. yersinia pestis virulence factors include: v and w antigens, lipopolysaccharide endotoxin, capsular envelope (antiphagocytic), coagulase, and fibrinolysin. the natural reservoir for yersinia pestis is rodents and the vector to humans is the oriental rat flea (xenopsylla cheopis). plague is highly communicable in the pneumonic form and may present as any of the following clinical syndromes: bubonic plague, primary pneumonic plague, primary septicemic plague, plague meningitis, plague pharyngitis, pestis minor, and subclinical infection. aerosolized yersinia pestis likely produces a clinical presentation identical to pneumonic plague. however, yersinia pestis does not produce spores and is susceptible to destruction by drying, heat, and ultraviolet light, therefore making it significantly more challenging to weaponize it then anthrax. it is more readily transmitted via an infected vector, such as the oriental rat flea, or by person-to-person transmission. the incubation period depends on the clinical presentation; bubonic plague takes 2 to 8 days and pneumonic plague as short as hours to 3 days. isolation for plague requires droplet precautions until 48 hours of effective antibiotic therapy. decontamination is not required specifically for plague. health care institutions should follow their usual procedures for cleaning after patient discharge from a room. tularemia francisella tularensis is the organism responsible for causing tularemia; it has often been associated with rabbits and various rodents and is occasionally referred to as rabbit fever and deer fly fever. 7, [19] [20] [21] francisella tularensis is a fastidious, small gram-negative, facultative intracellular coccobacillus able to live in soil, water, and decomposing carcasses for long periods. francisella tularensis can be transmitted by direct contact with mucous membranes, cutaneous inoculation through broken skin or bites from infected ticks (or other arthropods), ingestion and inhalation (although human-to-human transmission has not been reported 20 ) . as discussed further in the epidemiology section, tularemia has been previously used as a biological weapon. the mode of deployment as a biological weapon in the past has often been through infected vectors 22 ; however, a modern bioterrorist would most like deploy the agent via aerosolization and it could present as: primary pneumonic tularemia (inhalation), oculoglandular tularemia (eye contact), ulceroglandular (broken skin contact), or oropharyngeal (mucous membrane contact without deep inhalation). tularemia is not communicable between humans, so specific isolation is not required; only standard universal precautions should be used. decontamination for aerosolized exposure should involve remove clothing and jewelry and washing the skin with soap and water. q fever the only category b bacterial organism to receive any extensive discussion in this article is the zoonotic rickettsial organism coxiella burnetii, the causative agent of q fever in humans. [23] [24] [25] coxiella burnetii is a small, obligate intracellular, gramnegative highly pleomorphic coccobacillus. it has a typical gram-negative cell wall structure, but does not stain well with gram stain although it can be seen with a gimenez stain. coxiella burnetii has 2 morphologic forms: large and small cell variants. the small cell form is the extracellular form, which is metabolically inactive and resistant to chemical agents as well as environmental and physical conditions. the large cell variant is the metabolically active and pathogenic intracellular form. the disease is called q fever for query fever, because the causative organism had not been identified at the time of the first documented large outbreaks. q fever occurs worldwide and is associated with contact with sheep, goats, or cattle, particularly during birthing when placental exposure occurs. human infection is through the inhalation of aerosolized organisms from infected animals. coxiella burnetii is highly infectious to humans, and its sporelike small cell variant form makes it a potentially viable biological weapon. most human cases of disease are zoonoses, with human-to-human infection only rarely reported; therefore, only standard infection control precautions are required in the clinical setting. however, coxiella burnetii is highly infectious from culture and therefore should always be handled under biosafety level 3 conditions. environmental decontamination should a culture spill or bioterrorism release occur is difficult given the highly resistant features of the small cell variant. 23 other bacterial agents in addition to the bacterial agents already discussed, there are several other potential organisms that could be used as bioweapons (see table 1 ). [24] [25] [26] rickettsia prowazekii is the causative agent of louse-borne typhus and, similar to q fever, efforts have been made in the past to develop it into a bioweapon. brucella sp are small, aerobic, intracellular gram-negative coccobacilli, which cause primarily zoonotic infections in sheep, cattle, goats, and other animals. brucellosis can manifest as either christian systemic or localized infections and is acquired by contact with or ingestion of fluids from infected animals, particularly by the consumption of unpasteurized milk or cheese. brucella suis was reportedly weaponized by the united states, and possibly other countries, in the past. 24 the organism was to have been aerosolized from a bomb. burkholderia pseudomallei is a gram-negative, facultative anaerobic, motile bacillus that causes melioidosis. there are naturally occurring cases of melioidosis in southeast asia and australia yearly. the disseminated form causes an acute illness, with high mortality. the disease is also known to cause abscesses, which, in some cases, do not present until many years after the initial exposure. burkholderia mallei is a gram-negative, aerobic, nonmotile bacillus that causes glanders. it exists only in living organisms and cannot survive in the external environment. glanders usually presents in humans as a nodular disease with regional lymphangitis; however, systemic dissemination of the organism does occur on occasion, producing septic shock, potentially leading to death. control measures have led to the eradication of glanders from most countries in the world, with the exception of ongoing zoonotic endemics in parts of the middle east, asia, africa, and south america. both burkholderia sp tend to be transmitted to humans through inoculation via a break in the skin, although inhalation is occasionally also a means of human infection. both organisms have been studied, and reportedly developed, as biological weapons in the past by the germans and russians. 26 given the possibility of human-to-human transmission, droplet precautions should at least initially be used for patients with systemic or pulmonary involvement. the more significant transmission risk is from cultured organism, and therefore, any cultures should be managed in a biosafety level 3 laboratory. smallpox once believed to be a disease of the past, smallpox is now one of the most significant bioterrorism threats to the world. 7, 19, [27] [28] [29] there are no animal reservoirs for smallpox and the last naturally occurring case was in somalia in 1977. 28 smallpox is caused by the dna variola virus, a member of the genus orthopoxvirus (family poxviridae), which also includes molluscum contagiosum, vaccinia (virus used in the smallpox vaccine), and monkeypox. a large vulnerable population has existed since smallpox vaccination was stopped in the late 1960s and early 1970s. variola is highly communicable, and can be transmitted by airborne, droplet, and fomite (bed linen and clothing) transmission in addition to direct contact. infection starts in mucosa of airway, then the virus replicates in regional lymph nodes before subsequently resulting in an asymptomatic primary viremia 3 to 4 days after infection with spread to the bone marrow, spleen, and lymph nodes. secondary viremia occurs (day [8] [9] [10] [11] [12] , in which the virus localizes in the dermis and oropharyngeal mucosa and is marked by the onset of symptoms and infectivity. five clinical syndromes of variola infection are seen: classic (unvaccinated only), modified (vaccinated or unvaccinated), flat (vaccinated or unvaccinated) hemorrhagic, and variola sine eruptione (vaccinated only). variola has several features that make it a potentially good bioweapon, including the ability to be made into a lyophilized form, which can be aerosolized and is heat resistant (box 2). however, the virus is easily killed, even in the lyophilized from, by ultraviolet light and disinfectants. the primary concern related to smallpox as a bioterrorism agent is that it was produced in large quantities as a bioweapon by the soviet union and the security of its viral stock remains uncertain after the collapse of the superpower. 29 mathematical models 30 suggest that even a moderate-sized attack infecting 100 to 1000 people would lead to a massive global pandemic that would require significant interventions to control, which in turn would carry with them significant economic and civic ramifications. 31 airborne and contract precautions should be used to manage patients with smallpox. clearly, in a large-scale attack or outbreak, isolation of all patients in negative pressure rooms would not be feasible. all fomites (bed linen and patient clothing) should be transported in sealed biohazard bags and autoclaved before washing or incineration. standard hospital procedures for room cleaning can be followed once a patient has been discharged from the room. viral hemorrhagic fevers viral hemorrhagic fevers (vhf) ( table 2 ) are a group of infections caused by 4 families of rna viruses (arenaviridae, bunyaviridae, filoviridae, and flaviviridae), which are believed to primarily exist within animal or arthropod reservoirs, but occasionally infect humans. 7, 19, 32 each virus is generally contained to a specific geographic region. all of the viruses have in common that they attack the vascular endothelium, leading to vascular leak and potentially producing shock or coagulopathies. with the exception of hantavirus, natural transmission is primarily by respiratory droplets and body fluids but not by airborne aerosol. aerosol transmission of hantaviruses from the urine of rodents has been well documented. most secondary (humanto-human) transmission of vhf is to family members or health care workers caring for patients with vhf. the primary mode of transmission in these cases is direct contact with infected body fluids and percutaneous exposures. however, questions still remain regarding the possibility of aerosol transmission, particularly for lassa fever or from aerosol-generating procedures. 33 several of the vhf viruses have reportedly been weaponized by the russian and the us militaries. isolation for all vhf is strict droplet and contact precautions. patients who are end stage or have significant pulmonary involvement may warrant airborne isolation in a negative pressure room, if available. for specific details regarding isolation and environmental decontamination procedures for vhf, see the cdc's interim guidance for managing patients with suspected viral hemorrhagic fever in us hospitals, available online at http://www.cdc. gov/hai/pdfs/bbp/vhfinterimguidance05_19_05.pdf (accessed january, 2013). in addition to clinical infection control precautions, it is important to minimize risk to box 2 why smallpox is a good candidate as a bioterrorism agent 1. it is transmissible by the aerosol route, both in a weaponized from and from infected to susceptible persons. 2. it is heat stable in a lyophilized form. 3. the populations of most countries contain a high proportion of susceptible persons. 4. smallpox is associated with high morbidity and about 30% mortality. 5. there is a significant psychological fear of smallpox among both the public and health care workers. 6. initial diagnosis of cases of the disease may be delayed, given that it has not been seen clinically for more than 20 years. 7. other than the vaccine, which may be effective only in the first few days after infection, there is no proven drug treatment for clinical smallpox. the laboratory and other health care workers by strictly limiting the number of samples and blood work collected. no fungal organisms are currently on the cdc list of category a or b agents, although it could be argued that some should be considered as potential category c agents. 34 coccidioidomycosis is caused by the soil-borne spore-forming organisms coccidioides immitis and coccidioides posadasii found in the americas. these organisms typically cause a self-limited respiratory illness in immunocompetent individuals. coccidioidomycosis is typically transmitted by inhalation, particularly after events such as earthquakes or dust storms, which generate airborne particulate mater from the soil. human-to-human transmission does not occur. overall, the long incubation period and mild clinical presentations in most individuals make coccidioides an unattractive agent as a bioweapon. however, given the ease of access to the organism and its natural propensity for aerosol transmission, its potential use should not be ignored. keeping an open mind about possible future bioterrorism agents is essential if we are truly going to be prepared. 10 biological toxins technically toxins are chemicals but because of their source (living organisms), biotoxins are typically classified as biological weapons. 7, 19, 24, 35 the cdc list of potential bioterrorism agents (see table 1 ) includes several toxins. in addition, there are several other toxins that also have the potential to be used as agents of bioterrorism (box 3). toxins can be deployed via aerosol dispersal devices or by contaminating food sources and have been used as bioweapons in the past ( table 4) . to be effectively deployed as an airborne agent, the toxin must be 1-3 mm for optimal aerosolization. ricin ricin is a protein toxin obtained from the organism ricinus communis (castor beans). castor beans are available worldwide and ricin is a natural by-product produced in the processing of castor beans to castor oil. the waste products resulting from the processing of castor beans contain approximately 5% ricin. 24 in its pure form, ricin is a white powder that is soluble in water and stable over a wide range of ph. chemically, ricin is a glycoprotein lectin with a and b chains joined by a disulfide bond. the b chain binds to galactose-containing proteins and lipids on cell surfaces, causing direct membrane damage and release of cytokines, whereas the a chain inhibits eukaryotic ribosomes by removal of adenine from the 28 rrna loop in the 60 subunit, therefore stopping protein synthesis, leading to cell death. ricin is less toxic by weight compared with botulinum toxin or staphylococcal enterotoxin type b (seb), but is more readily available and easy to produce in large quantities than either of these agents. ricin can be deployed as a bioweapon via several routes, including inhalation, ingestion, or injection. clearly, as a bioterrorism agent with the intent to cause mass casualties, inhalation or ingestion is more likely than injection. after exposure, decontamination should include removal of clothing and jewelry, washing with soap and water, followed by rinsing with copious amounts of water. clostridium botulinum toxin (botulism) botulinum toxin is the most poisonous substance known to man with a dose lethal to half those exposed (ld 50 ) of 1 ng/kg. clostridium botulinum is a gram-positive, spore-forming, anaerobic bacillus found in soil and water globally. rarely, the toxin is also produced by clostridium butyricum and clostridium baratii. botulinum toxin is produced in pharmaceutical grade and is commercially available for cosmetic and other medical purposes. seven types of toxins exist and are identified as types a to g, with a, b, and e causing most cases of human disease. the toxin contains 3 chains (heavy, light, nontoxic hemaglutinin). the heavy chain permits the toxin to bind with the cell, and the light chain contains zinc-dependent endopeptidase, which stops calcium release. botulinum toxin binds to peripheral presynaptic cholinergic terminals (including muscarinic and nicotinic) and blocks the calcium-dependent exocytosis process. the central nervous system is not affected by the toxin. botulism is most often considered a food-borne illness, given that most of the naturally occurring cases are the result of ingesting contaminated food products. other naturally occurring forms of toxicity are wound botulism (soil-contaminated wounds) and infantile botulism (ingestion of spores). the spores themselves are heat resistant, tolerating temperatures up to 100 c, but the toxin itself denatures and becomes inactivated with even brief exposures to temperatures higher then 85 c. use as a potential bioweapon includes contaminating food sources or via aerosolization. however, the toxin quickly degrades when exposed to environmental conditions, thus limiting its usefulness as a bioterrorism agent. botulinum toxin cannot cross the skin but can be absorbed by mucous membranes. should exposure to the toxin occur via aerosolization, decontamination should include removing clothing and jewelry, washing with soap and water, and rinsing with copious amounts of water. wash contaminated surfaces with a 0.1% hypochlorite bleach solution to destroy the toxin. micotoxins several hundred varieties of toxins are derived from fungi, with the trichothecenes and aflatoxins being most concerning. aflatoxins are produced by aspergillus flavus or aspergillus parasiticus and are common contaminants in harvested food. aflatoxins bind to and damage dna and cellular proteins. trichothecenes are produced by a large number of fungi including: fusarium, stachybotrys, trichoderma, myrothecium, and cephalosporium. the most likely toxins from the class trichothecenes to be used as bioweapons are: t2 (trichothecene) and deoxynivalenol (vomitoxin). t2 causes skin irritation/pain and can be absorbed through the skin or inhaled, and then disseminated systemically, binding to peptidyltransferase inhibiting protein synthesis. it also interferes with dna polymerase and monoamine oxidase. the organs affected first are those are with rapidly reproducing cell lines (gi tract, bone marrow, and skin) as well as impairing proteins involved in coagulation and the krebs cycle. in addition, the breakdown of serotonin, epinephrine, and norepinephrine is impaired. t2 is a yellow droplet in appearance. should exposure occur, decontamination includes removal of clothing and jewelry, scrubbing the skin with soap and water, and isolating clothing or other contaminated objects. staphylococcal enterotoxins staphylococcal enterotoxins are heat-stable toxins produced by the common staphylococcus aureus bacteria. there are more than 20 types of enterotoxins, with type a commonly known for causing food poisoning and type f causing toxic shock syndrome. the genes for staphylococcal enterotoxins are found on plasmids and bacteriophages, allowing transfer between different strains. seb can be aerosolized and has been studied as a potential incapacitating biological agent. seb is a superantigen, which produces massive stimulation of t cells and cytokine storm, which is rarely fatal but is significantly incapacitating to the victim. if there is exposure, decontamination requires removing clothing and jewelry, followed by washing skin with soap and water. clostridium perfringens epsilon toxin clostridium perfringens is an anaerobic, grampositive, spore-forming bacillus that is found in soil in all parts of the world. clostridium sp produce several toxins, one of which is the epsilon toxin, which commonly causes food poisoning. similar to seb, epsilon toxin could be another potential incapacitating bioweapon. insects were some of the earliest bioweapons ever used against an enemy (see table 4 ). 6, 22 there are 3 potential ways to use insects in bioterrorism: direct attacks, agents of agroterrorism, or as vectors of disease. direct attacks most often use stinging insects and have been used in the past to defend fortifications and rout enemies from entrenched positions (the bee hive in the log, as seen in cartoons). another insect that has been studied by the indian military as a potential bioweapon is the paederus beetle, found in the middle east, which produces the toxin pederin. pederin is potent, causing intense pain, festering lesions, and blindness if it contacts the eyes. ingestion of the beetle can be lethal, as is injecting pederin into the bloodstream. agroterrorism is "the deliberate introduction of an animal or plant disease as well as damage to crops and livestock with the goal of generating fear, causing economic losses and/or undermining social stability." 6 an example of insects used as an agent of agroterrorism is the medfly (ceratitis capitata), a fruit fly found in parts of the world but not the united states. their larvae eat many plants, causing significant crop destruction. in 1989, a group threatened to release medflies in california if the government did not stop a pesticide-spraying program. if the insects had been released, the estimated damage to the economy would have been $13.4 billion. 6 insects can be used in bioterrorism as disease vectors. table 3 lists potential vector-borne diseases of concern. epidemiology it is challenging to describe the epidemiology of agents of bioterrorism in the traditional manner for several reasons. first, as discussed in the introduction, theoretic risks are largely being discussed, with many unknown factors involved. further, given that most of the work done in the area of bioweapons has been conducted by military or state organizations, only a small percentage of the overall activities have been publically reported. to provide the best possible overall perspective on the epidemiology of bioterrorism agents, table 4 offers a comprehensive overview of past use of bioweapons as compiled from several sources. 1, 4, 6, 7, 13, 19, 22, 24, [35] [36] [37] in addition to the general risks discussed in the introduction, academic health centers potentially play a unique role in the epidemiology of bioterrorism. any public area where mass gatherings occur or any infrastructure critical for the smooth functioning of society are potential targets for bioterrorism attacks. not only do hospitals in general, and academic health centers specifically, fit this profile, but academic health centers may also be at an increased risk because they are also potential sources of agents of opportunity (ao). the term ao is used to connote the use of a routine and unregulated chemical, biological, or radiologic agent by terrorists. 36 table 3 potential bioterrorism vector-borne diseases of concern for humans vector disease two key features of an ao are (1) its availability, and (2) a practical means of dissemination. box 4 lists potential biological aos available in many academic health centers. an ao does not have to cause significant morbidity or mortality to be effective; the psychological effects on the public and impact on society/critical infrastructure functioning are sufficient to have a serious effect. aos are of significant concern to law enforcement officials because potential barriers to the development of a highly effective biological weapon are the cost and expertise required to develop them, whereas aos mitigate both of these factors. in the past, the development of bioweapons has required significant funding and labor, which typically only states can mobilize. it has been said that the us military bioweapon program employed more than 3000 staff and the ussr bioweapons sections employed more than 60,000 staff, both at costs of millions of dollars. 7 although presenting a significant obstacle in bioweapon development, cost does not always prevent nonstate actors from pursuing bioweapons; the aum shunrikyo cult may have spent more than $10 million dollars in its failed attempt to develop weapon-grade anthrax. 7 the diagnosis, or identification, of a bioterrorism event is likely the most challenging step in the response. 38 recognition of a bioterrorism event has 2 components: (1) identifying that an intentional rather then natural phenomenon has produced several cases of illness, and (2) diagnosing the specific organism or agent causing the illness. they may occur in either order depending on the circumstances. bedside clinicians, specifically critical care physicians, 39 play a crucial role in both aspects of recognizing that a bioterrorism event has occurred. 2, 39, 40 recently, a great deal of attention and money has been directed toward bioterrorism detection technologies, such as environment sampling and syndromic surveillance. however, these efforts are highly unlikely to replace the bedside clinician in recognizing the event. 2, 41 in a simulated anthrax attack designed to test the performance of a syndromic surveillance system currently in use, nordin and colleagues 41 found that performance of syndromic surveillance varies with the infection rate. based on their analysis in a metropolitan area if there was a release of anthrax at a large public venue, a syndromic surveillance system that monitored approximately 9% of the local population would detect the event most of the time if the infection rate was 20% and all of the time if the infection rate was more than 40%, but it would take 3 to 6 days in both situations. therefore, these investigators concluded that "a suspicious clinician may detect the first case of anthrax before a syndromic surveillance system sounds an alarm and public health determines it is an anthrax release." 41 identifying the important role that the beside clinician plays in recognizing a bioterrorism event is not intended to diminish or exclude the role of others in the process, especially not public health. the rapid recognition, and effective response, to a bioterrorism event requires that clinicians and public health officials work together. [42] [43] [44] in some circumstances, such as smallpox, the diagnosis of a single case triggers the alarm that a bioterrorism event has occurred. 45 however, for any of the diseases with a naturally occurring incidence, as well as with a novel organism or illness that has not yet been identified, it is the existence of a cluster of cases in a geographic area that triggers suspicion of a possible bioterrorism attack or emerging epidemic ( table 5) . recognition of a cluster of patients typically requires a level of situational awareness that is beyond what an individual clinician can acquire and is usually attainable only by a regional, state, or federal public health agency depending on how widely distributed the cases are. for example, if an anthrax attack took place in a busy international airport, the cases would be widely distributed across a nation or internationally (eg severe acute respiratory syndrome [sars]). 46 in this case, to the individual clinicians receiving the cases of inhalational anthrax, each would be an unusual, but not necessarily alarming, occurrence. however, the reporting of several cases of inhalational anthrax to a federal or international public health agency simultaneously would certainly trigger an alarm. however, we are often left with trying to distinguish between a naturally occurring event (epidemic) and an intentional event (bioterrorism). it can be difficult to distinguish between an artificial incident (terrorism) and a natural occurrence (epidemic). 47,48 box 5 lists some of the characteristics that might help christian table 5 case number triggering criteria for considering a potential bioterrorism event disease(s) a a this factor is in part based on the background incidence of disease in a geographic area and therefore varies from location to location as well as based on seasonal variations. for example, a viral encephalitis that is naturally transmitted by an endemic vector occurring in the summer when mosquitoes are prevalent would not necessarily raise concerns; however, a single case occurring in the winter or in a geographic area where that infection is not typically seen may raise concerns immediately. features that suggest a bioterrorism event rather than an epidemic 1. an epidemic curve that suggests a point source (common source) outbreak or extended source rather then a naturally propagated (transmitted) source (fig. 2) 2. identification of a cluster of cases (large numbers of patients from a similar geographic area with similar symptoms) distinguish between the 2 events based on epidemiologic features. recently, radosavljevic and belojevic 38 have developed a scoring system to help distinguish between a bioterrorism incident and an epidemic ( table 6) . these investigators use an interesting approach, which scores several qualitative and quantitative features of the incident categorized under 3 groupings: person (cases), place (spatial distribution), and time. a score of 8 or higher on this system is said to suggest that an artificial (intentional or accidental) event is more likely then a naturally occurring epidemic. the questions considered in this score have to be answered at the public health level, not the individual clinician level, because they require a level of situational awareness not attainable by a clinician in a hospital. the importance of clinicians working effectively with public health agencies is further reinforced by computer modeling work by hupert and colleagues, 49 which suggests that even minor delays in detecting (diagnosing) and initiating the response to a large-scale anthrax attack would make even perfectly executed prophylaxis campaigns ineffective in preventing the health care system from being overwhelmed by cases. as noted in the earlier discussion, identifying the disease or organism involved can be key to identifying the event as a bioterrorism incident. this section provides an overview of the clinical and investigational findings that assist in diagnosing the specific diseases. key to any biological diagnosis are laboratory, particularly microbiological, tests. 50 table 7 summarizes the appropriate specimens and tests for diagnosing potential agents of bioterrorism. however, as with many aspects in critical care, it is important not to be overly reliant on technology. the college of american pathologists regularly tests the ability of laboratories to accurately diagnose, or appropriately refer to a reference laboratory, clinical potential agents of bioterrorism. 51 in their recent testing of laboratories, the college of american pathologists found rates of acceptable identification responses were as follows: bacillus anthracis, 90% (2007) 51 although the rates of appropriate diagnosis are improving, there still remains a delay in notification of the reference laboratory and a not insignificant failure rate for diagnosing category a organisms other then anthrax. anthrax as discussed earlier, anthrax presents as 3 typically distinct clinical syndrome: cutaneous, gi, and thoracic. 7, 11, 12, 19 diagnosis of anthrax often involves a combination of clinical, radiographic, and microbiological data. the clinical presentation of the 3 anthrax syndromes is outlined in table 8 . cutaneous anthrax most often affects the hands, arms, and face. one of the main differential diagnoses for cutaneous anthrax are spider bites, and table 9 presents clues to help distinguish between the 2. the diagnosis of cutaneous anthrax can often be made clinically followed by laboratory confirmation. other laboratory and radiologic investigations are of limited use in cutaneous anthrax. conversely, in the cases of gi and thoracic anthrax, the clinical presentation is relatively nonspecific, and radiologic investigations, especially for inhalational anthrax, 52 along with laboratory and microbiological testing are essential for making the diagnosis. with thoracic anthrax, sputum cultures are rarely positive but first blood cultures are almost always positive. 7 with the relatively nonspecific findings associated with thoracic anthrax, it can be challenging for clinicians to differentiate it from the more commonly seen community-acquired pneumonia (cap). kyriacou and colleagues 53 conducted a retrospective review of cases of bioterrorism-acquired thoracic anthrax and compared them with cases of cap or influenza. these investigators found that the most accurate predictor of anthrax was mediastinal widening or pleural effusions on chest radiograph, which were 100% sensitive, 71.8% specific compared with cap, and 95.6% specific compared with influenza (see fig. 2 ). other features more common in anthrax were: nausea, vomiting, pallor, cyanosis, diaphoresis, altered level of consciousness, and increased hematocrit level. two groups 54, 55 have developed screening criteria to help diagnose anthrax after a known bioterrorism incident. however, a costeffectiveness analysis showed that although both criteria would have identified cases abbreviations: ct, computed tomography scan; cxr, chest radiograph; mri, magnetic resonance imaging. a for microbiological findings, refer to table 7 . data from refs. 7, 11, 12, 19, 52 of anthrax, the mayer criteria 55 would have screened only 4 patients (cost $1900 usd), whereas the hupert criteria 54 would have screened 273 patients ($126,025 usd). 56 although it is rare with cutaneous anthrax, any case of anthrax has the potential to develop hemorrhagic meningitis. therefore, any evidence to suggest meningitis should prompt appropriate investigations such as neuroimaging and a lumbar puncture. plague as with anthrax, the diagnosis of plague varies significantly based on the clinical syndrome as which it presents. 7, 18, 19 plague may present as any of the following clinical syndromes: bubonic plague, primary pneumonic plague, primary septicemic plague, plague meningitis, plague pharyngitis, pestis minor, and subclinical infection. the first 3 syndromes are the most common presentations and their diagnostic features are outlined in table 10 . bubonic plagues is characterized by initial lymphadenitis, most commonly inguinal, followed by the development of a systemic illness, which may include secondary plague pneumonia. primary septicemic plague is similar to bubonic plague, without the initial appearance of buboes. it remains unclear if it is the same entity with subclinical lymphadenitis or a distinct pathophysiologic process. pneumonic plague may be primary, which presents as an acute respiratory infection, or may occur as a result of hematologic spread (secondary) from bubonic or primary septicemic plague. hemoptysis is frequently seen with pneumonic plague and can help differentiate it from anthrax or tularemia. 7 general laboratory and radiologic investigations are nonspecific and the diagnosis is primarily made via microbiology testing (see table 7 ). automated culture detection systems may not detect or can misidentify yersinia pestis, and therefore, if there is a high level of suspicion for plague, the microbiology laboratory should be made aware of this when the cultures are ordered. tularemia tularemia is another disease with several distinct presentations that are variable and dependent on the mode of transmission. 7, 19 the clinical spectrum of disease includes: typhoidal, ulceroglandular, glandular, oculoglandular, oropharyngeal, and pneumonic tularemia, with the last being the most likely presentation from a bioterrorism incident. the average incubation period is 3 to 5 days, but ranges from 1 to 21 days. the pneumonic form typically presents with: fever, chills, headache, malaise, coryza, cough, chest pain, pharyngitis, abdominal pain, arthralgia, septic shock, respiratory failure, and acute respiratory distress syndrome (ards). the ulceroglandular form presents with cutaneous ulcers 0.5 to 3 cm with heaped-up edges and regional lymphadenopathy. ulceroglandular infections may progress to systemic disease similar to the pneumonic form. the typhoidal form does not involve lymphadenopathy. the typhoidal form usually involves the lung primarily, whereas the ulceroglandular form first affects the mediastinal lymph nodes, then progresses to parenchymal involvement. microbiological testing is required to make the diagnosis, although cultures are generally low yield given the difficulty growing the organism. therefore, alternative techniques for identification such as immunofluorescence and polymerase chain reaction (pcr) are preferred. 7 radiologic findings are generally nonspecific, although approximately 75% of patients with tularemia present with bronchopneumonia, typically bilateral, and may develop cavities. only approximately 33% have lymphadenopathy and pleural effusions, which is significantly lower than would be expected with thoracic anthrax. 52 q fever q fever, when seen in naturally occurring infections in humans, causes a spectrum of disease from asymptomatic fevers and fevers of unknown origin through to life-threatening infections, typically associated with endocarditis or pneumonia. 23, 24 the primary concern in a bioterrorism setting is inhaled coxiella burnetii, which presents with fever and cough alone in cases of lower inoculation through to severe pneumonia and respiratory distress with high inoculums. the diagnosis of q fever requires microbiological testing. the chest radiograph and computed tomography (ct) finding are nonspecific and consistent with any other type of pneumonia. 52 there is no significant adenopathy seen on the chest radiograph or ct, which may help to rule out anthrax. other bacteria the 2 other bacterial agents that are discussed are brucella sp and burkholderia sp. 24 brucellosis has multiple clinical presentations and can be difficult to diagnose. typically brucellosis infects organs (lung, liver, spleen), bone marrow, bones, or central nervous system and is an insidious infection, presenting primarily with constitutional symptoms and organomegaly. the incubation period ranges from 2 to 8 weeks. diagnosis of brucellosis is best made by bone marrow cultures or blood cultures, although they are less sensitive. burkholderia mallei causes the clinical disease glanders, which presents as skin nodules and lymphadenopathy after inoculation through the skin, but in a bioterrorism event with inhalation exposure, the most likely presentation would be a nonspecific systemic febrile illness, which may or may not be accompanied by a pustular rash. burkholderia pseudomallei causes the clinical syndrome of melioidosis, a disease with a broad spectrum of presentations, including skin ulcers, pneumonia, acute fulminate sepsis, and chronic abscesses. if used in a bioterrorism setting with inhalational exposure, the acute presentation would most likely be either pneumonia or sepsis. smallpox smallpox can present as 1 of 5 clinical syndromes (table 11) : classic, modified, flat, hemorrhagic, and variola sine eruptione. 7, 19, 28 the primary challenge in the diagnosis of smallpox is the lack of clinicians who have experience with the disease. the cdc has published a case definition to aid in the diagnosis of cases (box 6). classic smallpox begins with a prodrome of fever and constitutional symptoms. after 2 to 4 days, the rash begins to emerge, initially as small red spots in the mouth and throat, which develop into sores that erupt and discharge virus, making the patient highly infectious at this point. at about the same time that the sores in the mouth begin to open, a rash develops on the skin. the rash is said to spread in a centrifugal pattern, starting on the face, then spreading to the extremities. the lesions progress from macules, to papules (day 2 of rash), umbilicated vesicles (day [4] [5] , and pustules (day 7) before crusting over (fig. 3) . in contrast to chickenpox (varicella), in smallpox, all the lesions progress at the same stage and the lesions are more densely concentrated on the face and extremities (figs. 4 and 5) . a single suspected case of smallpox is a public health emergency and the local public health agency should be notified immediately if there is a suspected case. imaging does not play a major role, because the respiratory symptoms tend to develop after the skin lesions. a few individuals who care for smallpox victims, or who have been vaccinated, develop a pulmonary form of smallpox without skin lesions. in these cases, chest radiography or ct may be helpful. the chest radiograph shows ill-defined nodular opacities in the upper lung fields, which may persist for months before calcifying. 52 vhf clinical presentation of vhf depends on several factors, including specific virus, virulence of the strain, route of exposure, dose, and host factors (table 12) . 32,57 the general feature that all vhf have in common is that they cause microvascular damage, leading to symptoms associated with increased vascular permeability, potentially resulting in hypovolemic shock or frank hemorrhage. medical imaging is not helpful in diagnosing these conditions, and the general laboratory findings are nonspecific, but often reveal evidence of hepatitis and hypovolemia. the specific diagnosis depends on microbiological testing (see table 7 ). the diagnosis of a bioterrorism event secondary to the use of biological toxins depends on the presentation of unusual clusters of cases. identifying the specific toxin can be challenging but the clinical presentation of some toxidromes can be useful in suggesting the agent involved. the clinical syndromes associated with ricin poisoning vary based on the mode of entry into the body. inhalation of ricin leads to symptoms within hours, which include dyspnea, fever, cough, pulmonary edema, and chest tightness, whereas ingestion of ricin results in vomiting and diarrhea, leading to dehydration, shock, hallucinations, seizures, and hematuria. the typical onset of symptoms after ricin poisoning averages from 4 to 6 hours up to 10 hours. laboratory findings associated with ricin poisoning are nonspecific but include metabolic acidosis, increased results for liver function tests, anemia, increased creatinine level, leukocytosis, and hematuria. aerosolized botulinum toxin, likely to be experienced in a bioterrorism setting, produces disease that mimics food-borne, infant, and wound botulism clinically. the clinical features that are anticipated with the inhalation of botulinum toxin include descending paralysis starting with diplopia, ptosis, fixed dilated pupils, dysphagia, respiratory failure, urinary retention, and constipation. with the ingestion of botulinum toxin, the clinical features begin with gi symptoms, including nausea, vomiting, diarrhea, bloating, and pain, before the development of the paralysis. onset of symptoms after exposure to botulinum toxin occurs as early as 6 hours after inhalation, but if ingested typically there is a 12-hour to 36-hour delay before symptoms begin. however, the onset may be delayed as much as 10 days. the diagnosis of botulinum intoxication is based on laboratory detection of the toxin in blood, food, or stool. the clinical presentations of mycotoxins vary based on the specific toxin. aflatoxin ingestion produces clinical features acutely, including hemorrhagic liver necrosis and pulmonary edema with chronic features, including the development of hepatic cancer. the effects and clinical presentation of inhaled aflatoxin are not known. t2 can be absorbed through the skin or inhaled. the clinical presentation in the acute phase includes pain on contact with skin or eyes, conjunctivitis, blurred vision, rhinorrhea, epistaxis, dyspnea, wheezing, tachycardia, shock, vomiting, diarrhea, erythema, and blistering. delayed symptoms present approximately 1 week after exposure and include thrombocytopenia, neutropenia, anemia, and coagulopathy. seb is a superantigen, which causes massive stimulation of t cells and cytokine storm, which produces the associated clinical features. ocular exposure results in conjunctivitis and eye swelling, whereas inhalation causes fever, chest pain, gi symptoms, pulmonary edema/ards, and shock. the respiratory effects may not be seen for up to 48 hours after exposure. as with most other infectious diseases, considerations for the management of bioweapons should include consideration of (1) vaccination to prevent infection/illness, (2) treatment of infection/illness, and (3) prophylaxis after exposure to prevent clinical infection/illness. also, as is common with most other infectious diseases, there are often several antimicrobial regimens that can effectively treat a specific bioterrorism agent, and for others there are no effective treatments. table 13 presents the generally accepted international recommendations for vaccination, treatment, and prophylaxis of bioterrorism agents. however, clinicians should always consult the most recent guidelines published by their public health authority when making clinical management decisions regarding bioterrorism agents. in addition to the current therapies, new drugs and vaccines are being developed specifically to address the threat of bioterrorism. 58 with the exception of possibly cutaneous anthrax, there are no specific indications for surgical management of any bioterrorism organisms. 11 even in the case of anthrax, surgical debridement has primarily been used for injection anthrax. 59,60 supportive care for victims of a bioterrorism event involves 2 components: the care of the individual patient and the response to a mass casualty event. despite the heightened awareness of the potential for bioterrorist events since 2001, many hospitals remain unprepared for biological threats. a study of uk emergency departments (eds) revealed that 24% did not have isolation facilities, and only 61% had departments with independent ventilations systems that would allow for the department to be isolated from the rest of the hospital. 61 in addition, the survey found that isolation procedures in many eds were poor; for example, 27% would not have isolated a patient with potential sars and 23% would not isolate a patient with chickenpox. 61 the task force for emergency mass critical care provides guidelines for hospitals to prepare for and manage mass critically ill casualties from events such as a bioterrorism attack. 62 critical care physicians should be familiar with these guidelines and prepared to respond in such circumstances, because if they are not, all of their skills and knowledge for treating individual patients are of little use as the system becomes overwhelmed. supportive care of individual patients can generally be grouped into categories based on the class of agent involved: bacterial, viral, or toxin. bacterial and viral agents typically produce a sepsis syndrome with vascular leak, particularly in the case of vhf, as well as variable degrees of a systemic inflammatory response. supportive management is similar to the standard best practices for managing septic patients (box 7). particular attention needs to be directed toward ventilatory support, with lung protective ventilatory strategies to minimize the development or exacerbation of adult respiratory distress syndrome. significant fluid shifts are to be expected given the vascular leak, requiring judicious volume resuscitation and cardiovascular support with vasopressors and inotropes based on goal-directed therapy. supportive therapy for patients involved in bioterrorism attacks with biotoxins varies based on the specific toxin. in the case of exposure to botulinum toxin, the primary support required is mechanical ventilation, hydration, and nutritional support until the paralysis resolves. toxins such as seb produce a response similar to sepsis and are supported by the sepsis protocols described earlier. ricin and mycotoxins both act at the cellular level, and although supportive therapies should be attempted, they may be of little benefit in altering the outcomes for patients. for mycotoxins specifically, there may be a role for the addition of steroids as a component of supportive care. 35 data regarding patient outcomes after exposure to bioweapons or bioterrorism agents are, for the most part, lacking, given the paucity of cases and that most research has been carried out by government or military organizations and the results cannot be shared publically. table 14 summarizes the published outcomes after exposure to bioterrorism agents when data are available. however, the outcomes from previous incidents, and in particular from naturally occurring illness, may not predict future outcomes, because bioterrorism agents may undergo genetic manipulation to increase their virulence or introduce antibiotic resistance, which decreases the efficacy of treatments. although to the average clinician, bioterrorism may seem to be only a remote possibility, it is not only a reality of the times in which we live but also bioweapons 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screening for inhalational anthrax after a bioterrorist attack inhalational anthrax due to bioterrorism: would current centers for disease control and prevention guidelines have identified the 11 patients with inhalational anthrax from screening for inhalational anthrax due to bioterrorism: evaluating proposed screening protocols the office of the surgeon general at tmm publications state-of-the-art therapeutic medical countermeasures for viral threat agents injection anthrax causing compartment syndrome and necrotising fasciitis the surgical management of injectional anthrax emergency departments (eds) in the united kingdom (uk) are not prepared for emerging biological threats and bioterrorism summary of suggestions from the task force for mass critical care summit prevention of laboratory-acquired brucellosis comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders key: cord-256553-iw5squ6n authors: neiva, mariane barros; carvalho, isabelle; costa, etevaldo dos santos; barbosa-junior, francisco; bernardi, filipe andrade; sanches, tiago lara michelin; de oliveira, lariza laura; lima, vinicius costa; miyoshi, newton shydeo brandão; alves, domingos title: brazil: the emerging epicenter of covid-19 pandemic date: 2020-10-21 journal: revista da sociedade brasileira de medicina tropical doi: 10.1590/0037-8682-0550-2020 sha: doc_id: 256553 cord_uid: iw5squ6n introduction: five months after the first confirmed case of covid-19 in brazil, the country has the second highest number of cases in the world. without any scientifically proven drug or vaccine available combined with covid-19’s high transmissivity, slowing down the spread of the infection is a challenge. in an attempt to save the economy, the brazilian government is slowly beginning to allow non-essential services to reopen for in-person customers. methods: in this study, we analyze, based on data analysis and statistics, how other countries evolve and under which conditions they decided to resume normal activity. in addition, due to the heterogeneity of brazil, we explore brazilian data of covid-19 from the state health secretaries to evaluate the situation of the pandemic within the states. results: results show that while other countries have flattened their curves and present low numbers of active cases, brazil continues to see an increase in covid-19 patients. furthermore, a number of important states are easing restrictions despite a high percentage of confirmed cases. conclusions: all analyses show that brazil is not ready for reopening, and the premature easing of restrictions may increase the number of covid-19-related deaths and cause the collapse of the public health system. with 13 million cases and 576,000 deaths, the largest pandemic since the spanish flu in 1918 has now reached over 200 countries in the world. covid-19, a disease caused by sars-cov-2, started in wuhan but rapidly spread across asia, europe, and america 1 . brazil, which had its first confirmed case on february 25, 2020, almost two months after the announcement of the outbreak of the disease in china, is now the country with the second highest total number of confirmed cases 2,3 . by july 14, 2020, we accounted for 1,888,000 cases and 72,000 deaths 1 . as a new disease, despite all advances in technology and rapid genome analysis, there are no scientifically proven treatments, such as drugs and vaccines, to protect people or cure infected patients. thus, the main recommendations from the world health organization (who) for the general population are to maintain social distance and hygiene habits such as the use of alcohol-based hand sanitizers and face masks, measures intended to slow the spread of the virus 4 . furthermore, social inequalities in brazil highlight the importance of higher isolation rates since the public health system cannot withstand high pressure. sus, the brazilian public health system, is responsible for the support of 80% of the population and could collapse if a considerable number of people contract the disease in a short period of time 5 . moreover, people's confidence in the government is referenced as one of the critical points in handling the pandemic successfully. nevertheless, brazil is going through a hard time in the political sphere. as examples that directly impacts the covid-19 battle, the health minister was replaced twice in two months and the country's president has been frequently seen in groups not complying with the medical community's recommendations, thereby influencing the population 6, 7 . understanding data is essential for the proper care of the population. therefore, in this study, we analyze the situation of the pandemic in other countries to explore how they dealt with the disease and under which conditions they decided to resume non-essential services 8 . by understanding the evolution of each country and the statistics in brazil, the goal of this study is to evaluate whether the tropical country is gathering knowledge from other regions and easing restrictions according to certain metrics such as the decrease in daily cases and deaths. this article is a descriptive observational study using classical statistical metrics, such as frequency and percentage. the analysis is divided into two parts. first, the aim was to characterize how brazil is evolving in the covid-19 war. for this, the brazilian data at the federal level were compared with data from other countries regarding three types of information: the number of new cases, the number of new deaths, and active cases. for this analysis, we collected data from john hopkins 9 , which allowed us to obtain the daily and total cases, deaths, and recovered patients, along with the evolution of the pandemic. active cases are not directly obtainable from the hospital database; thus, we computed them using the following equation: active_cases(x) = total_cases(x) -total_deaths(x) -total_recoveries(x), where total_cases(x), total_deaths(x), and total_recoveries(x) are the total number of positive cases, deaths, and recovered patients, respectively, until day x. the measure is a good track to understand how many patients are currently transmitting the viruses or might require hospitalization, which is important for public health policies. furthermore, we also compared the total number of cases in each country according to their size. the relative comparison is essential because countries with different sizes probably have more cases compared to smaller regions. therefore, by dividing the cases by the population size, one can understand the proportion of inhabitants of each country that contracted covid-19 and the differences and similarities among couples of countries. besides brazil, seven countries are evaluated: south korea, the united states of america, spain, italy, new zealand, germany, and china. along with the comparison of countries, we also compared the first and second positions in the worldwide rank of case numbers provided by 1 . to do this, we used the epidemiological knowledge that virus propagation within the population can fit an exponential model 10 . thus, we used computational simulations in python to fit the total number of cases of brazil and the us as an exponential curve. to perform this analysis, we used the data from the previous two weeks, the approximate period of time sars-cov-2 remains in an individual organism 11 , to obtain the exponential parameters. then, we projected the data for the next 15 days. another statistic is the testing rate for each confirmed case. testing suspected cases reduces the under-detection probability and enhances the reliability of the data. data were retrieved from our world in data 12 for each country. the results are shown in section 3.1. finally, in section 3.2, we seek to understand how covid-19 performed within the brazilian territory. disaggregating geographically by state, the analyses aimed to characterize the number of cases, the number of deaths, and their influences on population rates over the time that the virus disseminated throughout the country over 5 months (from february to july). the behavior of dissemination and danger of the virus was characterized by the contamination rate, which describes the speed of spread of the virus through the number of cases normalized by the population of each place per 100 thousand inhabitants, and the lethality rate, which describes the proportion of people who die among all those infected 13 . data were obtained from the state health secretaries' databases and along with information of absolute and relative cases for each of the main states in brazil, we also evaluated the spatial evolution of the pandemic in the state of são paulo, the most populated location in brazil. finally, we discuss the evolution of reopening measures in brazil, showing a map with coping policies for each country. the coping information was retrieved from the news and collected from https://ciis.fmrp.usp.br/covid19/. as previously mentioned, it took two months for brazil to have its first confirmed case of covid-19, which gave the country a foundation of information and knowledge to better understand the evolution of the disease, as well as the opportunity to see how other countries proceeded with reopening. first, one can start by understanding the situation in wuhan, the breakpoint of the pandemic that previously suffered from sars and the avian flu 14 . despite the recent history of respiratory diseases, covid-19 is a new challenge that is not yet fully described by science, and the prognosis of patients can be unpredictable. in order to contain the spread of the disease and a high number of deaths, the chinese government ordered a lockdown in hubei, applying severe restrictions regarding social isolation which were widely obeyed 1 . furthermore, the culture of using face masks as a routine probably helped to decrease the rate of transmission 15 . by the beginning of march, china had already less than 100 new cases per day, and, by mid-june, there were under 10 new cases per day 1 . the country was one of the first to reopen and is currently tracking new cases by applying social distancing in workplaces, constant disinfection, and temperature measuring more than once a day 16, 17 . these actions appear to be effective, considering the number of new cases shown in figure 1f . new zealand is another country that has succeeded in beating covid-19. after 24 days with no detection of covid-19, a patient was diagnosed on june 16, having contracted it in the united kingdom, meaning no local transmission had occurred in three weeks 18 . the reason for such success? travel restrictions and quarantine measures with a low level of confirmed cases 19 . prime minister jacinda ardern has demonstrated strong leadership, convincing almost five million people to respect the stay-at-home guidelines 20 . furthermore, testing and tracking were applied all over the country with the support of mobile applications that use qr codes to scan buildings and alert possible infected people 21 . after one week with zero new cases, new zealand began to ease its restrictions 22 . south korea also used technology, isolation restrictions, and extensive temperature measuring 23, 24 . in europe, germany also applied consistent rules and strategies to fight the virus, such as intensive care unit (icu) monitoring, testing, and patient tracking 25, 26 . italy was one of the countries most affected by covid-19 due to a delay in establishing restrictions in many points of the country. the 'milan does not stop' campaign, for example, ignored the isolation instructions from who in an attempt to prevent the economy from collapsing 22 . after losing control of the disease spread, a lockdown began on march 9 and lasted until may 18 27, 28 . although these countries may have established different strategies, most of them relied on who instructions, scientific evidence, and strong leadership. figure 1 shows that almost all countries are on the downside of the curve of new cases. the database of the graphs was retrieved from the john hopkins hospital 9 . the number of active cases in italy, around 13,000, is still high, which requires intense observation of health services capacity since isolation restrictions are being loosened. with no drug or vaccine, all countries must keep monitoring its cases to understand whether it is time to re-apply restrictions or if one proceed to new phases of reopening. however, one can see that brazil and the united states, the second and first countries, respectively, in the number of confirmed cases, do not follow the same patterns in the new cases/deaths and active case graphs, as shown in figures 1g and 1h . furthermore, the graph of active cases shows a steep incline, while new cases and deaths are increasing. this is also evident in figure 2a , where most countries' curves are flattening and brazil and the united states curves continue to increase. as the us and brazil rank as first and second in numbers of active cases 1 , we compared both exponential projections in an attempt to predict the future. in figure 2b , it appears that brazil will overtake the us in numbers of active cases by the end of august. such analysis considers the relative number of cases in both countries to remove population size influence. this means that besides being the country in south america with the most covid-19 cases, brazil will also have the highest number of cases worldwide. the situation is even more worrying, as brazil has a low test rate and, consequently, a high likelihood of under detection (figure 3 ) compared with countries that are coming out of lockdown. furthermore, several places in brazil are easing isolation rules and opening non-essential services, such as malls, which are usually environments with low air circulation and, therefore, conducive to the spread of the virus. it should be taken into account that brazil is a very heterogeneous country with high social inequality. the pandemic is affecting the public health system and the population in a non-uniform way, as approximately 11.4 million people live in shanty houses. the following section highlights these differences and shows more in-depth features of this enormous country. brazil, with 26 states and one federal district, is the seventh most populated country, and the fifth with the greatest territorial extension worldwide 29 . therefore, the country is inconsistently dealing with the coronavirus epidemic. each brazilian state behaves like a small country, and the segregation of analysis aims to strengthen the action plans. the following analyses considered the five brazilian states with the highest numbers in each indicator. figure 4a shows the accumulated number of cases over time. in order, the five states with the highest number of cases were são paulo, ceará, rio de janeiro, pará, and bahia. at the end of may, they recorded approximately 880,000 cases, 47% of all brazilian cases. são paulo has been the leader in the number of cases since the beginning, with approximately 173% more cases than the second ranked state, ceará. this is because são paulo is the industrial center figure 3: besides the high number of cases in brazil, the country is known for its lower testing rate. this has direct consequences on the under detection covid-19, meaning that brazil possibly has more cases than those noted by health care centers. conversely, south korea and new zealand are among the countries with the highest rates of testing and are considered successful cases in the pandemic. and the most populated area of the country, with approximately 45 million people. however, the absolute number of cases alone does not provide a complete picture of the severity and speed of dissemination. thus, figure 4b shows the contamination rate. the latter four states did not have a large accumulated number of cases, but showed high daily growth in relation to the other brazilian states. a different scenario is observed when analyzing the contamination rate, as shown in figure 4b . the measure refers to the number of cases normalized by the population of each state per 100 thousand inhabitants. since the end of april, the states of amapá, roraima, amazonas, and acre have presented high community transmission of the coronavirus. this has resulted in a new focus on the north region of brazil. none of these regions were even mentioned in previous analyses. this negative impact of this statistic was clearly seen in mid-april when the state of amazonas became the first brazilian state to be on red alert, asking for international support due to the collapse of the funeral and health system 30, 31 . with the diversity and immensity of the country, brazil has several areas and different needs that require attention. thus, it is essential to explore different indicators to capture these needs as quickly as possible. figure 4c presents the death analysis. it is expected that the number of deaths will be proportional to the number of cases. four of the states with the highest absolute number of deaths have already been mentioned as critical in previous analyses; however, the state of pernambuco is unprecedented and had more than 2,500 deaths by the end of may. together, the five states with the highest number of cases have reported almost 20,000 deaths and represent 75% of total brazilian deaths. regarding daily case information, the highest number in the country occurred in são paulo on june 3 when 609 deaths due to covid-19 were recorded in 24 hours. hospital morgues located in rio de janeiro, which also has a high number of daily deaths, are dealing with overcrowding and accumulating bodies day after day 32 . the last indicator explored in this subsection is the lethality rate, as shown in figure 4d . the lethality rate is the ratio between the number of deaths and the total number of cases of a disease in a given period; it represents the risk that people with the disease have of dying from the disease 13 . brazil has a high rate of covid-19 transmission. within 15 days, its number of cases had doubled, which translated to 500,000 new cases 9 . the heterogeneity and extensiveness show a burdensome scenario within the decision-making process and the need for protective measures. social inequalities enhance the necessity of deep analysis prior to easing restrictions based on intensive care units, infrastructure, and infection rates presented in section 3. for instance, the north region required significant medical support and structure in order to prevent a worse collapse in the health system. the northeast also showed high lethality and death rates, mainly in ceará and pernambuco. são paulo and rio de janeiro appear in almost all covid-19 analysis indicator rankings. both states represent the main economic poles in the country, and the high density of people in both areas highlights the importance of continuous tracking and testing. the brazilian government does not seem to be learning lessons from other countries, such as those referenced in section 3.1. as previously mentioned, all of them, except for the united states, only eased restrictions after intensive tracking, testing, and a descending curve in the number of daily cases was shown. são paulo and rio de janeiro, along with other states in brazil, do not follow the same approach (see figure 3a ). in addition, the number of active cases is still high, along with all other indicator analyses concerning each state. this is an urgent issue and must be addressed in all public spheres so that we can improve our response to the pandemic. in public health, prevention is always the least costly option, especially in this case, where an individual's response to the infection could be death. supported by the quantitative information in figure 2b demonstrating that brazil is expected to overtake the us in total number of cases (proportionally by population size), the current low isolation rates, low population testing percentage with consequently high under detection, high daily cases, and death rates all suggest that brazil has eased its restrictions prematurely and may face severe consequences in the near future as a result, such as becoming the next global epicenter of the pandemic. coronavirus cases [internet]. worldometers covid-19 cases in brazil surge to 330,890 ranking it as second place to america worldwide coronavírus brasil [internet]. brazilian government covid-19 lota hospitais e gera colapso funerário em belém demand for hospitalization services for covid-19 patients in brazil brazil loses second health minister in less than a month as covid-19 deaths rise [internet]. the guardian sob vaias e frases de apoio, bolsonaro come cachorro-quente em brasília doria anuncia flexibilização gradual da quarentena em são paulo agência brasil an interactive web-based dashboard to track covid-19 in real time a systematic review of covid-19 epidemiology based on current evidence science forum: sars-cov-2 (covid-19) by the numbers coronavirus disease (covid-19) -statistics and research epidemiologia clínica: elementos essenciais, artmed the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china the state council information office -the people's republic of china. full text: fighting covid-19: china in action asia may have been right about coronavirus and face masks, and the rest of the world is coming around life after lockdown: how china went back to work zealand's first covid cases in 24 days came from uk how new zealand's 'eliminate' strategy brought new coronavirus cases down to zero new zealand's prime minister may be the most effective leader on the planet coronavirus: aucklanders have to scan qr code to enter council buildings more covid-19 lockdown restrictions to be eased in new zealand south korea is relying on technology to contain covid-19, including measures that would break privacy laws in the us -and so far, it's working south korea's health minister on how his country is beating coronavirus without a lockdown how germany contained the coronavirus oversupply of hospital beds helps germany to fight virus italy's virus shutdown came too late. what happens now? learning from the italian experience in coping with covid-19 wikipedia contributors. brazil -wikipedia, the free encyclopedia com saúde em colapso, governo do amazonas usará contêineres frigoríficos para mortos do coronavírus governador do am escreve à onu e pede ajuda contra covid-19 na amazônia [internet]. uol notícias com necrotério lotado, corpos se acumulam em corredor de hospital no rj we thank the university of são paulo for technical support. the study did not require ethical approval since it is a descriptive study and there are no experiments involving humans. the authors declare that there is no conflict of interest. conception and design of the study, performed the computations, performed the analysis, wrote the paper; 7/8 ic: conception and design of the study, performed the computations, performed the analysis, wrote the paper; escf: performed the analysis, wrote the paper, reviewed the paper; fbj: interpretation of data, drafted the article, wrote the paper, reviewed the data; fab: model of data server, drafted the article, wrote the paper, reviewed the data; tlms: wrote the paper, reviewed the data; llo: wrote the paper, reviewed the paper; vcl: model of data server, drafted the article, wrote the paper, reviewed the data; nsbm: wrote the paper, reviewed the paper; da: conception and design of the study, final approval of the version to be submitted. key: cord-265049-uwzmvlr4 authors: tuge deressa, chernet; file duressa, gemechis title: modeling and optimal control analysis of transmission dynamics of covid-19: the case of ethiopia date: 2020-10-09 journal: nan doi: 10.1016/j.aej.2020.10.004 sha: doc_id: 265049 cord_uid: uwzmvlr4 a mathematical model to estimate transmission dynamics of covid-19 is developed. a real data of confirmed cases for ethiopia is used for parameter estimation via model fitting. results showed that, the diseases free and endemic equilibrium points are found to be locally and globally asymptotically stable for ro<1 and ro>1 respectively. the basic reproduction number is ro=1.5085. optimal control analysis also showed that, combination of optimal preventive strategies such as public health education, personal protective measures and treatment of hospitalized cases are effective to significantly decrease the number of covid-19 cases in different compartments of the model. it is reported that [1] up to the present, the world has witnessed the happening of seven strains of human coronaviruses including sars-cov,mers-cov and the current coronavirus 2019-ncov named covid-19. covid-19 was declared a pandemic by the world health organization(who) on 11 march,2020 [2] . as of july 27 th 2020, covid-19 pandemic has claimed several hundreds of deaths globally and become the greatest threat of its kind in our generation [3] . it also posed a huge risk to public health and economics all over the world [4, 5] . the ethiopian federal ministry of health confirmed its first case of covid-19 on 13 march, 2020, in addis ababa [6] . despite the fact that the number of confirmed cases and deaths reported varies from time to time, the outbreak has taken the lives of several hundreds and affected different livelihoods in ethiopia. since the first case of covid 19, various mathematicians around the world develop different mathematical models to understand the transmission dynamics of the virus, estimated the basic reproductive number and investigated effects of different intervention strategies via optimal control analysis. for instance, in [7] real data of confirmed cases of covid-19 in wuhan, china from january 21,2020 to january 28,2020 is used in a model developed to estimate the basic reproductive number the authors developed a 0 2.4829. r  fractional model and solved it numerically. a mathematical model for predicting transmission dynamics of the pandemic for italy is considered in [8] .the result indicated that the combination of restrictive social distancing measures with widespread testing and contact tracing can result in ending the covid-19 transmission in italy. analysis of covid-19 infection, based on real data is made in [9] by developing a mathematical model for wuhan, china. in [10] fractional order covid-19 sidarthe model is developed to predict the evolution of the pandemic and investigated the impact of different plans to reduce the transmission of the disease with different values of fractional order. there are also many other mathematical models that reported on covid-19 pandemics, refer [11] [12] [13] [14] . mathematical models developed were mainly used to investigate the effects of different non-pharmaceutical intervention strategies via simulation using different computing soft-wares [15] . different scholars used different type of mathematical models in their analysis. for instance, an seir model was introduced by wu et al. [16] for estimating the spread of the pandemic and obtained the basic reproductive number to . tang et al. [17] used a deterministic 0 2.68 r  model to estimate the basic reproductive number to be as high as and concluded 0 6.47 r  that, contact tracing followed by quarantining and then isolation can mitigate the spread of the pandemic via reducing the basic reproductive number. kiesha et al. [18] used seir model to simulate the outbreak of coronavirus in wuhan and indicated that imposing restriction on wuhan people movement could help in delaying the peak time of the pandemic. roda et al. [19] claimed that sir model performs better than seir model and used it to predict covid-19 epidemic in wuhan and predicted the potential of a second outbreak after the return-towork in the city. pang et al. used seihr mathematical model to investigate the effectiveness of quarantine measures applied in wuhan city and factors affecting its effectiveness [20] . it is not only for covid-19 that models were used to study the dynamics but also for several other infectious diseases. in [21] a mathematical model for the deadly disease named ebola hemorrhage fever is developed and investigated the detail of endemic equilibrium points. sir model with delay in the context of fractional derivative with mittag-leffler is considered in [22] . the author established the global and local stability of disease free and endemic equilibrium points using lyapunov direct method. mathematical models that investigated the dynamics of various infectious diseases are numerous in the literature [1, 23] and the references there in. most of these studies have either restricted their domain of study to a particular country or they used data from a certain particular region. some of the studies also overlooked the issue of optimal control analysis. moreover, to the best of authors' knowledge, no study has been conducted on transmission dynamics of covid-19 for the case of ethiopia. to this end, the main objective of this work is to develop a mathematical model for the transmission dynamics of covid-19 followed by mathematical analysis in order to come up with evidences that may help for designing mitigation strategies relevant to the context of ethiopian. in this paper a mathematical model for covid-19 relevant to study the transmission dynamics of coronavirus in ethiopia is developed. the existence and uniqueness of solution of the model is proved. local and global stability analysis of the diseases free and endemic equilibrium points are established. parameter estimation based on real data of confirmed cases is made. the basic reproductive number suggested by the model is calculated. the importance of the basic reproductive number in target setting to control the pandemic is discussed by calculating sensitivity indices of the parameters of the basic reproductive number. optimal control analysis of the model with three control strategies namely: public health education, personal protective measures and treatment of hospitalized covid-19 cases were investigated followed by numerical simulation. discussions, conclusions and limitation of the model are well specified. in this work, a mathematical model for covid-19 transmission dynamics is developed based on compartmental approach of six groups named, s-susceptible, e-exposed, isymptomatically infected, a-asymptomatically infected, h-hospitalized and r-recovered/ immune compartments. the total population at any time is given by humans are recruited into the compartment  considered in the model. the above mentioned is the scenario that governmet of ethiopia is using in combating the deadly covid-19 pandemic, in the sense that the data of confirmed cases of covid-19 in ethiopia is organized incorporating all the compartments we have used in developing the model of this study. it is these data that we translated into a mathematical model. it seems logical to divide infected cases of covid-19 into symptomatic and asymptomatic cases, in agreement with the observed property of coronavirus infection (majority are asymptomatic and the remaining symptomatic) in ethiopia and elsewhere in the world. moreover, the isolation/hospitalization is considered to be effective in reducing the transmission of covid-19 [24] . in addition, it is known that coronavirus infected people don't get infectious immediately after contraction of the virus and this ensures the importance of considering the compartment of exposed cases. as a result, the aforementioned scenario and modification of seir [18, 23] model are used to develop the present model. the model developed and the parameters used are indicated in system of differential equation (1) and table 1 . . the biological validity of the mathematical model depends on the solution of the dynamic system being positive and bounded for all values of time . the boundedness and positivity of the solution is proved in the subsequent lemmas. remain positive in + . proof: it is assumed that all the parameters used in the model are positive and hence we can set a lower bound for each of the equations in (1) as follows: . solving the above inequalities respectively leads to: . thus, for all and are positive in , of (1) there applying groonwall's inequality leads to, ( ) and are all bounded since, , thus, the region 6 ( , , , , , ) is positively invariant region for model (1) . then either the solution of (1) enters in a finite time or asymptotically. hence the region attracts all solution of (1) in  since has a continuous first derivative in ,it is then locally lipschitz. as a result, by the f ℝ 6 + well known fundamental existence and uniqueness theorem [25] and lemma 1 and 2 proved above, there exists a unique, positive and bounded solution for the system of differential equation (1) in ℝ 6 + . and that satisfies the quadratic equation where, the basic reproductive number, denoted by , is defined as number of secondary infections 0 r appears from one infected individual. it provides a threshold condition for the stability of the system. finding through jacobian approach using linearization of the dynamic system 0 r often doesn't work for complex systems [26] . thus, we obtained by establishing the next 0 r generation matrix, , and calculating a spectral radius of the matrix at [27] . the matrices and are obtained by linearizing the mathematical model (1) about dfe that t 1 v  resulted in the jacobian matrix given as: after constructing a matrix (m) from infectious components of the and partitioning it into dfe j transmission (v) and transition (t) matrices, we have: the spectral radius of the next generation matrix is the basic reproductive number note that, can also be written as where this section deals with the local stability analysis of the diseases free and the endemic equilibrium points. necessary condition: since both and , then from the 1 2 and are positive r r , for the two negative eigenvalues of the jacobian matrix are . the sign of the real part of the remaining four eigenvalues are determined from a characteristic equation (5) by routh-hurwitz stability criteria: necessary condition: the coefficient is positive and can be shown to be positive as follows, in this subsection we showed global asymptotical stability of the dfe and eep using lyapunov function method. i. global stability analysis of dfe the locally asymptotically stability of the dfe proved earlier indicates that covid-19 can be eliminated from ethiopia when if the initial sizes of the cases in each of the 0 1 r  compartments or in the society is very close to the dfe; initial size is in the basin of attraction of dfe, . in order to show that the stable in the positively invariant region defined in section2.  consider a lyapunov function candidate defined by, ( , , , , , ) differentiating with respect to time in the direction of the solution of ( , , , , , , ) (1), and then substituting the appropriate values from (1) and leads to, note that, by the inequality of arithmetic and geometric means we have   differentiating in the direction of the solution of (1) leads to, l replacing by their respective expression from model (1) we get, , , , , , we can now write since all the parameters used in model (1) eep is globally asymptotically stable. note the implication of theorem 4 is that, covid-19 will institute itself in the society whenever regardless of the initial size of infectious individuals in the population. parameter estimation is made based on real data of covid-19 confirmed cases of ethiopia for the first 140 days from 13 march to 31 july 2020 available online at [28] https://en.wikipedia.org/wiki/covid-19_pandemic_in_ethiopia. some of the parameters are calculated from the data as follows: the total population of ethiopia is approximately and the life expectancy of ethiopians [29] one symptomatic case was confirmed, no individual was confirmed asymptomatic, no hospitalized and no recovery cases. consequently, assuming initial number of exposed cases to be 200, the initial number of population is assumed where it is also learnt from the data that, in the last 140 days of the pandemic 5700 hospitalized or isolated cases were recovered from the diseases. hence, the recovery rate of hospitalized  cases is calculated as, =6700/17530 =0.00273/day.  some of remaining parameters are estimated from the mathematical model (1) via model fit using a computing soft-ware matlab2018a by optimal estimation of parameters; least square curve fit along with trust-region-reflective to find bounded estimated parameters. the result of model fit versus confirmed cases of covid-19 is portrayed in figure 1 . thus, values of the parameters used in this work are obtained from assumption, calculation, model fitting and from the literature and are indicated in table 2 . all negative and hence the endemic equilibrium point is locally asymptotically stable, and is in agreement with the analytical proof made in subsection 3.2. in order to determine the best control measures using the basic reproductive number, the normalized sensitivity index, which measures the the relative change of with respect to 0 r  denoted by ,and defined as is useful [24, 27] . the magnitude of sensitivity analysis of the reproductive number (3) shows that the most sensitive parameters in descending order are and so on. increasing the , , , , , ,... it then follows that, if the transmission from asymptomatic cases to suspected cases is reduced by a fraction of at least ( i.e. ), then the transmission of covid . now, the objective is to find an optimal control for the preventive strategies/measures for the relatively reduced coasts of the prevention strategies. the necessary 1 2 3 , and u u u and sufficient conditions for the existence of optimal control are established by pontryagin maximum principle [34] . the function that minimizes the number of exposed cases , number of symptomatically infected cases , number of asymptomatically infected cases and number of hospitalized cases over a time interval of can be defined as, where the control set is given by constraint given by system of differential equation (6). the necessary conditions that need to be satisfied by optimal control called pontryagin's maximum principle converts equation (6) and (7) . differentiating the hamiltonian function with respect to the compartment variables gives the adjoint variables corresponding to the system given as follows: and are the adjoint variables , , , , , ( , , , , , ) , . now, setting the transversality condition: we obtain the optimal controls and the optimality conditions respectively as: note that, state equation (6), the adjoint equation (9) together with the characterization of the optimal control (13) and the transversality condition (11) are said to be optimality system. the sections and subsection treated above describe analytical behaviors of the optimal control. the corresponding numerical simulation of the optimal control is treated in the next section. in this section numerical solution for the optimality system is presented. the initial conditions used in the simulation are as can be seen from figure 2 above, the peak for the spread of the virus is observed around 238 th day of the pandemic. the result indicated that there are about 5095000 exposed, 603500 asymptotic, 479100 symptomatic and 1154000 hospitalized cases at the peak. case ii: in this case, all the three intervention strategies namely: educating the public , using of personal protective measures , and treating the hospitalized case iii: this case considered the combination of optimal personal protective measure and optimal treatment of hospitalized cases with no any public health education intervention . the simulation result of the effect of these two intervention 1 2 3 ( ( ) 0, ( ) 0, ( ) 0 ) u t u t u t    strategies is almost identically the same as in figure 4 . in mitigating the transmission of covid-19 i.e. case ii. but as to case iii, the full intensity of using personal protective measures has to be prolonged to the first 50 days before it slowly gets down to the lower bound at the end of the pandemic. note that, it has been checked that cases where and as can be seen from figure 6 , using the intervention strategy 'treating hospitalized covid-19 cases' alone with the maximum effort couldn't reduce the number of exposed, symptomatic and asymptomatic cases as the number of cases in figure 6 is the same as number of cases in figure 2 for the compartments . applying this control strategy , and e i a alone reduced the number of hospitalized cases from 1154000 ( figure 2) to 27130 ( figure 6 ) at the peak. further, the control profile of this case is the same as case ii for of figure 3 ( ) u t case v: in this case, optimal practice of public health education is considered without the intervention of personal protective measures and treatment of hospitalized cases . the simulation result for the effect of optimally practicing this intervention strategy is presented in figure 7 for the optimal profile of and in 1 ( ) u t as can be seen from figure 7 , the control profile that the optimal time range for public education is ranging from 176 th -186 th only for 10 days. further, figure 8 reveals that the peak of the pandemic for this case is around 176 th day but it is around 238 th day in figure 2 . one can also anlyze from figures 2 and 8 that using optimal public education alone could have reduced the duration of the pandemic, the peak period and number of cases in each of the compartments. case vi: this case considered the optimal practice of personal protective measure without intervention of the other two control strategies: public health education and treatment of hospitalized cases . the simulation result of the control profile is depicted in figure 9 where as its effect on the number of cases in the compartments identically the same as in case ii. it can be observed from figure 9 above, the usage of personal protective measures is optimal for the first 120 days of the duration of the pandemic. comparing cases ii and vi, one can learn that both cases lead to the required result of mitigating the transmission of the pandemic independently. however, in case vi the practices of personal protective measures need to be applied optimally for the first 129 days as compared to 8 days for case ii. despite the effort made by the gvernment to teach the public and many ethiopians effort to maintain personal protective measures and treatments of hospitalized cases being underway, the transmission of covid-19 is increasing from time to time (see figure1) . consequently, it seems reasonable to make numerical investigation of nonoptimal intensity of practicing personal protective stratgies to appreciate their effect in decreasing the number of cases in the compartments of model (6) . accordingly, we assumed and simulted the 1 3 ( ) 0 ( ) u t u t   model for different percentage values of pesonal protective measure 2 ( ) 1%, 5%, 9% u t  and the result is shown in figure 10 below. figure 10 it can be inferred that, if at least 1% from each of the symptomatic, asymptomatic and hospitalized cases maintained practicing personal protective measures at an optimal level from the onset of the pandemic, then the number of cases in the compartments would have reduced (black) as compared to the graph with no personal , , and e i a h protective (red) measures . the figure further reveals that a pretty improved result 2 ( ( ) 0) u t  would have been obtained for in the different compartments. 2 5% and 9% u  in this study, a mathematical model for transmission dynamics of covid-19 for the case of ethiopia is developed and its different properties including local stability analysis of the diseases free and endemic equilibrium points have been checked. some of the parameter estimates were taken from spencer et al., [12] and the remaining parameters were computed via model fitting based on real daily data of covid-19 confirmed cases of ethiopia from 13 march to 31 july, 2020. it is then calculated that the basic reproductive is . 0 1.5085 r  an optimal analysis of the model for the purpose of assessing the effect of public health education, effect of personal protective measures and effect of treating hospitalized or isolated cases in mitigating transmission of covid-19 was conducted. the result showed that the optimal practice of combination of all the three intervention strategies significantly reduces the number of exposed, symptomatic, asymptomatic and hospitalized cases (see figures 3 and 4) . likewise, optimal usage of personal protective measures alone led to the required decreases in the number of cases in the compartments except that the optimal application of the control measure needs to be maintained relatively for a longer period of time (see figure 9 ). it is also found that combining control strategies personal protective measures and treatment of hospitalized cases (case iii) is as good as combining the three strategies (case i) in combating the deadly covid-19 pandemic in ethiopia. in this work, the parameters that need to be targeted for suppression of covid-19 pandemic by decreasing the basic reproductive number, are identified. for instance, it is shown that if the target is to decreasing the rate of transmission from asymptotically infected to suspected individuals then reducing this parameter to at least can suppress coronavirus from , ,  0.47   ethiopia. the same type of work can be conducted in different countries by adapting to their situation. moreover, optimal combination of the three intervention strategies proposed in this work or optimal combination of any two of the strategies or optimal practice of personal protective measures alone reduced the number of covid-19 cases in the compartments as shown in the simulation results. hence, the result of this study can be used as a policy input for the government of ethiopia and other countries. the government of ethiopia has to take necessary measures to make personal protective measures a mandatory practice throughout the period of the pandemic. personal protective measures such as wearing facemask, regular hand washing and social distancing if practiced with optimum effort, can significantly decrease the disturbing effect of covid-19 and safeguarded the nation. the government can also target its control strategy on reducing the basic reproductive number, taking into account the sensitivity analysis results of this work while considering the limitations of this work given below. it is ethical to make clear that mathematical models are in general approximations of real phenomena and as such they are naturally inaccurate. moreover, the parameters used in any mathematical models are determined based on observations and experimentations using different numerical methods of computing software and hence are uncertain. therefore, readers should take into account the limitation of the models while interpreting the findings. the dynamics of covid-19 with quarantined and isolation, advances in difference equations situation report -51, data as reported by 11 a mathematical model for simulating the phase-based transmissibility of a novel coronavirus pneumonia of unknown aetiology in wuhan, china: potential for international spread via commercial air travel nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a 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differential equations and dynamical systems a nonlinear epidemiological model considering asymptotic and quarantine classes for sars cov-2 virus on the definition and the computation of the basic reproduction ratio r 0 in models for infectious diseases in heterogeneous populations wikipedia, the free encyclopedia epidemiological parameter review and comparative dynamics of influenza, respiratory syncytial virus, rhinovirus, human coronavirus, and adenovirus the reproductive number of covid-19 is higher compared to sars coronavirus high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 extending the type reproduction number to infectious disease control targeting contacts between types uncertainty and sensitivity analysis of the basic reproductive rate: tuberculosis as an example an introduction to mathematical epidemiology we, the authors of the manuscript, thank jimma university college of natural sciences for all the supports. key: cord-023186-gqltd6u0 authors: nan title: poster sessions date: 2019-06-27 journal: pediatr pulmonol doi: 10.1002/ppul.24373 sha: doc_id: 23186 cord_uid: gqltd6u0 nan in this study. the near-fatal group accounted for 19%. the 12 months to 5 years age group accounted for 58%. female/male ≈ 1.2/1. sixtyone percent of children were from hồ chí minh city. admission to icu increased recently. dyspnea was the chief complaint, accounting for 88%. cough, wheezing, dyspnea were essential symptoms. fiftly-one percent had a history of asthma. preventer medication ownership was recorded in 34% of cases. the rate of impaired consciousness was 59.4%. 27% had a ph < 7.3% and 33% had a paco2 > 40 mm hg. chest x-rays showed hyperinflation, infiltrates and atelectasis. time of treatment was 7 days (6,11), days in icu was 2 days (2, 4). ten children were intubated, 43.5% received ncpap; 72.6% of children older than 1 year were given mgso4, 65% children were given diaphyllin, 20% children were given salbutamol and 6% children were given adrenaline subcutaneously. all intubated patients received pressure control ventilation. inspiratory pressure was 14 to 16 cmh2o to achieve tidal volume of approximately 6ml/kg, mean peep was 7 cmh2o, fio2 was approximately 80% and i/e was 1/2 -1/3. conclusions: appropriate treatment at first is crucial to lower the number of children to be intubated. mechanical ventilation still remains the last recourse in the treatment of near fatal exacerbations of asthma after mgso4, diaphyllin and salbutamol. a-132 | peanut and brazil nut sensitization profile among asthmatic patients in southern taiwan data on its importance in asian populations. the relevance of cdhr3 to childhood lung functions is also unknown. this cross-sectional study investigated the associations between cdhr3 and wheezing illnesses and lung function of preschool children in hong kong. methods: chinese children younger than 6 years of age were recruited from randomly selected nurseries and kindergartens throughout hong kong. the demographic, early-life exposures and allergy phenotypes of these preschool children were recorded by validated and modified isaac questionnaire. these children underwent incentive spirometry to measure their forced expiratory indices. their buccal swabs were collected for dna extraction and tagging single-nucleotide polymorphisms (snps) of cdhr3 were determined by taqman genotyping assays. genotypic and haplotypic associations between these snps and wheezing and lung function traits were analyzed by multivariable regression and r-package haplo.stats 1.7.7, respectively. results: the mean (sd) age of a total of 1341 children was 4.7 (1.0) years. forty-one percent of them had domestic exposure to cigarette smoking, whereas 17% and 11% had wheeze ever and current wheeze respectively. current wheeze was associated with rs6967330 and rs140154310 of cdhr3, with the respective odds ratios (ors) being 1.63 and 2.20. these two snps were also associated with frequency of wheezing illnesses over the past 12 months, with ors being 1.57 (p = 0.034) for rs6967330 and 2.14 (p = 0.024) for rs140154310. current wheeze was also associated with the 3-locus gac haplotype of cdhr3 (or, 1.54 and p = 0.048). two cdhr3, snps rs408223 and rs140154310 were associated with fev0.5, fvc and fev0.5/fvc, and the ggg haplotype was associated with fev0.5. a-174 | development and validation of an adherence questionnaire for adolescents with asthma on controller inhaled corticosteroids with asthma on ics at multiple hospitals and clinics in japan. adherence to ics medication was separately evaluated by "confidential" questions to ask frequency of "forgetting to take medicine" by nonmedical study staff. the best model to predict adherence was formed by multivariate logistic analysis. validation of the model was performed using answers to the questions from a separate group of ics-treated asthma patients of the same age. results: responses from 445 adolescents were used as development data set and those from separate 275 adolescents were used as validation data set. a 6-item logistic model was selected from the development data set. it showed good statistical fit and well discriminated poor adherence with auc at 0.814 and 0.759 in development and validation datasets, respectively. probability of adherence was calculated as propensity score in the logistic regression model and named as the pediatric asthma adherence questionnaire (paaq) score. the paaq scores for the physicians' ratings of adherence differed significantly in the hypothetical direction. the paaq may be a useful tool to evaluate adherence in adolescents with ics-treated asthma. a-208 | effect of long-term inhaled corticosteroid therapy on adrenal suppression growth and bone health in children with asthma years diagnosed as having asthma on inhaled corticosteroid therapy for more than 6 months were included as cases. children who were on oral steroids for exacerbations a week before the study, asthma with other chronic illnesses and children over 9 years old to avoid the confounding effects of pubertal growth acceleration seen at this age, were excluded. comparison group consisted of 70 age-matched children with asthma who were not on ics. heights were assessed according to their mid parental heights (mph). serum calcium, alkaline phosphatase and vitamin d levels were assayed in both groups. low dose short synacthen test was performed on cases and serum cortisol at 0, 30, 60 minutes of the test was performed to assess hpa axis function. peak cortisol level > 500nmol/l at 30 minutes was considered to have passed the test and exclude adrenal suppression. the average daily dose of ics was categorized as low, medium and high according to published literature. results: both the associations between long-term ics and growth (chi square value = 0.785, p = 0.376) and calcium levels (p = 0.88) were not statistically significant. a significant association was found between long-term inhaled corticosteroid therapy and alp level (p < 0.01) although the interquartile ranges of serum alp in both groups were within the normal range for the age. there was no statistically significant difference in vitamin d levels in both groups (p = 0.886), although vitamin d levels were deficient in 34% of cases and 41% of controls (< 50 nmol/l). interestingly suppressed cortisol levels were seen in 24% of cases. cumulative doses of ics in 70 cases were low, medium and high in 41%, 41% and 17% of children respectively. results: total antibiotic use appeared to have decreased 1 year after follow-up in a specialized asthma clinic. both the overall use (p 0.08) and the use of amoxycillin / clavulanate (p 0.01) were statistically significant. the use of amoxycillin, cephalosporins and macrolides was also found to be reduced. a statistically significant reduction was also found in the use of antihistamines (p 0.048). in terms of quality of life, a statistically significant improvement was found in the worry domain of asthma both in children (p 0.012) and their parents (p 0.013) as well as in the impact domain for parents (p 0.05). methods: in this prospective cross sectional study, we enrolled consecutive children between 5 to 15 years of age with poorly controlled asthma (partly controlled and uncontrolled as per gina guidelines) between july 2016 to march 2018. we excluded children with cystic fibrosis. the enrolled children were investigated for abpa that included total ige, aspergillus-specific ige, aspergillus-specific igg, skin prick test for aspergillus, serum precipitins, absolute eosinophil count (aec), chest x-ray, and chest ct (in selected patients). abpa was diagnosed as per recent criteria: if both of the following criteria were fulfilled: (1) total ige > 1000 iu/ml and positive skin prick test (spt) to aspergillus or aspergillus-specific ige > 0.35 kua/l; (2) at least of two of following three: presence of precipitating antibody or aspergillus-specific igg > 27 mg/l; chest radiology suggestive of abpa; and total eosinophil count > 500 cells/mm3. data were analyzed using stata 12.0. we used the receiver operating characteristic (roc) curve along with area under the curve (auc) to determine the utility of various parameters to differentiate children with abpa from those without. to determine best cut-offs, youden's index was used (sensitivity + specificity -1). results: we included 106 asthmatic children (male: female 72:34) with mean (sd) age of 10.2±2.6 years. the prevalence of abpa was 12/106 (11.3%; 95% ci, 5.2%, 17.5%). among baseline characteristics including spirometry of included children, only the presence of brownish sputum s88 | abstracts was greater in abpa children. among diagnostic criteria, all were significantly different between children with abpa and without abpa except aspergillus-specific igg and positive spt. the auc (95% ci) of rocs are shown in figure 1 . the difference between auc of total ige and aspergillus-specific ige vs. aspergillus-specific igg was significant. the sensitivity and specificity of total ige (>1000 iu/ml), aspergillus-specific ige (> 0.35 kua/l), aec (> 500/ mm3) and aspergillus-specific igg (> 27 mg/l) cut-offs as defined by diagnostic criteria was 100% and 65.9%, 75.0% and 79.8%, 91.7% and 57.0%, and 27.3% and 87.5% respectively. the best cut-off values as per youde's index of total ige, aspergillus-specific ige, aec, and aspergillus-specific igg were 1806 iu/ml, 0.63 kua/l, 786/mm3, and 16.8 mg/l, respectively with corresponding sensitivities and specificities of 75% and 76.6%; 75.0% and 88.3%; 83.3% and 74.2%; and 63.4% and 71.5%, respectively. conclusions: aspergillus-specific ige levels had the best discriminative value followed by total ige, aec, and aspergillus-specific igg for abpa in asthmatic children. the currently proposed cut-off values may not be appropriate for children. reflections and concrete proposals for action: there is need to develop childhood-specific diagnostic criteria for abpa. methods: this is a one-year retrospective study that was conducted in our pediatric intensive care unit (picu) comparing the intervention failure rate of three different noninvasive respiratory support modalities (bi-level positive airway pressure (bipap), continuous positive airway pressure (cpap) and hfnc) for infants and young children between the ages of 1 month and 2 years admitted with the diagnosis of bronchiolitis. a sample size of 137 patients was collected with a median age of 2 months. children who required hfnc were older (mean 4.5 months) than children who required bipap (2.7 months) while the mean age for children who required cpap was (2.8 months). results: hfnc carried a higher failure rate in comparison with the other two respiratory support modalities (50.6% for hfnc n39/77 vs. zero % for cpap n0/10% and 8% for bipap n4/50, p < 0.01). among the 39 patients who failed hfnc, (90%) were successfully shifted to bipap and weaned off later, while the other 4 were intubated and needed mechanical ventilation. on the other hand, all 4 patients who failed bipap were intubated and mechanically ventilated. no difference was found between the three groups in terms of gender or the causative virus. no respiratory complications or mortality was reported in the three groups. in the bipap group, oxygen requirement was significantly reduced at 24 hours from the start of the intervention and afterward in comparison with the hfnc group (p < 0.01 -0.02), although not statistically significant with the cpap group. no difference was observed in length of picu stay or hospital stay between the three groups. we did not observe any association between causative virus(es) and the number of er or pulmonology clinic visits for wheezing or recurrent bronchodilator use after discharge from the hospital. in the ba group, we found virus infection only in 25%, predominantly adenovirus, followed by rv and rsv. in 56% of the cases, streptococcus pneumoniae was confirmed in the throat swabs vs. only 33% for isolated moraxella catarrhalis. there were no other bacteria isolated in the ba group. in the cwc group, we found only in 10% of viral infections mainly hmpv, followed by adenovirus and rv. results: 105 children had been taking antibiotics before hospitalization while the remaining 180 patients were antibiotic naïve. in 44.9% of the cases, we could not prove etiological agent, while bacteria were confirmed in 41.6% and viruses in 16.2% (in 4.6%, we found combined virus and bacteria), and fungi were found in 2.5%. when dividing the cases according to prior antibiotic use, the distribution was: 38.8%, 48.3%, 15% and 2.2% for antibiotic naïve vs. 55.23%, 28.5%, 18.9% and 2.8% for the others respectively (p = 0.002). when looking at the bacterial isolates and prior antibiotic use, we did not find any significant difference regarding mycoplasma pneumoniae isolation (p = 0.36) although there was one for streptococcus pneumoniae (p = 0.015), mainly due to the standard guidelines to gps to start with penicillin antibiotic for pneumonia. as expected mycoplasma was isolated in older children, while streptococcus was mainly isolated in younger patients. mean age for children with bacterial pneumonia was 6.54 years, for those with viral pneumonia 4.34 years., for combined (viral+bacterial) 3.08 years. and for fungal pneumonia 10.42 years (p = 0.000). the values for crp were lower in cases with mycoplasma and in cases with non-compact infiltrate changes on x-rays (p = 0.002 and p = 0.000). almost 2/3 of the children have been immunized with pneumococcal vaccine (66%). the immunized patients had higher numbers of viral and lower numbers of bacterial isolates − 25.7% and 37.23% vs. non-immunized patients − 4.12% and 48.45%, respectively (p = 0.002). there was no difference in streptococcus pneumoniae isolation and vaccination status, but there was a major drop in mycoplasma isolates in vaccinated patients (6.9% vs. 32.98%, p = 0.000). for the patients with asthma only, we could not identify the microorganism in 14%, while in 48.5% and 51.2%, we found viruses and bacteria, respectively (co-infection was found in 23.26%). in 50% of children without asthma, we did not isolate the etiological agent and there was co-infection only in one case, while viral pneumonia and bacterial pneumonia was confirmed in 10.6% and 39.3% respectively (p = 0.008). all children but 3 were discharged healthy for mean 6.54 days in hospital stay. the mentioned 3 patients had severe complications and required surgical intervention. conclusion: in the future, we could expect more viral pneumonia with increasing vaccination coverage and maybe we should reevaluate our treatment guidelines. objective: to compare the need for pediatric critical care in a tertiary children's hospital with a diagnosis of community-acquired pneumonia (cap) or hospital-acquired pneumonia (hap). furthermore, we conducted a pilot study to evaluate the possible medical biomarkers which are associated with longer pediatric intensive care unit stay. methods: an observational, retrospective cohort analysis was conducted in children who were admitted to our tertiary children's hospital with a diagnosis of cap or hap. patient demographics, clinical characteristics, and comorbidity were collected between january 2012 and december 2013. the following prospective pilot study was conducted in children who were admitted to our pediatric intensive care unit (picu) due to pneumonia progression. we evaluated the clinical profiles and medical biomarkers. the primary endpoint was the duration of picu stay with associated predicting factors. results: a total of 548 patients with 598 episodes of pneumonia (310 males; 288 females) requiring admission to our children's hospital were included. the mean age at admission was 59.6 ± 1.95 months and the average length of stay was 11.4 ± 0.70 days. 530 episodes were identified as cap and the other 68 episodes were hap. patients with cap had significantly shorter lengths of hospital stay and duration of icu stay than those with hap (8.2±10.5 vs. 36.5±31.5 days, p < 0.001; 2±6.9 vs. 10±18 days, p < 0.001). the most common co-morbidities in cap were neurological diseases and atopy history. among the cap patients, 90 episodes (17%) led to icu admission during treatment course with the most common comorbidities being neurological diseases. however, in hap patients, cardiovascular diseases were the most common co-morbidities as well as those (38.2 %) who required picu care. the overall mortality rate was 3.8%, with the mortality rate being significantly higher in the hap group (p < 0.001). the pilot study included 8 children with the diagnosis of pneumonia in picu from jan 2015 to dec 2015. neither progressive ards mobility nor mortality occurred. the mean age at diagnosis was 37.5 ± 30.4 months. the average number of days of picu stay was 6.9 ± 4.7 days. the median duration of hospital stay was 14 days. patients were divided into two groups: picu stay more than 7 days and less than 7days. the values of pro-bnp, aado2, platelets, crp and ci in patients with picu stay more than 7 days showed a significant difference with those less than 7 days in the initial picu admission (p < 0.05). however, the values of thoracic fluid content (tfc) or even tfc corrected by cardiac output were not significantly different between the two groups. the levels of sputum 8-isoprostane and urinary 8ohdg revealed a trend of decreasing level after disease relief. conclusions: in this study, we found that (1) hap resulted in significantly longer lengths of hospital stay and picu stay than cap. the possible risk factors for the need of critical care are associated neurological disease in cap and heart disease in hap; (2) the possible biomarkers of pro-bnp, platelet, crp, ci, 8-isoprostane and 8ohdg may predict the duration of picu stay in our pilot study. these results not only help further our understanding of the risk of pneumonia in children who require critical care but also provide chances for better intensive respiratory care. background: pneumonia is a leading cause of respiratory morbidity and mortality in children younger than 5 years of age. 1 the incidence of severe cases of community-acquired pneumonia (cap) in low-and middle-income countries is still high. 2 although severe cap can be diagnosed by clinical features and chest x-ray, it could be useful to measure biomarkers to predict the outcome. the aim of the study was to determine the association between methods: ninety children aged between > 28 days to < 5 years hospitalized with diagnosis of severe cap in the pediatric department of west nusa tenggara province general hospital from january to october 2018 were enrolled. data on demographic and clinical characteristics, and laboratory examination were recorded. all subjects were treated in accordance with the hospital cap protocol and prospectively monitored until discharged. results: out of 90 subjects, 68% were ages < 1 year, 59% male, 87% passive smoke exposure, 56% lived in crowded environment, 62% came from low family income, 97% showed infiltrate on chest x-ray 81% with comorbidity and fe deficiency anemia was the most common (64%). c-reactive protein, esr, lc, and nlr were not significantly associated with hospital length of stay and duration of oxygen consumption (p > 0.05). after adjustment for crp, lc, nlr, and age, esr was found associated with mortality, with every increase in one log of esr decreasing the log odds of death about 3.3 (p = 0.043). conclusion: higher esr was associated with lower risk of death. however, none of the biomarkers were associated with hospital length of stay or duration of oxygen consumption. keywords: community-acquired pneumonia, outcome, children, biomarker. recurrent episode of bronchial obstruction were enrolled in this study. detailed history, physical examination, blood sample and nasopharyngeal aspirate (npa) collection were performed. the viral etiology of the respiratory tract infections was determined using polymerase chain reaction (pcr) and the concentration of ifn-γ in npa by elisa kits. results: the mean npa levels of ifn-γ in rsv (+) infants -4.3 (0-30.1) pg/ml was lower than rsv (-) infants -14.43(0-49) pg/ml, (p = 0.12). a gender difference in ifn-γ was detected with significant higher values in girls (or, 1.95; [ci] 0.85-4.26; p = 0.05). the cytokine ratio did not differ between infants with or without atopic status and family history of asthma. moderate-to-severe bronchiolitis in 7 cases (20%) was associated with lower ifn-γ level (or, 0.8; [ci] 0.64-1.13; p = 0.06), none of them required mechanical ventilation. decreased ifn-γ production correlated with the recurrent episodes of wheezing (p = 0.05). objectives: to test the hypothesis that high amplitude bcpap support after acute lung injury may have different effects on gas exchange efficiency and lung injury protection compared to high flow cpap (mimicking hfnc) support in rats with ali. methods: after normal saline lavage lung injury, all rats initially received high tidal volume mechanical ventilation (9 ml/kg) for 30 minutes, then were randomly divided into three groups: high amplitude bcpap group using the bubble technique with 135 degrees of expiratory limb (n = 8); standard bcpap group using the bubble technique with 0 degree of expiratory limb (n = 4); and high flow cpap (mimicking hfnc) group using the high flow technique (2 l/min, n = 8). all groups were killed 2.5 hours after bcpap or high flow cpap (mimicking hfnc) support. arterial blood gases, respiratory rate, peak inspiratory pressure (pip) and mean airway pressure (map) of rat lung during respiratory support, wetto-dry lung weight ratio, lung homogenate and/or bronchoalveolar lavage fluid tumor necrosis factor-α, macrophage inflammatory protein-2, interleukin-6 and total protein levels were measured and compared among groups after study completion. results: the high amplitude bcpap group exhibited a significantly higher pao2, lower paco2 and significantly lower alveolar protein, pip, map, wet-to-dry lung weight ratio and cytokine level compared to high flow cpap (mimicking hfnc) group. high amplitude bcpap group also exhibited a lower cytokine level compared to the standard bcpap group. no difference in gas exchange efficiency was observed between the two bcpap groups. conclusion: high amplitude bcpap support decreases lung inflammation, increases gas exchange efficiency and lung compliance compared to high flow cpap (mimicking hfnc) support in rats with ali, and may have a better lung protective effect than standard bcpap. keywords: bubble continuous positive airway pressure, high-flow nasal cannula, respiratory support, acute lung injury. nontraumatic chylothorax in children is rare and more difficult to treat than traumatic chylothorax. in some cases, they are refractory to conventional treatment, leading to severe morbidity and mortality. thus, the purpose of this study is to analyze the clinical features of chylothorax in pediatric patients in our hospital and seek appropriate therapeutic management. methods: a retrospective review was performed in 63 patients with chylothorax from january 2000 to december 2018 in the children's hospital of seoul national university. traumatic or postoperative chylothorax was excluded. a total of20 patients with nontraumatic chylothorax were included in our study. etiology, treatment, and outcome of chylothorax were analyzed. results: nontraumatic chylothorax was diagnosed in 20 patients. male patients (14/20 = 70%) were more frequently affected than female (6/ 20 = 30%) patients. eighteen patients were diagnosed before 1 year of age (90%), only two patients were diagnosed after 1 year of age (6 years old, 12 years old respectively). the most common cause of spontaneous chylothorax was idiopathic factors, constituting 45.5% (13), three cases were related to high central venous pressure due to venous thrombosis and recurrent sepsis, 2 cases were related to down syndrome, 1 case was noonan syndrome, and the remaining case was gorham stout syndrome. seventeen patients needed a respiratory support device, 6 of the latter received low flow oxygen supplementation, 11 patients received ventilator support. dietary modification (npo or mct base feeding), conventional medication (somatostatin or octreotide), sirolimus, surgical management were administrated to our patients. in the neonate and infant group, three patients who were related to venous thrombosis died because of recurrent septic shock before chylothorax management was administered. fifteen patients received a dietary modification (npo or mct base feeding) and nine patients improved by conservative management. one patient died due to heart failure before medical treatment. somatostatin or octreotide was used in 5 patients who failed dietary modification, but only one patient improved with octreotide. among the somatostatin or octreotide failure group, 4 patients received surgical management (pleurodesis or thoracic duct ligation). three of these patients improved, although one patient died after thoracic duct ligation operation because of post-op ards. two patients who were diagnosed after 1-year of age were refractory to nutritional modification and conventional medication such as somatostatin or octreotide. however, lymphatic intervention and surgical treatment were not suitable for these two patients. considering that their underlying disorder consisted of noonan syndrome and gorham stout syndrome, we used sirolimus to treat the refractory chylothorax. after administration of sirolimus, their chylothorax improved compared to before. conclusions: most of the nontraumatic spontaneous chylothorax in pediatric patients occur in newborns and the most common cause of chylothorax in the neonatal and infantile period is idiopathic. on the other hand, nontraumatic chylothorax in childhood is rare and tends more to be accompanied by the underlying syndrome. moreover, the treatment failure rate is higher in the childhood group. in such cases, sirolimus which is an mtor inhibitor, can be beneficial to patients who tend to be refractory and cannot be treated with lymphatic intervention or operation. purpose: specific genetic causes for childhood interstitial lung disease (child) in immunocompetent patients have been identified within the past decade. however, little is known about the pathogenesis of many forms of child, and treatment approach has not been standardized. a national survey was carried out by the japanese society of pediatric pulmonology (jspp) to identify the histopathology and response to current treatment, especially hydroxychloroquine, which is contraindicated for children younger than 6 years old in japan. methods: a questionnaire was sent to pediatricians who registered a child patient to the jspp. we conducted the survey over a period of 9 years, between 2010 and 2018. children (0-15 years) were included in the survey with persistent hypoxemia (pao2 less than 60 torr or spo2 less than 90%) for more than 2 weeks, diffuse infiltrates on ct scanning, and elevated serum markers such as kl-6, sp-a, or sp-d. immunodeficiency and other diseases which present with similar symptoms to child were excluded. the questionnaire included information on the patients' clinical symptoms, family history, pathological histology, clinical genetic findings, treatments and clinical outcomes. informed consent was obtained by all patients' guardians before participating in the survey. results: twenty-six cases were identified, including 15 males and 11 females. age of onset was between 0 months and 8 years. fourteen (53%) cases presented in the first year of life. lung biopsy was performed in 11 (42%) cases. five cases showed changes of nonspecific interstitial pneumonia (nsip), 1 case with desquamative interstitial pneumonia (dip), 1 case with cryptogenic organizing pneumonia (cop), 1 case with usual interstitial pneumonia (uip), 1 case with acute lung injury (ali), and 2 cases untagged. genetic testing was performed in 21 (80%) cases. mutation in sp-c gene (sftpc) was detected in 8 cases, atp binding cassette subfamily a member 3 (abca3) in 1 case, nk2 homeobox 1 (nkx2-1) in 1 case, coatomer associated protein subunit alpha (copα) in 1 case, and no mutation was detected in 10 cases. in the first half of the study period, only 5 out of 16 (31%) cases were diagnosed with genetic testing without lung biopsy, which increased to 8 out of 10 (80%) cases in the latter half. prednisolone was used in 24 (92%) cases and hydroxychloroquine in 20 (76%) cases with no onset of retinopathy. conventional treatment with prednisolone or hydroxychloroquine, monotherapy or in combination, resulted in a good response in 17 (65%) cases. three (11%) children died despite all therapies. in addition, it turned out that 1 case was diagnosed as juvenile idiopathic arthritis 4 years after registration. conclusions: this is the first nationwide prospective study regarding child in japan. the histopathology in this study is similar to that reported previously. there is increasing emphasis on genetic studies in the diagnosis of child as it can help avoid unnecessary lung biopsy. corticosteroid and hydroxychloroquine were the main therapeutic agents in our study. hydroxychloroquine therapy was tolerated in many cases, with no significant side effects. methods: twenty three children aged 11.8±4.9 years with bos after allogeneic hematopoietic cell transplantation were enrolled, and their clinical data were reviewed retrospectively. all subjects repeated the pulmonary function test at an interval of 1 month after occurrence of results: among 23 subjects with bos, 6 (25.0%) subjects expired due to respiratory failure, 4 (17.4%) subjects underwent lung transplantation, and 16 (69.6%) subjects needed o2 therapy. the mean value of fev1% predicted at the diagnosis of bos was 37.0±13.0%, and it rose after 12 months (47.0±24.9%). fev1 % predicted at diagnosis of bos tended to be lower in subjects with oxygen therapy (34.7±12.2) than in subjects without oxygen therapy (45.8±11.1), [yj1] although there was no statistical significance. the changes in fev1 % predicted at 3 months after bos diagnosis were significantly lower in the subjects with oxygen therapy (-19.4±24.3%) than in subjects without oxygen therapy (8.6±21.9%). however, there was no significant difference in the change over 3 months of fev1% predicted values between the two groups at 6, 9, and 12 months. in addition, the group with a negative slope of fev1% predicted change during the first 3 months had a higher likelihood of o2 therapy, compared to the group with a positive slope of fev1 change during the period (hr of 3.57, p = 0.059). the change in fev1 during the first 3 months after bos was significantly different between the subjects with and without oxygen therapy. these results suggest active intervention strategy is needed during the first 3 months after bos to improve the prognosis. this study is aimed to assess the effectiveness of hs nebulizations before physiotherapy over conventional physiotherapy in children with non-cf bronchiectasis. objectives: primarily to compare the change in fev1 from pretreatment phase to posttreatment phase between the two groups. method: we performed a retrospective cohort study of extremely preterm infants born before 28 weeks of gestational age in our nicu. study subjects were divided into two groups: auto group who underwent the automated control of inspired oxygen concentration (auto) with cpap, and manual group who underwent manual control of inspired oxygen concentration (manual) with cpap before introduction of auto in our nicu. we retrospectively investigated the patient characteristics and short-term outcome related to the respiratory system (reintubation rate, duration of cpap, introduction rate of home oxygen therapy) and retinopathy of prematurity (incidence, stage, and therapy rate). the mann-whitney u test and chi-squared test were used for statistical analysis, and logistic regression was used for multivariable analysis. result: a total of 47 infants were eligible for this study. there were 25 and 22 infants in the auto group and manual groups, respectively. in the auto and manual groups, the median (range) of their gestational age in weeks was 25.1 (23.3-27.9) and 25.5 (23.7-27.9, p = 0.25), birth weight in grams was 690 (461-990) and 789 (442-1178, p = 0.02), the rate of maternal steroid administration was 73% and 36% (p = 0.01), age at study entry in days was 2 (54-48) and 31 (2-67, p = 0.31). reintubation rate was 56% and 50% (p = 0.68), duration of cpap (day) was 28 (6-62) and 26 (3-38, p = 0.29), and introduction rate of home oxygen therapy was 20% and 27% (p = 0.56). the incidence of total retinopathy of prematurity (rop) was 64% and 77% (p = 0.32), the incidence of rop over stage ⅱ was 24% and 59% (p = 0.02), and therapy rate for rop was 8% and 14% (p = 0.53) in each group. there was a statistically significant association between the decreasing risk of rop over stage ⅱ and use of auto by multivariable analysis adjusted for confounding factors (odds ratio: 0.22 [95% ci: 0.06-0.76], p = 0.02). discussion: in this study, the incidence of rop over stage ⅱ was significantly decreased in extremely preterm infants who underwent auto with cpap. it is suggested that exposure to the excessive oxygen levels and the fluctuation of oxygenation are related to the incidence of rop. large studies indicate the strict spo2 target range. but in fact, the manual control of inspired oxygen concentration during respiratory support is not sufficient for the maintenance of spo2 within the latter. auto can maintain spo2 within the target range compared to the manual control of inspired oxygen concentration. auto has the potential to prevent rop by decreasing the exposure to excessive oxygen levels and the fluctuation of oxygenation. several limitations should be considered in our study. this was a retrospective, single-centered, non-randomized study; a further multi-centered prospective study is needed. since their pulmonary function is decreased, lower respiratory tract infections in this population may have a more severe clinical course. in our study, in addition to lung function measurement, we also wanted to see whether there is a connection between mental abilities and pulmonary function. methods: before the examination, a license was obtained from the ethical committee of the university of szeged. conclusions: we could also detect in our smaller population that vlbw patients at the age of 6 to 8 years have lung function test values in the lower normal region of the reference range. this may raise the presumption of the higher susceptibility of these children to respiratory tract diseases also in the later decades of life. bpd patients have lower pulmonary function test results than non-bpd patients. on the other hand, we could not find any connection between low raven test performance and lung function results. background: esophageal atresia (ea) and/or tracheoesophageal fistula (tef) is one of the rare congenital anomalies occurring in 1 out of 3,000-5,000 births. there has been improvement in the survival of these infants during recent decades. the diagnosis of tef with ea is commonly made during the first 24 hours after birth. preoperative flexible endoscopy (fe) is not yet routinely included in the diagnostic and postoperative assessment. this study aimed to evaluate the predictive factors that affected patients' prognosis and the role of flexible endoscopy application in managing infants with ea and/or tef in a tertiary medical center. methods: we enrolled patients who were admitted into our hospital due to suspected ea and/or tef and accepted an fe examination for one or more times between jan. 2000 and dec. 2017. all associated medical and surgical records were retrospectively reviewed. the analyzed data included basic characteristics, diagnosis, age of surgical repair, associated anomaly, timing of fe before and after surgical repair, and mortality. factors related to patient's mortality were analyzed. results: a total of 33 patients were enrolled, including 28 (84.8 %) cases referred from other hospitals. their mean birth weight was 2448 ± 603 gm, including 19 (57.6%) low-birth-weight infants, 17 (51.5%) cases with cardiac anomalies, 12 (36.4%) cases aged > 90 days, and 12 (36.4%) cases underwent fe before reconstruction. the most common classification of enrolled cases was type c (84.8%). additional other airway anomalies were found in 23 (69.7%) cases, including tracheomalacia, bronchostenosis, lung hypoplasia, and laryngeal cleft. one case underwent nasaltracheo-fistula-gastric catheter insertion before surgery. the mean age of receiving surgical reconstruction was 5 ± 7 days. the most common postsurgical complication was anastomotic stenosis (25, 75.8%) that required laser therapy (9, 27.3%), balloon dilatation (17, 51.5%), or stent implantation (2, 6.1%). gastroesophageal reflux was also commonly found in 21 (63.6%) cases. the overall 2-year survival rate was 72.7% (27/33). significant factors related to 1-year mortality were post-reconstruction referral (p = 0.004), age of reconstruction > 7 days (p < 0.001), and cardiovascular surgery requirement (p = 0.032). conclusions: in infants with ea and/or tef, fe is feasible for the early identification of associated airway and esophageal anomaly, as well as postoperative diagnosis and therapeutic interventions. postreconstruction referral, age of reconstruction > 7 days, and cardiovascular surgery requirement were significantly related to 1-year mortality of infants with ea and/or tef. background: patent ductus arteriosus (pda) is a common cardiovascular complication among premature infants and may be responsible for prematurity-related complications. surgical ligation is considered when medical treatment has either failed or was contraindicated. transcatheter occlusion, which was established in term infants, has recently been applied to premature population. previous reports stated that the complication and success rates were not statistically different between the transcatheter technique and surgical ligation. in this study, our aim was to compare the oxygenation status and oxygen dependence between these two techniques. the basic demographic data, fio2 change, pulmonary score, intubation days, ventilator-dependent days, oxygen-dependent days and mortality within 1 year were evaluated. results: the birth body weight, gestational age, post-menstrual age on procedure day, body weight on procedure day, pulmonary score and fio2 before procedure were not different between these two groups. the range of body weight on procedure day was from 478 to 1602gm in group a and from 551 to 1646gm in group b. the overall mortality within 1 year was similar (p = 0.360). the overall incidence of chronic lung disease was not significantly different (2/10 vs. 8/16, p = 0.218). when comparing the fio2 change before and 5 days after the procedure, the transcatheter closure group had a significant improveare rare disorders of which the incidence is not precisely known. this malformation might threaten the fetus, but it can also disappear spontaneously, or neonates can be asymptomatic with the malformation. according to previous studies, cpam may increase the risk for lung infections and lung malignancies, which is also the argument used when treating asymptomatic cpam-patients with surgery. purpose of the study: the purpose of the present study was to investigate whether there are differences between the treatment protocols between nordic countries. material and methods: a questionnaire was sent to 16 nordic centers dealing with pre-and postnatal management of patients with congenital lung malformation. this questionnaire was aimed to collect information on pre-and postnatal treatment protocols in the centers involved. results: the treatment protocols of this malformation vary largely in nordic centers. prenatal ultrasound was the primary examination in all centers. magnetic resonance imaging (mri) was used routinely as next prenatal examination in four centers for every cpam-patient with persistent finding in ultrasound during whole pregnancy (4 of 11). five centers (5 of 11) used mri if needed for differential diagnosis. various prenatal interventions were used in cases of fetal hydrops caused by cpam. shunting macrocystic lesion was used in 4 out of 11 centers, maternal cortisone in 9 out of 11. over half of the centers (7/11) consulted and co-operated with centers that performed fetal surgery and 3 out of 11 did consider ex utero intrapartum (exit) surgery. postnatally surgery was performed in every center (100%) for symptomatic cpam-patients. one center (14%) did not remove cpam but instead performed follow-up on asymptomatic patients. four infants used precision flow: three used single prong cannula (tip od1.9 mm), and the remaining infant used double prong, infant cannula (tip od1.9 mm). pharyngeal pressure tended to rise to flow rate dependence with any type of cannula. in patients managed with the optiflow junior, the mean pharyngeal pressure at the flow rates of 1, 2, 3, and 4 l/kg/min for premature size cannula were 2.3, 3.0, 3.1, and 3.6 cmh2o, respectively, and for neonatal size cannula were 0.5, 1.9, 3.0, and 3.7 cmh2o, respectively. in patients managed with precision flow, the mean pharyngeal pressure for each cannula at flow rates of 1, 2, 3, and 4 l/kg/min for single prong cannula were 1.8, 3.9, 4.7, and 5.0 cmh2o, respectively, and for double prong, infant size cannula were 2.6, 5.7, 6.0, and 7.0 cmh2o, respectively. background: even though it is considered as a screening test, sweat conductivity (sc) analysis seems to be an alternative diagnostic method to the coulometric quantitative test (cqt) for the diagnosis of cystic fibrosis (cf). it is widely accepted that coulometry requires specialized technicians, in addition to being performed only in referral centers. on the other hand, sc sweat analysis is a semiautomated procedure, simpler and faster than the conventional cqt. specially in poor-resource settings, it allows the decentralization of the diagnostic network and, consequently, a wider accessibility to cf diagnosis. to date, there are no studies comparing two concomitant conductivity tests performed in the same patient. objective: to assess the agreement between two sweat conductivity results performed concomitantly in young infants. methods: this was a prospective, cross-sectional study in which two sweat samples were obtained from the two arms among 100 consecutive patients, using the wescor macroduct collection system conclusion: since the strength of agreement was perfect, it reveals a high reliability of sc. apart from its role as a screening test, it seems that it has a place as a diagnostic tool in cf. background: because of frequent exacerbations and pulmonary deterioration in cystic fibrosis (cf), the radiological exposure to chest x-ray and ct scan is frequent in patients with cf, with subsequent potential secondary effect, although also required. lung ultrasound is, abstracts | s103 currently, a useful method of evaluation in multiples diseases such as: pneumonia, pleural effusion, interstitial syndrome or pulmonary fibrosis; therefore, the use of lus in cf would be of interest aim of the study: evaluation of thoracic ultrasound utility in cf exacerbations. methods: fifty cf patients were included in the study and monitored for 2 years of age. lung ultrasound was performed every 3 months, at clinical and biological evaluation. ct scan was performed during exacerbations and for stable patients, every 2 years timetable (aged over 8 years). ultrasound was performed using a linear high frequency 8 to 12 mhz probe, using a score based on specific artifacts which quantified the presence of consolidation, interstitial syndrome, saccular bronchiectasis and pleural effusion. ct was interpreted using the bhalla scoring system, independently of the lus score. pearson's correlation was used for the evaluation of the relationship between lus score and ct. results: median ct bhalla score was 14.3 + -4.5, and the average lus score = 5.13 + -2, consistent for moderate morphological lung injury. a good correlation was found in patients with increased lus (> 4) and ct score, r= 0.75, p < 0.001. there was no reliable correlation between the lung ultrasound score and ct, therefore, validation of the lus-cf score (r= 0.37, p = 0.26) was impossible with the currently identified artifacts. for patients in acute exacerbations, alveolarinterstitial syndrome described by multiple b-line artifacts and the presence of lake signs quantifying cystic bronchiectasis were accurately identified by lus and confirmed by ct. methods: we conducted a retrospective descriptive study at the university hospital of montpellier between 30/08/2000 and 30/08/2018. we collected the various respiratory disorders presented during the follow-up of these children. results: one hundred and two files were examined and 76 patients were included in our study. asthma concerned 26 patients in our cohort (34%) which is more frequent than in our general population (9% in occitania region, france). it was significantly related with atopy (p < 0.01). respiratory tract disorders were identified in 10 patients (13%). the most common were: tracheomalacia and laryngomalacia. lower respiratory infections were found in 18 patients (24%) and were significantly related to lymphopenia (p < 0.05) and swallowing disorders (p < 0.05). infants were tested for hiv using pcr and antibody testing. the association between hiv and arv exposure and lung function during 2 years was assessed using linear regression, adjusted for bmi for age z-score, sex, ethnicity, socioeconomic status (ses) abstracts | s105 quartile at enrolment and pre-and postnatal smoke exposure (based on urine cotinine). results: a total of 1036 infants had at least one lung function measurement and were followed over 2 years, 226 (22%) were heu; 535 (52%) male, 560 (54%) black african ancestry, 330 (33%) mothers smoked during pregnancy, 775 (71%) household tobacco smoke exposure. nine hundred and ten (88%) infants had lung function tested at 6 weeks and 743 (72%) children at 2 years. the majority of heu infants were black african (93% vs. 43% hu, p < 0.001), heu infants had less household smoke exposure (69% vs. 81%, p = 0.01), lower ses (p = 0.001) and had higher bmi zscore at 2 years (p = 0.001) compared to hu; other demographics were similar. at 6 weeks, heu infants had higher tidal volume compared to hu (1.1ml, ci, 0.02; 2.2, p = 0.045). amongst the heu infants, those whose mothers had triple therapy arvs had lower expiratory flow ratios, tptef/te, compared to those whose mothers had zidovudine (azt) only (-0.26, ci -0.4; -0.1, p < 0.001). at 2 years, tidal volume differences were no longer seen, but heu infants had a higher lung clearance index compared to hu (0.12, ci, 0.02; 0.23, p = 0.019). low antenatal maternal cd4 count was associated with an average 6.5ml lower tidal volume at 2 years compared to infants whose mothers had cd4 counts> 500 cells/ mm3 (ci, 1.0, 12.1; p = 0.02). background: chronic wet cough, which is prevalent in indigenous children, is the most common symptom of bronchiectasis and its precursors. early recognition of chronic wet cough leading to prompt and appropriate treatment may potentially prevent progression to bronchiectasis. therefore, timely health seeking for chronic wet cough by families, and its effective management by health practitioners is potentially important to prevent development of bronchiectasis. purpose: to identify the barriers to and enablers for timely health seeking for chronic wet cough by families, and optimal management by health practitioners, for chronic wet cough in aboriginal children. method: a qualitative study was conducted in two communities (one remote town and one very remote community) in the kimberley region of northern, western australia, of which 43% of the population is aboriginal. data were gathered through individual semi-structured in-depth interviews and focus groups to ascertain: the key enabler described was the provision of culturally appropriate health literacy information. all participants reported that they would seek help for chronic wet cough once they were informed that it could signify underlying disease. furthermore, families suggested that improved health practitioner knowledge of chronic wet cough database. complete monitoring data for the air pollutants (so2, no2, o3, co, pm2.5 and pm10) and meteorological factors (relative humidity, rainfall, and daily average temperature) were collected from 74 environmental protection agency monitoring stations of the taiwanese government. in the present study, each er visit for asthma ae was defined as case day. in bidirectional control samplings, the same weekdays 1, 2 or 3 weeks before and after er visit for asthma were defined as the control days. multiple correlation coefficients (r) (multiple regression analysis) were used to explain how much of the variance in the ed visits could be explained by a given set of air pollutants. result: as the study cases were divided as male and female, a 1 ug/m3 increase in the 48-h averages of pm2.5 and a 1℃ increase in temperature were associated with asthma er visit [or= conclusion: in taiwan, asthma ae is closely related to low temperature and indicated air pollution. there is an obvious bias if only a single air pollutant is being considered and neglects the influence of meteorological factors in studying the effects of the environment on asthma. with the result of this study, we can predict asthma exacerbation precisely according to individual age, gender and local air pollution/ meteorological conditions. in days with high risk for asthma exacerbation, patients with asthma should avoid/decrease outdoor activity, dress warmly and maintain inhaled corticosteroid therapy. however, while the path length of the right dorsum manus was not statistically significant, there was a significant difference in completion time between the two groups. the reason considered was that their left hand was their nondominant hand, while their dominant hand needed to be kept clean to touch an asepstic suction catheter. therefore, novice participants were unfamiliar with manipulating suctioning equipment with their left hand, which appeared as a difference in their proficiency. benefits of 3d cg were reported that the function of a virtual camera improved the accessibility to aois by screen transition, whereas operation was difficult, so they could not realize the effect of learning. advantages of 2d video was described that it is easy to concentrate because the device was familiar, while there was a negative impression that it was hard to observe aois compared to 3d cg. there was no significant difference between the groups who watched 2d video or 3d cg animation in the frequency distribution of fixation point movement speed analysis. however, considering the unignorable potential of 3d cg animation as a training tool, we concluded that defining visual criteria for assessing learning outcome of digital contents requires more research in the future. graduate school of nursing, the jikei university school of medicine -tokyo, japan the aim of this study is to compare the level of endotracheal suctioning proficiency between experts and novices, using an eyemark recorder. a head-mounted eye-tracking device (pupil, pupil lab, usa) was used to capture eye movement of the participants. twelve experienced nurses from three healthcare facilities who had more than 3 years of experiences of suctioning, and twelve nursing students from a university who had no experience of providing suctioning for real patients, participated in a simulation scenario of a patient with endotracheal intubation. we utilized cephalothorax realia with a simulated upper respiratory tract from mouth, nose, to a tracheal bifurcation, which did not represent any biological/patholoshowed that 57.3% of students are exposed to environmental tobacco smoke, which causes inflammation of respiratory tracts and a decrease in lung function. urinary cotinine can be used as a biomarker for cigarette smoke exposure. objectives: to examine the effects of environmental tobacco smoke exposure on urinary cotinine level and lung function test in children. methods: subjects were students aged 11 to 16 years-old in jakarta. data were obtained from questionnaire, spirometry and urinary cotinine test using elisa method. results: there were 92 subjects, consisting of 46 in the case group and 46 in the control group. urinary cotinine level > 10 ng/ml was found in 37.0% of the case group and 4.3% of the control group; p = 0.000; or= 8.50 (ci, 95% 2.08-34.71). there were significant differences between urinary cotinine level with number of smokers (p = 0.027) and number of cigarettes per day (p = 0.037). no association was found between cigarette smoke exposure and lung function test. there was a significant difference in school absenteeism between the case group and control group; p = 0.004; or= 6.00 (ci, 95% 1.42-25.33). conclusions: children exposed to environmental tobacco smoke there are relatively little data in healthy children and no data that compared these techniques. in 100 children without respiratory disease, we described (i) the bal differential cellular count and its correlation with age, and (ii) compared the differential cytology versus b-bal (10%, iqr 4-13%)). however, when adjusted for age using regression statistics, these differences were not significant. there was no significant difference in the total cell count (nb-bal 9.3x104/ml, iqr 4.5-15.3x104/ml versus b-bal 11.8x104/ml, iqr 8-22.1x104/ml) and neutrophil percentage (nb-bal 4.5%, iqr 2.8%-7% versus b-bal 3.5%, iqr 1.8-8%) between the two groups. there was no significant difference in the differential cytology with age in both b-bal and nb-bal. conclusion: nb-bal provides comparable information on the cellularity components of bal when compared to b-bal and should be considered an alternative. as age influences cellular differential count, age-matched data are required for comparative studies on bal in children. j-99 | spirometry assessment in children with congenital heart disease after open heart surgery in these spirometry assessments, obstructive type was more common than restrictive type (31.5% vs. 20.7%). however, in tof patients, restrictive type was slightly more common than obstructive type. j-115 | to squeeze or not to squeeze? can the raised -volume passive expiration flow-volume curve be used to estimate pulmonary functions in infants? background: spirometry is an essential tool for assessing patients with lung disease. however, it necessitates cooperation and thus, it cannot be performed in infants. one of the established alternative techniques in infants is the raised-volume rapid thoraco-abdominal compression technique (rvtct) which uses a compression vest to squeeze the chest forcedly after inflating the lungs to near total lung capacity (tlc). although it is an acceptable method, it is far from being perfect since it is not easy to perform. obtaining the raisedvolume passive expiration curve (rvpe) from passive deflation of the lungs from tlc is an easier technique (figure 1) . however, to our knowledge, data obtained from the rvpe curve were not assessed for their ability to estimate expiratory airway function in infants. tst was considered positive if induration > 10 mm and qft-git however, anesthetic choice as a risk factor for fever has not yet been reported. objectives: the aim of our study was to explore the role of sedative agents as a risk factor for fever during the 24 hours after the procedure. additional risk factors were investigated as well. multivariate analysis of the data (comorbidities, sedation choice, age, indication for performing the procedure, bal performed and its findings, post-prematurity, ftt, and medications) suggested that only sevoflurane and young age were statistically significant risk factors for fever. chronic treatment with montelukast was found to be a protective factor against fever. we conclude that post-bronchoscopy fever is probably an inflammatory noninfectious process. sevoflurane is a significant risk factor for developing post-bronchoscopy fever by generating such inflammation. the same mechanism may also explain why montelukst has a protective role. conditions were mostly present in young children less than 5 years old while obesity was more common in adolescents. of the treated patients, 75.6% had allergic rhinitis. comparing symptoms before and within 6 months after pap trial, there were significantly less patients with complaints of habitual snoring (95.6% vs. 8.9%, p < 0.01) and excessive daytime sleepiness (42.2% vs. 11.1%, p < 0.01). there was significant improvement in psg parameters with mean oahi decreasing from 21.5±18.5/hour tst to 1.6±1.9/hour tst (p < 0.05) and mean spo2 (oxygen saturation) nadir increasing from 80.6% ±10.4% to 90.9%±5.7% (p < 0.05) after pap therapy. within 6 months of follow up, 57.8% of the patients adhered to pap satisfactorily while the adherence dropped to 37.8% at the time of the latest follow-up. satisfactory usage established before 6 months was associated with favorable long-term outcomes of satisfactory usage or cessation of therapy due to improvement at the latest follow-up (chi square test p = 0.008). the commonest reported reason for non-adherence was due to nasal obstruction. results: forty-three bemss were placed in 26 infants. the mean bw and mean age were 4.0 ± 0.8 kg and 4.9 ± 2.7 months, respectively. there were 21, 9 and 13 stents located in trachea, carina and mainstem bronchi, respectively. seven infants with 13 stents died without obvious stent-related mortality. among them, 12 stents in 8 children were successfully retrieved by rigid endoscopy. at placement, the diameters of 30 tracheal and 22 bronchial stents were 7.5 ± 1.1 (4-10) and 5.4 ± 0.9 (4-8) mm, respectively. these implanted bemss could be gradually and significantly (p < 0.01) expanded. at the end of this study, all the remaining 18 stents in 12 infants could be kept functional. the diameters of the 11 remaining tracheal and 11 remaining bronchial stents were 9.7 ± 2.0 (8-14) and 7.0 ± 1.4 (4-10) mm, respectively. results: eithty -three children were included. males represented 48.5%, average age was 31.7 ± 4 months. the rate of ards was 39.8%. the mortality rate in children with this syndrome was 78.8%. septic shock with ards had a higher mortality rate than septic shock without this syndrome (26%). most children with this syndrome were in the moderate and severity range (78.8%); 81.8% were ventilated. the tidal volume was 8.3 ± 1.6 ml/kg and average peep was 8.6 ± 2.2 cmh2o, the medium fio2 was 90.7 ± 16.4 %; 36.4% of cases had a tidal volume of 8 to 10 ml/kg. there were no differences in tidal volume, peep and fio2 between survivor and non-survivor cases. conclusions: ards had a high prevalence in pediatric septic shock and the mortality rate among septic shock cases with this syndrome is still very high. according to the unit policy to transfer to icu which is based on idsa guidelines, 2% of the patients needed intensive care. hdu care was given for 38% of patients who required supplemental oxygen to maintain pulse oximetry above 92%. analysis of length of hospital stay indicated that 25% of the patients were discharged by the 4th day and 61% were hospitalized for 5 to 9 days. there was no mortality among the patients evaluated. conclusions: this retrospective analysis revealed that childhood community-acquired pneumonia is associated with significant morbidity and is an economic burden. the majority (65%) needed therapy beyond first line antibiotics which may indicate significant antibiotic resistance. central sleep apnea (csa) and periodic breathing are unusual findings described in pediatric patients with congestive heart failure. however, csa has not been reported in children with pulmonary hypertension. we hereby report on a 10-year-old girl with idiopathic pulmonary arterial hypertension (ipah) who had frequent central events in a periodic breathing fashion seen in her polysomnography, which was normalized following medical treatment leading to improvement of the pulmonary pressures. this case supports the importance of psg in pediatric patients with ipah, not only to exclude osa as a potential cause but also to assess for the presence of pb. we also show that the presence of pb might be a sign of disease severity and can be a marker of response to we present two children from a tb endemic region, with microbiologically confirmed tb presenting with parapneumonic effusion containing chyle, that were misdiagnosed initially as pleural empyema. tuberculous pleural effusion occurring with chylothorax is uncommon. the first case is a 12-year-old girl who presented with localized left-sided chest pain and parapneumonic effusion and a previous liver transplant and long-term intravenous catheter, who was on chronic immunosuppressive therapy. the second case was a 10-year-old boy who was hiv-exposed but uninfected and presented reports, to our knowledge, on using gastric lavage pcr to diagnose pjp. we report two cases in which p. jirovecii was identified by gastric lavage pcr and was treated appropriately. case 1 was a 1-year-old preterm, low-birth-weight male infant with down syndrome who had undergone operation for necrotizing enterocolitis and was being administered central venous nutrition. he was started on prednisolone for pericardial effusion because of postpericardiotomy syndrome when he was 7 months old and had been in an nicu for a year. on the day of onset of pjp, he required increased oxygen levels, and infiltrative shadows were observed in both lung fields on chest radiography. further, his (1→3)-d glucan (bdg) levels were elevated. p. jirovecii was detected on gastric lavage polymerase chain reaction (pcr), and trimethoprim-sulfamethoxazole was administered for 3 weeks, following which his condition improved. case 2 was an 8-month-old preterm, very-low-birth-weight male infant who was under central venous nutrition because of digestive tract disease. he was under treatment with hydrocortisone for 5 months because of refractory hypoglycemia and had been in an nicu. on the day of pjp onset, he showed increased oxygen demand, and chest radiography showed infiltration in both lung fields. further, his bdg level was elevated. p. jirovecii was detected on gastric lavage pcr, and trimethoprim-sulfamethoxazole was administered for 3 weeks, following which his condition improved. discussion: about 10%-30% of immunocompetent children carry p. jirovecii in their respiratory tract, and this percentage, depending on the underlying disease, is 60%-70% in immunosuppressed patients. therefore, when p. jirovecii is detected, it is important to distinguish infection from colonization. in these two cases, we suspected pjp on the basis of the clinical symptoms, backgrounds, bdg levels, and observations from image examination, and p. jirovecii was detected on gastric lavage pcr analysis. the patients' condition improved after definitive therapy. to our knowledge, there has been no report of pjp diagnosis by gastric lavage pcr to date, and this diagnostic technique may be useful if it is difficult to collect lower respiratory tract specimens. both patients had cellular immunodeficiency because of long-term steroid administration, although prophylactic treatment with trimethoprim-sulfamethoxazole for pjp had not been administered. this suggests the necessity of prophylaxis. we are reporting a 7-month-old boy, who was previously hospitalized with recurrent monthly pneumonia since birth until presentation to a regional tertiary center with pediatric respiratracheal agenesis (ta) is extremely rare and usually fatal. complete or partial absence of the trachea below the larynx can be found, and a tracheoesophageal fistula may exist. there is usually no prenatal symptom, but other congenital anomalies are commonly found. we present a low-birth-weight male infant (gestational age 36 weeks; birth weight 2100g) born with respiratory distress. physical and radiological examination disclosed single umbilical artery, bilateral malalignment of thumbs, sacral dimple, and hemi vertebra. difficult intubation was noted soon after birth. after being transferred to our center with an intubated endotracheal tube, flexible bronchoscopy was performed and found it was an esophageal intubation. a blind pouch at the subglottic level of the trachea without a fistula was observed. diagnosis by computed tomography (ct) with 3d-reconstruction revealed ta (floyd type ii) with a small esophago-bronchial (eb) fistula at the lower esophagus and linked to the right upper bronchus. at the age of 3 days, esophageal ligation and gastrostomy were performed. at the age of 21 days, with the assistance of 3dprinting simulation and veno-venous extracorporeal membrane oxygenation (ecmo) support, cervical esophagostomy and tracheoplasty were executed. the cervical esophagus was end-to-end anastomosed to the larynx. after dividing and excision of the eb fistula, the proximal end of the lower thoracic esophagus just above the fistula was end-to-side anastomosed to the right main bronchus. the distal end of the lower thoracic esophagus was ligated. however, pneumothorax, anastomosis dehiscence and repair, narrowing of anastomosis, collapse of esophageal-consisted airway, and bilateral pulmonary atelectasis developed in the following 2 weeks. flexible bronchoscopy aid balloon dilatation and stent implantation were planned. however, massive air-leak to mediastinum and peritoneum, shock and disseminated intravascular coagulation (dic) occurred. initially, nasal cpap (continuous positive airway pressure) was provided for respiratory support; however, intubation was finally performed with high-frequency oscillation use due to fluctuating respiratory condition. series of chest plain film showed progressive diffused infiltration, while high-resolution computed tomography showed bilateral ground glass appearance. in combination of her clinical features and image presentation, lung biopsy was performed due to the high suspicion of diffuse lung disease. oral steroid and azithromycin was administered but her improvement was poor. we did not administer hydroxychloroquine because of her g6pd (glucose-6-phosphate dehydrogenase) deficiency. recurrent ventilator-associated pneumonia was noted which needed several types of antibiotics. at 3 months of age, a tracheostomy was performed. lung transplantation was offered to the family but they decided to proceed with mechanical ventilation use. due to worsening of clinical condition and resistance to medication treatment, she received palliative extubation after full discussion with her family at the 5th month. a blood sample was collected for genetic testing before the patient passed away. revolutionized imaging of the "forgotten third circulation", improving our understanding of lymphatic diseases and paving the way for a new treatment modality, lymphatic embolization. we present a 7-year-old girl with kla whose pleural effusion stabilized following two lymphatic embolization procedures. case report: our patient presented at 2 years with recurrent severe non pulmonary infections. chest radiographs showed prominent diffuse reticular opacities despite the absence of respiratory symptoms or signs. comprehensive immunology and rheumatology investigations were normal, as were exhaled and nasal nitric oxide, ciliary biopsy, bronchoscopy and lavage, echocardiography and oxygen saturation at rest, on exertion and overnight. a chest ct showed thickening of interlobular septae with preservation of parenchymal architecture. a bone scan showed reduced uptake at l4, as well as t10 and the left clavicle in keeping with "vanishing bone disease". at has no effect on osa caused by ca, although rapid maxillary expansion (rme) is reported to be useful for it. objectives: herein, we report the case of a patient with osa likely due to ca that was successfully treated with rme. nights. respiratory event index (rei) were 24.2/h and 17.0/h on nights 1 and 2, respectively; nadir spo2 were 70% and 76%. as a result, severe osa was diagnosed. since his family did not wish for at, rme was performed. snoring and retractive breathing during sleep disappeared 3 weeks after the start of rme, and 8 months later, ocst was performed again on two consecutive nights. rei had improved to 5.7/h and 3.7/h on nights 1 and 2, respectively, and nadir spo2 had improved to 81% and 85%. no evidence on the usefulness of rme for osa in children has been established. however, rme was useful for osa likely due to moderate ath and ca in this patient, suggesting that tongue space expansion by rme can be a useful therapeutic method for osa. gorham-stout disease is also known as vanishing bone disease. it is a rare disease of unknown etiology characterized by progressive osteolysis and proliferation of lymphatic vessels. we report 2 cases of gorham-stout disease presenting with recurrent chylothorax and lytic bone lesion. we present a 2-month-old female infant with fever, cough, and vomit for 4 days. she was transferred to our medical center due to rapid progression to pards within 1 day, and her throat swab cmv pcr test revealed positive. her chest radiograph showed bilateral white-out, and very high ventilator settings were required to maintain oxygenation. cardiac disease was excluded by ultrasonography. intravenous ganciclovir, broad-spectrum antibiotics, and methylprednisolone were given, but the gas exchanges were still poor (oxygenation index= 31 at 38 hours after hospitalization). hfov was then applied (fio2 = 1.0, map= 20 cmh2o, amplitude = 30 cmh2o, frequency = 10, i time = 33%). after using hfov, the lungs were recruited and the settings could be weaned down gradually. intravenous immunoglobulin was given for suspected viral sepsis. case report: patient a was an 18-month-old girl with down syndrome, congenital hypothyroidism, small patent ductus arteriosus and anorectal malformation who presented with persistent fever and cough for 2 weeks post stoma closure. on examination, she was tachypneic with a respiratory rate of 50 breaths/minute and subcostal recessions. her breath sounds were reduced over the right lower zone with stony dullness. chest x-ray (cxr) showed right lower lobe consolidation with a loculated parapneumonic effusion. minimal thick stale blood-stained fluid was aspirated during an ultrasound guided thoracocentesis and an unusual mass was seen. hence the chest drainage was aborted and a contrast-enhanced ct angiography revealed a pulmonary pseudoaneurysm within the consolidated lung, adjacent to the right pleural collection. blood investigations showed a drop in hemoglobin from 12g/l to 8.8g/l. chest x-ray showed an extensive right pleural effusion and ultrasound described a right basal consolidation with a pleural effusion of 353ml. pleural fluid sample obtained by thoracentesis was purulent with exudate characteristics and a chest tube was inserted for drainage. due to these findings, a thoracic ct scan was performed and revealed atelectasis of the right lung and severe loculated pleural effusion with fluid-filled cavities in the right middle and lower lobes. since the patient was stable, a conservative management was preferred and empiric iv antibiotics, cefotaxime and clindamycin, were started. after 7 days of treatment, pleural fluid culture was positive for s. mitis. s. mitis was sensitive to ampicillin, thus treatment was completed with 10 days of ampicillin and 7 days of cefotaxime and clindamycin with a satisfactory outcome. considering that s. mitis is an innocuous commensal organism of the oropharynx, skin, and gastrointestinal and genitourinary tracts, we looked for primary infection sites. the origin of the infection was a cavity in the third lower molar that extended to the root. the patient had a history of a dental procedure 4 weeks before diagnosis. case report: a nine-month-old male preterm bpd infant with maternal premature rupture of membranes delivered by emergency caesarean section at 28 + weeks gestation due to fetal distress. apgar score was 4 at 1-minute and 7 at 5-minutes and birth weight was 1500 g. because of respiratory distress syndrome, endotracheal intubation was performed and the patient was transferred to the neonatal intensive care unit. after 3 months of mechanical ventilation support, the endotracheal tube was removed and the patient was treated under niv support. after 9 months of mechanical ventilation support since birth, we decided to make an assessment of the neurodevelopment of the patient by brain mri. conclusion: most of the time, preterm bpd infants with vd who are receiving niv support will be considered as high risk and follow-up for mri delayed because of two reasons: (1) need of oral intubation with endotracheal tube to guarantee the safety of the respiratory system; (2) the neonatal mri-ventilator which includes niv mode is too expensive and requires a well-trained respiratory therapist to operate the mri-ventilator. we believed that once we well-prepared the mri-ventilator, infants with vd who are receiving niv support could undergo mri more safely. by sharing this case experience, we hope the profession of neonatal respiratory therapy could receive more attention. we present two cases that were diagnosed after multiple consultations: case 1. a 2-year-old girl presented with a history of recurrent episodes of cyanosis during her first months of life, worsening respiratory symptoms and poor exercise tolerance. she was assessed on several occasions by a general practitioner (gp) who did not consider that further investigations were necessary. she also had recurrent epistaxis and was being followed by the local ent specialist who considered the episodes as normal. at the age of two, she was admitted to her local hospital with a lrti, cyanosis and persistent low oxygen saturation levels (70%). she was treated with antibiotics, but given her slow improvement she was referred to our center for further workup. on examination, she presented with failure to thrive, general cyanosis, multiple telangiectasia in oral cavity and nostrils, and clubbing fingers. initial chest x-ray showed abstracts | s139 ct angiography revealed an arteriovenous malformation (avm) in the middle segment of the right lower pulmonary lobe, multiple intraparenchymal small avms in both lungs and an aberrant artery from the descending aorta that irrigated the right upper lobe. embolization of the main pavm was proposed, however, it was not possible due to its large extension since there was no embolic device according to its size. further discussion in several international forums, a thoracotomy and pulmonary segmentectomy of the right lower lobe was suggested and then performed 7 months after presentation. since then, the patient has been asymptomatic, despite the fact that spo2 levels have not improved as expected. background: children with cystic fibrosis (cf) have frequent respiratory exacerbations, therefore the occurrence of cough and fever in a cf child raises the suspicion of an acute infectious event, while in a non-cf child this fever/cough association would suggest a pneumonia. what about the presence of tachycardia, which would suggest a different diagnosis, from myocarditis or hyperthyroidism. we herein present the case of a 3-year-old girl with cystic fibrosis with frequent acute cough, persistent fever and tachycardia attacks. a 3-year-old girl was diagnosed with cystic fibrosis in infancy, because of a pseudo-barterr syndrome. the child's evolution was very good, with good clinical and biological status, normal pulmonary lung function described by a normal lung clearance index and germ-free cough swab and negative induced sputum. she was monitored according to the national romanian guidelines every 3 months in the regional cf center and annually at the national center, by clinical and biological investigation. no ct or bronchoalveolar lavage was performed until age of 3 years. results: in evolution, her mother noticed episodes of tachycardia 5 months before the actual admission, supposed secondary to play or hospital anxiety, without any electrical ecg alteration. four months before admission, she had a mild exacerbation associated with insignificant chest x-ray findings and she received intravenous cephalosporin. after 1 month, she started to productively cough and fever, with a relatively strong persistence despite ains. her first sixteen emergency situations will be presented in detail using the above format. purpose: obstructive sleep apnea (osa) is a common disorder estimated at 1%-5% in school-aged children. with the obesity prevalence reaching staggering rates globally, osa in obese adolescents is estimated to be 4 to 5 fold higher than their lean peers. there is a paucity of data regarding obesity-related osa in children 6 years and less. this is particularly relevant as osa is associated with neurocognitive deficits. the aim of this study is to evaluate the prevalence of osa among obese toddlers and preschool children and further to determine what other factors may be associated with the presence of osa. methods: a retrospective study involving children < 6 years, identified from two canadian pediatric tertiary care centers who had an in-lab polysomnography (psg). obesity was defined by a bmi of > 95th percentile for age and gender or a z-score of > 2. osa was diagnosed if the obstructive apnea-hypopnea index (oahi) was greater than 2 events per hour. results: there were 60 participants included; the mean age was 4.4 years (standard deviation [sd] + 1.7), mean bmi z-score was 3.0 (sd + 1.2). of these, 22/60 (36.6%) had osa. compared with the non-osa group, the osa group had a higher epworth sleepiness score (p = 0.03) and were more likely to snore (p = 0.01). the present study "comparative study of nebulized beta 2 agonist, nebulized adrenaline and any added advantage of 3 % hypertonic saline in bronchiolitis" was undertaken to assess the efficacy. a total number of 150 patients (50 patients in each group) were examined below 24 months of age. group a was nebulized with salbutamol, group b was nebulized with adrenaline and similarly, group c was nebulized with 3% hypertonic saline. comparison of mean ± s.d. of rdai score before and after nebulization was performed in all 3 groups. in each patient from all groups (ie a, b and c), rdai scores were recorded at the time of admission (ie at 0 minutes), at 20 minutes and 40 minutes (after nebulization) and on subsequent days. the mean value of rdai score at admission and discharge was 10.78± 1.03 and 6.58±1.54 respectively in group a. in group b, rdai score at the time of admission and discharge was 11.20 ± 1.05 and 5.9 ± 1.61, respectively. similarly in group c, rdai score at the time of admission and discharge was 8.14 ± 1.80 and 3.18 ± 0.94, respectively. all the parameters before treatment had a p value > 0.05 (not significant at 0 minutes.) and at the time of discharge, the p value became < 0.0001 (extremely significant). mean differences in hr, rr, rdai score and spo2 were assessed after subsequent nebulization in all 3 groups. the post treatment values for group a (salbutamol), group b (adrenaline) and group c (3% hypertonic saline) were compared. all groups showed an increase in hr, but group b (mean difference of 14.80) showed a more increasing trend than group a (mean difference of 4.84) and group c (mean difference of 7.04) and had a p < 0.05. regarding rr, all 3 groups showed decreased rr, but group b (mean difference of 15.96) was more significant than group a (mean difference of 14.80) and group c (mean difference of 12.20) and had a p < 0.05. regarding rdai score, all 3 groups showed improved score, but group b (mean difference of 5.3) was more significant than group a (mean difference of 4.2) and group c (mean difference of 4.96) and had a p < 0.05. similarly, all 3 groups showed improved spo2 %, but group b (mean difference of 10.24) showed more improvement than group a (mean difference of 9.26) and group c (mean difference of 6.38) and had a p < 0.05. clinical parameters (heart rate, respiratory rate, rdai score and spo2 %) in both groups were comparable before nebulization. nebulized salbutamol was given to group a, nebulized adrenaline to group b and nebulized hypertonic saline to group c. different clinical parameters in the groups were compared before and after subsequent nebulization. mean respiratory rates, rdai scores and spo2 (%) significantly improved in all 3 groups. however, improvement was more significant in the adrenaline group. shorter length of hospitalization was noted in group c (hypertonic saline). no undesirable side effects were noted in all 3 groups. all 3 groups showed a transient increase in heart rate, although more so in the adrenaline group. objectives: endothelial dysfunction (ed) is one of the initial pathological changes ultimately leading to atherosclerosis and consequent cardiovascular disease. children with endothelial dysfunction are at higher risk of developing systemic and pulmonary hypertension, atherosclerosis and cardiac remodeling, with potential long-term adverse outcomes into adulthood. obstructive sleep apnea syndrome (osas) has been found to cause impaired endothelial function in adults. however, the evidence in pediatric osas is limited. the aim of the study is to evaluate endothelial function in a large cohort of children clinically referred for suspected osas, and to identify risk factors contributing to the presence of ed. methods: children aged 3 to 11 years old with habitual snoring (snoring ≥ 3 nights per week) were recruited to this study between june 1st 2015 -march 1st 2016. all subjects underwent an overnight polysomnography (psg), as well as endothelial function testing using peripheral arterial tonometry (pat) to derive the reactive hyperemic index (rhi). subjects were then divided into osas and primary snorers (ps) groups according to their obstructive apnea-hypopnea index (oahi). results: a total of 355 cases completed the study, with 248 children being diagnosed as osas, and 107 children assigned to the ps group. there were no differences in age, gender and bmi z-score between the two groups (all p > 0.05). the osa group had lower rhi than that of ps (p < 0.05). univariate correlation analysis showed that rhi was linearly correlated with age, gender, oahi, oxygen desaturation index, respiratory related arousal index, and oxygen saturation nadir. the relationship between bmi z-sore and rhi was quadratic. rhi and bmi z-sore were positively correlated when bmi z-score< 1. year follow-up, we regularly performed ekg, cardiac echogram, 6 minute walk testing (smwt) and monitoring of serum bun, bnp and creatinine. also, cardiac catheterization and hemodynamic studies, pet study with uptake of glucose analog 2-deoxy-2-﹝18f﹞fluoroglucose (18f-fdg) and pulmonary perfusion-ventilation scan were performed. we enrolled six patients with es including atrial septal defect type ii (3 cases), atrial septal defect type i (1case), right atrial isomerism after total cavopulmonary connection (tcpc) (1case) and ventricular septal defect (1case); the male:female ratio was 3:3. hemodynamic studies revealed that mean pulmonary arterial pressure was 52±7.3 mm hg; pulmonary vascular resistance index was 26.1±7.9 wu*m2, except the case receiving tcpc. in addition, three cases of intrapulmonary thrombus in right pulmonary artery, three cases of cerebral stroke and three cases of atrial fibrillation were found. the lung scan revealed that there were nonspecific findings noted in the es patients with or without intrapulmonary thrombus. very interestingly, an increased uptake of 18f-fdg in the right ventricle was noted in three cases. subsequently, there were two mortalities because of right ventricular failure. and pediatric settings which corresponds to a child of 15kg weight (tidal volume 150 ml, 25 breaths/min, ratio between inspiratory and expiratory 50/50 and peep 5 cm h2o). a filter was placed between the endotracheal tube and the test lung model (smartlung adult, imt medical, switzerland) to collect the drug. all components were tested for drug deposition. deposited doses were quantified by spectrophotometry. all measurements were performed five times. results: the percentages of the nominal dose of salbutamol deposited on the filter with the adult model after nebulization and aerosolization, were similar when the device was located before or after the y piece results: flow analysis. during inhalation, the pressure differential (y)/ flow rate (x) relationship was y = 0.1015x2 + 0.8343x (r2 = 0,9985). during exhalation, the pressure differential (y)/flow rate (x) relationship was y = -0.0106x2 + 0.0155x (r2 = 0,9873). this indicated that the flow rate closely followed the pressure differential (n = 5). furthermore, the ap was able to measure flow rate with great stability and reproducibility during all the experiments. results: valve resistance generated at low inspiratory flow rate (5 l/min) varied greatly between the different valves ranging from 9.5 to 0.27 cmh2o/l/s; however as the flow rate increased, variations were reduced (3.98 to 0.43 cmh2o/l/s at 60 l/min). the ability to close varied strongly from one valve to another: for some valves, expiratory flow did not pass through the inspiratory valve while for others, only partial closing could be achieved (85% of expiratory flow passed through the inspiratory valve). these results were confirmed by measuring the flow through the expiratory valve, maximum (100% of the initial flow rate) when inspiratory valves were completely closed. tilting the chamber did not seem to affect this result. ed measured with pediatric breathing parameters in coordinated and uncoordinated use was similar for four of the custom vhcs while with the other two vhcs, ed was lower. , and a high-speed camera (hxlink64,nac) and two different shapes of membrane models were used in this study. five catheter motions: 1) quiescent technique, 2) piston technique, 3) rolling technique, 4) twist technique, and 5) dynamic pressure technique, were performed 10 times each and the weight of secretion was measured by a digital scale. the negative pressure was set at 20kpa, suctioning time was counted 10 seconds. one-way anova and tukey test were used to analyze the obtained data. in experiment 1, the mean amount of secretion for 1) to 5) were 0.31, 0.48, 0.32, 0.43, and 0.45, respectively. there were significant differences between 1) quiescent technique and 2) piston technique (p = 0.025), 2) piston technique and 3) rolling technique(p = 0.039) on a flat membrane model with high viscous sputum. the high-speed camera showed that absorption was started when a catheter tip aperture and two side apertures were sealed with the high viscous sputum. as dynamic pressure technique, sputum was quickly absorbed right after the researcher released her thumb which bent the suctioning catheter. in experiment 2 with high viscous sputum on a tracheal membrane model, mean secretion weight 1) to 5) were 0.36, 0.31, 0.30, 0.42, and 0.43 respectively. there were no significant differences between these five techniques. in experiment 3 with low viscous secretion on a tracheal membrane model, mean amounts of suctioned secretion were 0.97, 0.76, 0.98, 1.01, and 1.15 respectively. there were significant differences between 2) piston technique and 4) twist technique (p = 0.042), 2) piston technique and 5) dynamic pressure technique (p = 0.000). in conclusion, the piston technique could absorb the most amount of secretion when a catheter tip was visible on a flat membrane model, however, the catheter motion which suctioned the most secretion was the dynamic pressure technique in a tracheal model despite the viscosity of the secretion. even though nurses had to carefully apply the level of negative pressure before their procedure, this result indicated that dynamic pressure technique is the most effective catheter motion to remove secretion on a tracheal model. background: respiratory syncytial virus (rsv) infection is the main cause leading to acute bronchiolitis in infants or children less than 2 years of age. children are more susceptible to rsv infection than adults, especially those who have hemodynamic-significant congenital heart disease or heart failure. in this study, we aimed to evaluate the possible risk factors leading to prolonged stay in the pediatric intensive care unit (picu) or hospital stay for chd children with rsv infection. methods: data were retrospectively retrieved of patients who had chd from jan 2011 to dec 2013. those patients who had concurrent rsv infections and underwent computed tomography imaging study were included as the study group. clinical presentations were recorded and classified. statistics used descriptive analysis and p < 0.05 was taken as significant. results: a total of 286 patients with associated congenital heart disease were included. the mortality rate was about 3% due to pulmonary edema, dysfunction or cyanosis. the total number of subjects whose age was below 1 year old was similar to those whose abstracts | s147 age was between 1 and 2 years old. there were 68 chd children with airway problems, including trachea-bronchomalacia, stenosis of the trachea or bronchus. patients aged less than 1 year old had longer hospital stay than those whose age was above 1 year old. the mean picu stay and the intubation period had a similar trend. in the comparison of groups without airway anomalies, the hospital stay in children who had hemodynamic-significant chd or heart failure (without associated airway anomalies) was 46.6 days compared to 11.6 days in children who did not have hemodynamic-significant chd or heart failure(without airway anomalies) (p < 0.05). in children with airway anomalies but did not fulfill the criteria of hemodynamic-significant chd or heart failure had longer hospital stay (22.8 days) than those without airway anomalies but with hemodynamicsignificant chd or heart failure (10.6 days) (p < 0.05). conclusions: patients with airway anomalies are not rare in children with chd, although the severity varies. lower airway anomalies were associated with longer hospital stay in chd children who were not hemodynamic-significant or heart failure. we found that airway problem is a risk factor for prolonged hospital or picu stay in chd children with rsv bronchiolitis. to achieve successful and sustainable outcome in chd children with rsv bronchiolitis, it is crucial to have early identification of concomitant airway anomalies in these children. childhood pneumonia and diarrhoea 1: global burden of childhood pneumonia and diarrhoea key: cord-266982-069pelqj authors: agua-agum, junerlyn; ariyarajah, archchun; aylward, bruce; bawo, luke; bilivogui, pepe; blake, isobel m.; brennan, richard j.; cawthorne, amy; cleary, eilish; clement, peter; conteh, roland; cori, anne; dafae, foday; dahl, benjamin; dangou, jean-marie; diallo, boubacar; donnelly, christl a.; dorigatti, ilaria; dye, christopher; eckmanns, tim; fallah, mosoka; ferguson, neil m.; fiebig, lena; fraser, christophe; garske, tini; gonzalez, lice; hamblion, esther; hamid, nuha; hersey, sara; hinsley, wes; jambei, amara; jombart, thibaut; kargbo, david; keita, sakoba; kinzer, michael; george, fred kuti; godefroy, beatrice; gutierrez, giovanna; kannangarage, niluka; mills, harriet l.; moller, thomas; meijers, sascha; mohamed, yasmine; morgan, oliver; nedjati-gilani, gemma; newton, emily; nouvellet, pierre; nyenswah, tolbert; perea, william; perkins, devin; riley, steven; rodier, guenael; rondy, marc; sagrado, maria; savulescu, camelia; schafer, ilana j.; schumacher, dirk; seyler, thomas; shah, anita; van kerkhove, maria d.; wesseh, c. samford; yoti, zabulon title: exposure patterns driving ebola transmission in west africa: a retrospective observational study date: 2016-11-15 journal: plos med doi: 10.1371/journal.pmed.1002170 sha: doc_id: 266982 cord_uid: 069pelqj background: the ongoing west african ebola epidemic began in december 2013 in guinea, probably from a single zoonotic introduction. as a result of ineffective initial control efforts, an ebola outbreak of unprecedented scale emerged. as of 4 may 2015, it had resulted in more than 19,000 probable and confirmed ebola cases, mainly in guinea (3,529), liberia (5,343), and sierra leone (10,746). here, we present analyses of data collected during the outbreak identifying drivers of transmission and highlighting areas where control could be improved. methods and findings: over 19,000 confirmed and probable ebola cases were reported in west africa by 4 may 2015. individuals with confirmed or probable ebola (“cases”) were asked if they had exposure to other potential ebola cases (“potential source contacts”) in a funeral or non-funeral context prior to becoming ill. we performed retrospective analyses of a case line-list, collated from national databases of case investigation forms that have been reported to who. these analyses were initially performed to assist who’s response during the epidemic, and have been updated for publication. we analysed data from 3,529 cases in guinea, 5,343 in liberia, and 10,746 in sierra leone; exposures were reported by 33% of cases. the proportion of cases reporting a funeral exposure decreased over time. we found a positive correlation (r = 0.35, p < 0.001) between this proportion in a given district for a given month and the within-district transmission intensity, quantified by the estimated reproduction number (r). we also found a negative correlation (r = −0.37, p < 0.001) between r and the district proportion of hospitalised cases admitted within ≤4 days of symptom onset. these two proportions were not correlated, suggesting that reduced funeral attendance and faster hospitalisation independently influenced local transmission intensity. we were able to identify 14% of potential source contacts as cases in the case line-list. linking cases to the contacts who potentially infected them provided information on the transmission network. this revealed a high degree of heterogeneity in inferred transmissions, with only 20% of cases accounting for at least 73% of new infections, a phenomenon often called super-spreading. multivariable regression models allowed us to identify predictors of being named as a potential source contact. these were similar for funeral and non-funeral contacts: severe symptoms, death, non-hospitalisation, older age, and travelling prior to symptom onset. non-funeral exposures were strongly peaked around the death of the contact. there was evidence that hospitalisation reduced but did not eliminate onward exposures. we found that ebola treatment units were better than other health care facilities at preventing exposure from hospitalised and deceased individuals. the principal limitation of our analysis is limited data quality, with cases not being entered into the database, cases not reporting exposures, or data being entered incorrectly (especially dates, and possible misclassifications). conclusions: achieving elimination of ebola is challenging, partly because of super-spreading. safe funeral practices and fast hospitalisation contributed to the containment of this ebola epidemic. continued real-time data capture, reporting, and analysis are vital to track transmission patterns, inform resource deployment, and thus hasten and maintain elimination of the virus from the human population. the ongoing west african ebola epidemic began in december 2013 in guinea, probably from a single zoonotic introduction [1, 2] . as a result of ineffective initial control efforts, an ebola outbreak of unprecedented scale emerged. as of 4 may 2015, it had resulted in more than 19 ,000 probable and confirmed ebola cases, mainly in guinea (3, 529) , liberia (5, 343) , and sierra leone (10,746) (see section 1.3 in s1 text for who case definitions). control measures for ebola are well known and based on past experience [3] [4] [5] [6] . however, the lack of local experience in handling ebola outbreaks coupled with severely limited health care resources and poor coordination in the international response led to an initial failure to prevent exponential spread of the outbreak [7, 8] . international partners, including the world health organization (who), emphasised four interventions [9] : (1) prompt identification and isolation of cases, (2) tracing of contacts, (3) safe and dignified burials, and (4) community awareness and social mobilisation. following several months of intensive efforts to enhance control measures and local community mobilisation, incidence dramatically fell in all three countries [10] (fig 1, top row) . to quantify the risk factors for transmission, we analysed reported exposure data collected during the epidemic in guinea, liberia, and sierra leone. we hypothesized that many reported exposures corresponded to transmission events, and that we could therefore discover correlates of transmission and properties of the transmission network by studying available exposure records. analyses were informed by epidemiological knowledge accrued during previous ebola outbreaks and by previous expertise in outbreak analysis. the descriptive analyses and association studies were not prespecified, but rather analyses were designed in response to preliminary examination of the accruing data, and in discussion amongst partners in the team. we also hypothesized that if the interventions deployed to interrupt transmission were effective in this epidemic, we should observe an association between the rate of epidemic spread at the district level and the proportion of cases reporting funeral attendance and/or slow hospitalisation. this latter analysis was decided upon before statistical implementation, and was thus hypothesis-driven. we did not have data to assess the effect of contact tracing or community mobilisation. data were collated during the outbreak to assist with surveillance and the response. national case databases were shared with who and were merged to form a joint line-list (one line of data for each case). the retrospective analyses presented here use data from the three main affected countries (guinea, liberia, and sierra leone) and were first conducted in real time in september and october 2014 as part of the response to the public health emergency. they were shared with who, national authorities, and international agencies working in the three countries and contributed to the planning and monitoring of the control effort. in summary, the objectives of these analyses are to understand the drivers of local transmission, the timing of transmission events relative to clinical timelines, and the characteristics of total number of confirmed and probable cases by week is shown in the top row. percentage of cases who reported a non-funeral exposure (triangles) or a funeral exposure (circles) is shown in the second row. the shaded regions represent the 95% confidence intervals around the proportions. note that cases can report more than one exposure, and so percentages need not add to 100%. exp., exposure. the transmission network. these are different from and complement the objectives of our previous papers [11] [12] [13] [14] . however, aspects of the process are the same as in those previous analyses: data cleaning, time series of incidence, and method of estimation for the reproduction number. the same exposure data were used to estimate the incubation period and serial interval in previous analyses [11, 12] . the dataset analysed is the most comprehensive available on the current epidemic, and represents a unique resource with which to increase our understanding of the outbreak and of ebola transmission in general. the analysis and presentation of the characteristics of exposures, transmission network, transmission risks, characteristics of potential transmitters and multiple transmitters, health care worker (hcw) exposures, and correlations between epidemic growth and behaviour are all novel. this paper is based on data collected during surveillance and response activities for ebola in guinea, liberia, and sierra leone. the work is based on routine national surveillance for ebola, for which informed consent is not needed. the surveillance was carried out under who integrated disease surveillance and response guidelines in africa (the recommended disease notification system). data were reported to who under the obligations of the international health regulations. all work was carried out under this legal framework. all information on individual patients has been anonymized for analysis and presentation. information from ebola cases was collected using a standardised case investigation form [12] and the epi info viral hemorrhagic fever application (https://epiinfovhf.codeplex.com/). the cleaning of the data has been described previously [11, 12] . the case investigation form records information on exposures that may have led to the infection of the case. cases were asked if in the month prior to symptom onset they had had contact with a potential ebola case and/or had attended a funeral. cases could report details of up to five exposure events with potential source contacts (up to three exposures with potential source contacts outside of a funeral context, and up to two exposures at funerals of potential source contacts); from autumn 2014, an abbreviated form was introduced with up to two non-funeral and one funeral exposure (see s2-s6 texts). cases reported the name of the person they were exposed to and provided details about their relationship to that person (e.g., family, friend, neighbour) and the nature of the exposure (e.g., touched bodily fluids or shared belongings). all names were anonymized using the sha-1 hashing algorithm [15] . here, we use "potential source contact", "source contact", or "contact" to describe the person who was named as a potential ebola source, and "exposure" to describe the potential transmission event that took place between the case and the potential source contact (see box 1 for terminology). the potential source contact may be alive or dead at the time of the exposure. ebola cases are classified as confirmed, probable, or suspected. here we analysed data from all cp cases unless otherwise stated. in practice, different countries implemented slightly different case definitions; hence, we also performed sensitivity analyses considering confirmed, probable, and suspected cases combined (see section 3 in s1 text). we assessed whether cases who reported at least one non-funeral or funeral exposure were different from cases who did not report that type of exposure. this allowed us to assess whether cases who report exposures are representative of the line-list. multivariable logistic regressions were performed using predictors identified as significant (p < 0.05) in univariable logistic regressions (see section 1.11 in s1 text for a list of predictors included in univariable analyses). the most parsimonious yet adequate multivariable model was then identified (using the akaike information criterion) through backwards stepwise model selection (see final results in tables b and c in s1 text). to identify potential transmission pairs and thus elucidate transmission networks, we examined whether named potential source contacts were themselves ebola cases. we searched for all named contacts among the names of cases in the line-list to find possible matches. multiple checks were undertaken to verify the consistency of all matched case-contact pairs according to a set of rules (see section 1.4 and figure a in s1 text), in particular comparing dates of symptom onset and exposure. importantly, the exposure data analysed here are retrospective and are completely distinct from prospective contact tracing data, where cases are asked who they had contact with after they became ill; we do not analyse prospective contact tracing data here, as such data were not available to us, but rather focus on identified potential source contacts. we hypothesized that the observed decrease in the proportion of hcws (the positions reported include medical staff and other health-care-facility-based workers) among cases over time (see figure f in s1 text) was due to decreased unprotected exposure to patients. to address this issue, we compared the estimated slopes from two linear regression models of (1) • case: an individual in the line-list with confirmed or probable (cp) ebola virus disease status using who case definitions [16] (see section 1.3 in s1 text). none of our results changed substantially when analyses were run on confirmed, probable, and suspected cases combined (see section 3 in s1 text). • exposure: an event reported by a case in which the case came into contact with an ill or dead person or attended their funeral. cases could report more than one exposure. • non-funeral exposure: exposure with an ill person, who may be alive or dead, but not at a funeral. cases could report up to three non-funeral exposures. • funeral exposure: attendance at a funeral, which could involve touching the corpse. cases could report up to two funeral exposures. • potential source contact, source contact, or contact: the person with whom the exposure was made. • matched contact: a potential source contact whom we could identify as a reported case in the line-list. • matched exposure: an exposure between a case and his or her matched potential source contact. the proportion of hcws among cases over time and (2) the proportion of hospitalised patients over time (see section 1.5 in s1 text). note that we define "hospitalisation" as admission to a number of different health care facilities (see box 2) . understanding when onward exposure occurred in the clinical course of infection in a primary case could inform the focus of interventions, for instance for early detection of cases or for improving the safety of funerals. we analysed the timing of non-funeral exposure events relative to the following clinical events of the named contacts: onset of symptoms, hospitalisation, and death. here, we describe the analysis of the time from symptom onset to onward exposure; the analyses for hospitalisation and death were similar. we fitted (by maximum likelihood) a distribution to the observed delays between symptom onset and exposure. in brief (see section 1.8 in s1 text for more detail), this likelihood accounted for (1) multiple exposures (when multiple exposures were reported, only one was likely to have led to infection, so exposures were weighted accordingly), (2) inaccurate recall of contacts, (3) inaccurate recall of dates of exposures, (4) errors in data entry. in order to address issues 2 to 4, we fitted a mixture of two offset lognormal distributions: the more peaked we regarded as the signal, and the broader we regarded as the noise (see section 1.8 in s1 text). we then interpreted the signal distribution as our best estimate of actual infection times. missing delays were imputed by random draws from the observed data. the analyses described above showed that the non-funeral exposure events were concentrated shortly after the onset of symptoms and around the day of death (see results). we performed an additional analysis to further explore whether one of these two clinical events was we use "hospitalisation" to mean the admission of any patient to a health care facility, as recorded in the case investigation form. this refers to a variety of events in a range of facilities, including • ebola treatment centres or units • referral centres if a case is hospitalised for ebola, who guidelines recommend that the case be isolated. the list above includes a variety of health care facilities where isolation capacity and quality will vary. it should be noted that the meaning of "date of hospitalisation" may be different among cases, depending on how many and which type of facilities they reported visiting in the course of their disease. however, the data do not allow us to distinguish between different meanings of hospitalisation. it should also be noted that not all transfers of patients between different types of health care units may have been recorded in the case investigation form. data cleaning for hospital type is described in section 1.4 in s1 text. more influential for the timing of onward non-funeral exposure events. we compared the ability to predict (1) the date of death of the source contact based on date of symptom onset of the source contact and the date of exposure reported by the case and (2) the date of symptom onset of the source contact based on date of death of the source contact and date of onward exposure (see figure o and section 1.9 in s1 text). these two models assume that the timing of onward exposure is mainly determined by the date of symptom onset (model 1) or by the date of death (model 2). the transmission network can be described by a network of nodes (cases) linked by directed edges (exposures leading to transmission). in order to characterise the heterogeneities in transmission, we analysed the properties of this network. we looked at separate networks for funeral and non-funeral exposures (see section 1.10 in s1 text). for each, we analysed the outdegree distribution, i.e., the distribution of the number of cases naming the same matched contact, which can be seen as a proxy for the distribution of the number of secondary cases per index case. several parametric distributions were fitted to each of the two observed out-degree distributions using maximum likelihood estimation (see section 1.10 in s1 text for a list of the distributions explored). the akaike information criterion corrected for finite sample sizes (aicc) [17] was then used to select the best distribution. for the best-fitting distribution, 95% confidence intervals for the distribution parameters were obtained using the likelihood ratio test, and the confidence interval for the corresponding distribution was obtained by numerical sampling. we derived the variance and the coefficient of variation for the offspring distribution (the distribution of the number of secondary cases infected by each case), based on the assumption that the network of exposures shown in figure p in s1 text is a sample of the full transmission network (see section 1.10 in s1 text). to obtain further insight into potential drivers of transmission, we analysed the data to see if there were predictors of being a potential source contact. we performed four analyses: (1) a logistic regression, which identified predictors of being named as a non-funeral contact, one or more times; (2) a logistic regression restricted to cases who died, which identified predictors of being named as a funeral contact, one or more times; (3) a negative binomial regression, which identified predictors of being named multiple times as a non-funeral contact, conditional on being named at least once; and (4) a negative binomial regression restricted to cases who died, which identified predictors of being named multiple times as a funeral contact, conditional on being named at least once. predictors included in the univariable regressions were as defined in section 1.11 of s1 text, and the method for identifying the final parsimonious multivariable model is described above. we performed these analyses on confirmed, probable, and suspected contacts who had been named by cp cases. this allowed us to understand the role of suspected contacts in onward transmission compared to cp contacts. we explored the relationship between district-level (second administrative division) transmission intensity and (1) the proportion of cases reporting funeral attendance amongst those reporting any exposure and (2) the proportion of cases ever hospitalised and the proportion of cases hospitalised 4 days (4 days was the median delay from symptom onset to hospitalisation; see figure m in s1 text for sensitivity analysis to that threshold). the transmission intensity was quantified by the reproduction number, r, which estimates the average number of secondary cases per index case [11, 18] (see section 1.6 in s1 text). we estimated the reproduction number (r d m ) and the proportion (p d m ), for every district (d) in all three countries over monthly intervals (m). (r d m was estimated with the r package "epiestim" [19] using districtlevel incidence, presented in fig 1 at the country level; see section 1.6 in s1 text.) the relationship between (r d m ) and the proportions was explored using linear regressions. as these quantities were estimated from a sample of data with a level of uncertainty, we used a custom linear regression method that accounts for measurement error [20, 21] . the method to compute the correlation coefficient was also adapted to account for measurement error (see section 1.7 in s1 text). all data cleaning and analyses were performed using r software [22] . of the 19,618 cp ebola cases in the line-list, 6,403 (33%) cases reported one or more exposures (table 1) , giving a total of 9,711 reported exposures. temporal trends in case incidence and reported exposures by country are shown in fig 1. the proportion of cases reporting exposures varied by country (table 1 ); in general, we found that exposure data were less complete for guinea. cases reporting exposures were broadly representative of all cases in the line-list (see tables b and c and figure c in s1 text for a comparison of characteristics between cases who reported exposures and those who did not). exposure at funerals is a known risk factor for ebola infection [23] [24] [25] , and 25% of cases who reported any exposure in the current outbreak reported exposures at funerals. most cases (89%) reporting a funeral exposure also reported one or more non-funeral exposures. cases were asked to provide details on the nature of their exposures and their relationships with the contacts, which are shown in table 1 . overall, 87% of exposures occurred between family members (of those where the relationship was reported). up until the introduction of the new case investigation form (see s2-s6 texts), non-funeral exposures were recorded as one or more types (e.g., an exposure could simultaneously involve shared belongings and exposure to bodily fluids). of those non-funeral exposures for which the type of exposure was reported, over 90% were reported to involve contact with bodily fluids and/or direct physical contact, and 38% were reported as occurring in a household (defined as having slept, eaten, or spent time in the same household or room as the contact). these patterns did not change significantly over time (see figure e in s1 text). for funeral exposures, cases were asked whether they had touched the corpse. of those giving a response, 65% reported having touched the corpse, with this proportion being greatest for guinea (71%) and least for liberia (61%). this proportion declined significantly after october 2014 (p < 0.001; see figure h in s1 text), most notably in sierra leone. we were able to identify 14% of potential source contacts as cases in the line-list. our ability to match contacts did not vary substantially over time (see figure i in s1 text) or geographically (see figure c in s1 text). the analysis of matched exposures provides information on the transmission network underpinning the ebola epidemic (fig 2) . there was evidence of modest assortativity in exposure patterns by sex (see table i in s1 text) and country-specific patterns by age (see table j in s1 text). the most important statistic characterising the transmission network is the out-degree distribution, the number of times each person was named as a contact by other ebola cases. the observed out-degree distribution was best fitted by a logarithmic probability distribution for both funeral and non-funeral contacts (fig 2a; see sections 1.10 and 2.8 in s1 text). since the network is not known in its entirety, but only through a sample of cases and their matched contacts, additional assumptions were needed to infer the true offspring distribution (the distribution of the number of secondary cases infected by each case) from the observed outdegree distribution. under the assumption that the matched exposures are representative of the underlying transmission network, we find high to extreme variability in the offspring distribution ( fig 2b; see section 1.10 in s1 text). the estimated coefficient of variation for the offspring distribution ranges from 1.6 to 5.6 depending on assumptions (see section 1.10 in s1 text). this implies that 5% of cases accounted for at least 30% of all new infections and that 20% of cases accounted for at least 73% of new infections (fig 2b; see figure q in s1 text), a phenomenon termed super-spreading [26] . super-spreading was found to affect both nonfuneral and funeral transmissions equally (see tables f and g in s1 text). one key potential determinant of the risk of onward transmission is the stage of progression of clinical illness. the risk of transmission was found to increase over time since symptom onset (fig 3a) , peaking 2 days after onset, with some exposures estimated to have occurred more than 2 weeks after onset. our model estimates a small probability of transmission before symptom onset; however, we do not regard this as strong evidence for pre-symptomatic transmission: all reported dates are prone to recall bias, but it is likely that the date of symptom onset is more uncertain than dates of hospitalisation and death, as it is subjective, so individuals may interpret symptom onset differently. transmission events from non-funeral exposures were estimated to be strongly peaked on the day of and the day after the death of the contact (fig 3b) . in all, 44% of non-funeral not all cases who reported funeral exposure explicitly reported whether they had touched the corpse. cases who reported non-funeral exposure could report multiple types of exposure: belongings-"touched or shared the linens, clothes, or dishes/eating utensils of the case [contact]"; bodily fluids -"touched the body fluids of the case (blood, vomit, saliva, urine, feces)"; in same household-"slept, ate, or spent time in the same household or room as the case"; direct physical-"had direct physical contact with the body of the case". relationship was not reported for every exposure. we grouped reported relationships into classes: "close family" is defined as siblings, marital, and parent-child relationships; other family members are considered "extended family"; "neighbour" is defined as tenants, lodgers, landlords, and neighbours; "health care" is defined as hcw-patient relationships and caregivers, or any reference to a patient; "other" includes traditional healers, contacts through religious practice, and transport contacts. type of exposure and relationship type are illustrated graphically in figure d in s1 text. we sought to determine whether date of death or date of symptom onset was the stronger predictor of transmission risk. we found that date of death was the stronger predictor, indicating that the timing of death is the strongest determinant of when transmission occurs (see figure o in s1 text). hospitalisation (see box 2 for definition of hospitalisation, which in this context is isolation from the community by admission to any health care facility) aims to improve the clinical outcomes of patients with ebola virus disease, and also to reduce onward exposures. we found that hospitalised cases (who were hospitalised at any stage of their infection) had a reduced risk of being named as a non-funeral source contact (univariable or = 0.78 [95% ci: 0.66, figure p in s1 text for full network) and the contacts they have named as having been exposed to. individuals (cases and contacts) are shown as nodes, and exposures as directed arrows from contacts to cases. arrows are red for funeral exposures, black for nonfuneral exposures, and blue for multiple exposures involving both non-funeral and funeral exposures. square nodes are males, round nodes females, and triangles unknown. red nodes are cases who have died, blue nodes are cases who have survived, and grey nodes are cases with no recorded outcome. of the contact to time of exposure. the green curves show the overall best fits, and the red curves show the best fits for the "signal" distribution (all obtained by maximum likelihood). the red-shaded areas indicate the 95% confidence intervals of the fitted "signal" distribution. the histogram shows a random set of exposure midpoints (in some instances, only a start or an end date of exposure is recorded; in those instances, the missing date is numerically imputed). note that the fitting procedure is not performed on the midpoints but fully incorporates the exposure window (see section 1.8 in s1 text). the inset panels are the observed cumulative distribution functions for the midpoint (black line) and start and end (grey lines) of the exposures. 0.92]) and as a funeral source contact (or = 0.43 [95% ci: 0.29, 0.63]) compared to cases never hospitalised. this indicates that hospitalisation reduced but did not eliminate onward exposures. the impact of hospitalisation on limiting onward exposure was stronger in guinea than in liberia (significantly so for non-funeral exposures, see tables m and n in s1 text). the protective effect of hospitalisation on exposure in sierra leone was not statistically significant. there was no significant increase in the effect of hospitalisation over time for any of the three countries (see tables m and n in s1 text). previously, we examined the timing of transmission risk relative to symptom onset and death. using the same method to characterise the timing of exposure events relative to hospitalisation, we found that a substantial proportion (42% [95% ci: 12%, 54%]) of non-funeral exposures from hospitalised source contacts occurred after their hospitalisation (fig 3c) . our definition of hospitalisation covers a broad range of health care facility types (box 2), each with varying levels of infection control [27] . in particular, as part of the international response to the epidemic, an unprecedented number of ebola treatment units (etus) were built to care for patients. the previous analysis considered all types of hospitalisation together. when we disaggregated hospitalisation by etu versus other facilities, we found that etus were better than other health care facilities at preventing exposure from hospitalised cases and at providing safer management of corpses. in etus, the observed proportion of non-funeral exposures from hospitalised cases occurring after hospitalisation was significantly lower than in non-etu facilities (p < 0.001, see figure n in s1 text). moreover, hospitalisation in an etu reduced the risk of being named as a funeral contact compared with non-hospitalised cases (univariable or = 0.30 [95% ci: 0.16, 0.50]) more than hospitalisation in a non-etu facility did (or = 0.72 [95% ci: 0.39, 1.23]; also see multivariable analyses in table l in s1 text and the next section). although for non-funeral contacts univariable ors were not significant, a multivariable analysis indicated similar effects (see table k in s1 text). overall, our analyses demonstrate that hospitalisation reduced infectious exposures to cases, but that the effectiveness of hospital isolation measures varied by country and facility type, with substantial scope for improvements. to further explore the characteristics of individuals who were named as potential source contacts (and therefore potential transmitters), we also performed systematic multivariable logistic regressions on the risk of being named as a potential source contact (see tables k and l in s1 text). based on the final multivariable model, individuals significantly more likely to be named as a non-funeral contact were those who were more severely affected at presentation ( to determine whether there were any factors associated with being multiply identified as a potential source contact, we conducted a multivariable negative binomial regression to explore predictors of the number of times cases were named as non-funeral and funeral source contacts, conditional on being named at least once (see tables p and q in s1 text). cases with reported haemorrhagic symptoms were found to be named as non-funeral contacts 1.67 times more often than cases not reporting these symptoms (95% ci: 1.07, 2.64). cases hospitalised in an etu were found to be named as non-funeral contacts 0.50 as many times as non-hospitalised cases (95% ci: 0.33, 0.73). also, hcws were found to be named as non-funeral contacts 0.48 as many times as non-hcws (95% ci: 0.23, 0.96). travel outside one's village of residence prior to becoming ill was the only significant predictor for a case to be named multiple times as a funeral contact (relative number of times being named compared to cases who did not report travelling was on average 4.41 [95% ci: 2.01, 10.55]). sex did not appear as an important predictor of exposure risk in any of the analyses that we performed. hcws have been at particularly high risk of infection, especially during the early part of the epidemic [28] . hcws reported that 49% of their non-funeral exposures were with other hcws or patients, versus 0.6% reported by non-hcws. consequently, hcws reported a lower percentage of exposures with family members (44%) compared to non-hcws (91%; see figure f in s1 text). this demonstrates that hcws were often infected whilst working. consistent with these data, the matched contacts reported by hcws were more likely to have evidence of hospitalisation at some point in their illness than the matched contacts of non-hcws (or = 2.91 [95% ci: 1.57, 5.81]). in the absence of data on the total number of hcws working in each of the three countries over time, we could not estimate the relative risk of infection of hcws compared to non-hcws. however, several indicators point to falling risk through time. since june 2014, the proportion of hcws among all cases has decreased over time (see figure f in s1 text). the rate of decrease in this proportion (estimated monthly relative decrease since june: 21% [95% ci: 18%, 24%]) was faster than that seen in the incidence of hospitalised cases (estimated monthly relative decrease: 12% [95% ci: 10%, 14%]; see also figures f and g in s1 text), suggesting an improvement in infection control measures and increased awareness of ebola among hcws over time. the analyses presented so far describe individual risks of infection. we next explored, in a hypothesis-driven manner, whether local epidemic transmission intensity was correlated with local population measures of presumed heightened risk of infection. we first examined whether the proportion of cases reporting funeral exposures in each district by month was correlated with the estimated reproduction number r in that district at that time. having accounted for uncertainty in the estimates of both quantities [20] (see section 1.7 in s1 text), we found a positive correlation for liberia (r 2 = 0.30, p = 0.013) and sierra leone (r 2 = 0.23, p < 0.001) and no significant correlation for guinea (r 2 = 0.001, p = 0.667) (fig 4; see figure j and table d in s1 text). this analysis suggests that across the three countries an absolute reduction of 10% in the proportion reporting funeral exposures would lead to an absolute reduction of 0.10 in r (95% ci: 0.05, 0.15; p < 0.001). in particular, reducing the proportion reporting funeral exposures to less than 29% (95% ci: 21%, 38%) would be sufficient to reduce r below 1, the threshold value for achieving control of the epidemic. however, this result varies by country, and is an association that does not prove causation, since reductions in funeral exposures could have been accompanied by changes in other control measures and other effects of community mobilisation. in that case, the measure is a surrogate for the total effect of interventions that correlate with reduced funeral exposures. another factor potentially influencing changes in transmission intensity is how promptly cases are hospitalised and isolated. we found no association between the proportion of cases that were hospitalised and the estimated district-level reproduction number r (see figure k in s1 text), likely because the line-list predominantly includes cases who have been hospitalised. however, we found a negative association between the estimated district-level r and the proportion of hospitalised cases admitted within 4 days after onset of symptoms in liberia (r 2 = 0.22, p = 0.039) and sierra leone (r 2 = 0.32, p < 0.001). no significant correlation was found in guinea (r 2 = 0.02, p = 0.377) (fig 4; see figures l and m in s1 text). this analysis quantifies the impact that earlier hospitalisation has in reducing transmission. across all three countries we estimate that increasing the proportion of cases hospitalised within 4 days of symptom onset by 10% would reduce r by 0.19 (95% ci: 0.12, 0.25; p < 0.001). in particular, if 64% (95% ci: 59%, 68%) of hospitalised cases are admitted within 4 days, r is predicted to be <1. again, the effect varied by country. we found no correlation between the two predictors we analysed, i.e., the proportion of cases reporting funeral exposure and the proportion of cases hospitalised within 4 d of symptom onset (naïve r 2 = 0.001, p = 0.711; see section 2.5 in s1 text). this shows that both factors are independently associated with the level of transmission, at least in liberia and sierra leone, and, together, they explain a substantial proportion (approximately 55%) of the variance in epidemic transmission intensity observed in these two countries. the analyses in this paper draw on 9,711 exposures to potential sources of infection, to provide a quantitative basis for understanding ebola epidemiology and to validate the choice of interventions used to combat ebola in the current and future outbreaks. these analyses were initially performed in september/october 2014 as part of the who ebola response during the epidemic and have been updated to be shared with the scientific community. the current ebola outbreak has been of unprecedented scale, occurring across three countries of diverse cultures with varying levels of urbanisation. our analysis provides a unique overview of ebola transmission that complements detailed investigations that have been performed in localised settings in this and previous outbreaks [29] [30] [31] [32] [33] . small studies may allow more detailed reconstruction of transmission and identification of risk factors for infection by comparing cases and non-cases, which is not feasible at a larger scale. however, analyses such as identifying district-level correlates for transmission intensity and determining when exposures occur with respect to clinical events are only possible with this unique large-scale dataset. our analysis confirms that exposure to ebola cases at funerals is an important amplifier of ebola transmission, in line with a study focused in sierra leone [32] . the significant correlation we found between the district-level reported frequency of funeral exposures and local transmission intensity provides support for the policy emphasis on safe and dignified burials. . the scatter plots show these monthly proportions against monthly estimated reproduction numbers r (method as previous [11] ) for the supplemented incidence (i.e., incidence based on two data sources including the line-list, see [12] ). each point is a district-month. trend lines are shown with 95% confidence intervals (shaded areas). we use a weighted regression method that takes account of the uncertainties in the data [20] . the area of each circle is proportional to the weight of that point (see section 1.7 in s1 text). in the bottom row, the black trend line is for the whole dataset. see figures j and l this effect was not replicated in guinea, which suggests we have less understanding of drivers of transmission in that country. our results also highlight the importance of exposure to individuals who are dead or dying from ebola outside the funeral context, as has been observed in past outbreaks [4, [23] [24] [25] 34, 35] . cases who died were more likely to be named as contacts than those who survived, with most transmission occurring within a few days on either side of the reported date of death-coincident with the timing of peak viral load [34, 36] . this reinforces existing evidence [4, [23] [24] [25] 34, 35] that exposures to individuals who are dead or dying from ebola contribute to transmission, even where those exposures occur outside the specific context of funerals. as in past outbreaks [3, 4, 23, 25] , and consistent with other analyses of the current outbreak [29] , such exposures have most often been between close family members, perhaps explaining why most such contacts have occurred in the household. early hospitalisation in facilities with the ability to isolate patients effectively-a key element of control efforts for ebola-is clearly a priority to reduce community transmission. investigations of the initial phase of this epidemic, as well as previous outbreaks, have highlighted the potential role of within-hospital transmission before or shortly after ebola is identified as the causative agent [37, 38] . here we found that hospitalisation, defined broadly as anything from visiting a clinic to admission to an etu, reduced transmission risk, showing that prompt hospitalisation may be an effective intervention as long as appropriate control measures are applied. however, hospitalisation did not eliminate transmission risk, indicating that improvements in infection control are needed in many health care settings. it should be noted that the meaning of date of hospitalisation may be different among cases, depending on how many and which types of facilities they visited in the course of their disease, and furthermore that there is potential for misclassification in the recording or interpretation of information on hospitalisation. in particular, while the health care facility of first admission was commonly reported, patient transfers to other facilities may not have always been recorded. thus, there may be ambiguity about the type of health care facility a case was in at a particular time. such missing information reduces our ability to resolve differences between types of facilities in their effectiveness at implementing case isolation, which could lead to underestimation of differences between facility types in isolation effectiveness. however, our results are broadly consistent with analyses of the outbreak in conakry [29] . half of reported non-funeral exposures where the matched contact was eventually hospitalised occurred after the reported date of hospitalisation. these results underscore the importance of further improving infection control in all health care facilities, but particularly in non-etus. however, in contexts where hospital bed capacity is insufficient or infection control in health care settings is imperfect, greater consideration might be given to measures to reduce within-family exposure-for example, via education and/or providing a home protection kit with hand disinfectant, protective equipment, and clear guidance for those caring for sick family members. another striking feature of the epidemic revealed by our analysis is the high level of heterogeneity in the number of times a case is named as a potential source contact. such heterogeneity has been observed for some other emerging infectious disease epidemics [26, [39] [40] [41] , in particular the middle east respiratory syndrome coronavirus outbreaks [42] . in principle, understanding the drivers of this heterogeneity might allow for the design of targeted interventions. however, our analysis found very few epidemiological predictors for being named as a source contact multiple times, suggesting that simple demographic characteristics are unlikely to pinpoint those most at risk of super-spreading. heterogeneity in transmission, particularly when associated with transmission in close communities, implies that epidemic trajectories may be difficult to predict at a local level [43, 44] . local flare-ups are possible when case numbers are low and declining. continued vigilance during the ongoing declining phase of the epidemic is essential. the data we have analysed have several limitations. not all ebola cases are recorded in the national databases shared with who. moreover, data on exposures were reported by only approximately one-third of cases. the remainder of cases may not have had the chance to report exposures or may have been unable to recall any. additionally, data collection teams and methods varied by country, district, and hospital facility, meaning observational or collection bias may have affected the data. all data (e.g., symptoms at presentation) were either selfreported or reported by friends/family or inferred by the interviewer and may therefore suffer from subjectivity and recall bias. in particular, there may be biases in the exposures reportedfor example, cases may recall funerals or exposures to family in more detail or have different perceptions of what constitutes exposure. recall and data entry errors (e.g., spelling mistakes, mistyped dates, and misclassification) and missing data may have affected our results, for example, they limited our ability to match the contacts named in the reported exposures. we accounted for this in our analyses as much as possible by discarding inconsistent data, imputing missing data, or explicitly accounting for noise. however, some biases may remain. performing these quantitative analyses on such a large scale was only possible due to the enormous commitment of ebola responders in the region throughout the epidemic, which is particularly remarkable given the very challenging circumstances in the affected countries. in generalising our findings, we need to recognise the relatively unique nature of this crisis. measures that were promoted, including emphasising bed capacity, safe funerals, behaviour change, and community mobilisation, followed the recognition that targeted case finding and contact tracing could not be scaled to meet the exponentially growing burden during the early phase of the epidemic. however, these latter measures, supported by continued community mobilisation, should be prioritised during new ebola outbreaks and during the end phase of the current epidemic, when capacity can meet need. our analyses provide a quantitative basis for prevention measures against the spread of ebola, but also highlight the challenges faced in the field. a compassionate response protocol needs to acknowledge that, as we found, most reported potential transmissions occurred between family members close to the time of death of the case. this provides evidence that ring vaccination methods, such as those trialled in guinea [45] , may be an effective way of delivering vaccines against ebola in the context of limited supply or in hard-to-reach populations. hospitalisation was found to be protective, but left substantial room for improvement. the association between reported funeral attendance and district-level epidemic trends highlights the continued need for improving access to and acceptability of safe burials of ebola cases. more robust associations could be detected using case-control studies, but these are hard to coordinate during an emergency situation. in their absence, the analyses reported here have already provided key insights into the drivers of the epidemic. heterogeneities in transmission made the road to ebola elimination likely to be marked by episodic flare-ups [46, 47] . ebola is controllable using the simple measures that have already been implemented in this outbreak, but complacency as case numbers decline could prolong the epidemic for months. continued real-time data capture, reporting, and analysis are vital to track transmission patterns, inform resource deployment, and thus hasten elimination of the virus from the human population. genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak emergence of zaire ebola virus disease in guinea ebola haemorrhagic fever outbreak in masindi district, uganda: outbreak description and lessons learned the epidemiology of ebola hemorrhagic fever in zaire ebola haemorrhagic fever in zaire report of a who/international study team what factors might have led to the emergence of ebola in west africa ebola virus disease in west africa-no early end to the outbreak geneva: world health organization geneva: world health organization who ebola response team. ebola virus disease in west africa-the first 9 months of the epidemic and forward projections who ebola response team. west african ebola epidemic after one year-slowing but not yet under control ebola virus disease among children in west africa who ebola response team. ebola virus disease among male and female persons in west africa fips 180-4. gaithersburg (maryland): national institute of standards and technology world health organization. case definition recommendations for ebola or marburg virus diseases. geneva: world health organization model selection and multimodel inference: a practical information-theoretic approach estimating individual and household reproduction numbers in an emerging epidemic a new framework and software to estimate time-varying reproduction numbers during epidemics regression techniques for the detection of analytical bias deming: deming, thiel-sen and passing-bablock regression. version 1.0-1. r project for statistical computing vienna: r foundation transmission of ebola hemorrhagic fever: a study of risk factors in family members ebola hemorrhagic fever associated with novel virus strain, uganda ebola hemorrhagic fever transmission and risk factors of contacts superspreading and the effect of individual variation on disease emergence rapid assessment of ebola infection prevention and control needs-six districts geneva: world health organization chains of transmission and control of ebola virus disease in conakry, guinea, in 2014: an observational study cluster of ebola virus disease controlling the last known cluster of ebola virus disease-liberia epidemiology and risk factors for ebola virus infection in sierra leone decreased ebola transmission after rapid response to outbreaks in remote areas containing hemorrhagic fever epidemic, the ebola experience in uganda ebola hemorrhagic fever, kikwit, democratic republic of the congo, 1995: risk factors for patients without a reported exposure the contribution of ebola viral load at admission and other patient characteristics to mortality in a médecins sans frontières (msf) ebola case management centre (cmc) transmission dynamics and control of ebola virus disease outbreak in nigeria the reemergence of ebola hemorrhagic fever, democratic republic of the congo temporal variability and social heterogeneity in disease transmission: the case of sars in hong kong predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore super-spreaders in infectious diseases middle east respiratory syndrome: a global health challenge multiscale, resurgent epidemics in a hierarchical metapopulation model dynamics and control of diseases in networks with community structure the ring vaccination trial: a novel cluster randomized controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to ebola ebola transmission linked to a single traditional funeral ceremony liberia confirms ebola case two months after being declared free of the disease the authors would like to thank the many field staff from guinea, liberia, and sierra leone involved in ebola case finding, detailed data collection, treatment and testing of patients, and data entry. this study was carried out in support of the ebola response in guinea, liberia, and sierra leone, and is based on data routinely collected by national and international staff in collaboration with who.the authors would also like to thank sir david cox for his helpful advice. the conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the u. s. department of health and human services, the public health service, the centers for disease control and prevention, or the authors' affiliated institutions. use of trade names is for identification only and does not imply endorsement by any of the groups named above. conceptualization: imb aco cad nmf cf hlm. key: cord-264486-o01s0upf authors: du, wenjun; yu, jinhong; wang, hui; zhang, xiaoguo; zhang, shouwei; li, qiang; zhang, zhongfa title: clinical characteristics of covid-19 in children compared with adults in shandong province, china date: 2020-04-16 journal: infection doi: 10.1007/s15010-020-01427-2 sha: doc_id: 264486 cord_uid: o01s0upf aims and background: the covid-19 outbreak spread in china and is a threat to the world. we reported on the epidemiological, clinical, laboratory, and radiological characteristics of children cases to help health workers better understand and provide timely diagnosis and treatment. methods: retrospectively, two research centers’ case series of 67 consecutive hospitalized cases including 53 adult and 14 children cases with covid-19 between 23 jan 2020 and 15 feb 2020 from jinan and rizhao were enrolled in this study. epidemiological, clinical, laboratory, and radiological characteristics of children and adults were analyzed and compared. results: most cases in children were mild (21.4%) and conventional cases (78.6%), with mild clinical signs and symptoms, and all cases were of family clusters. fever (35.7%) and dry cough (21.4%) were described as clinical manifestations in children cases. dry cough and phlegm were not the most common symptoms in children compared with adults (p = 0.03). in the early stages of the disease, lymphocyte counts did not significantly decline but neutrophils count did in children compared with adults (p = 0.02). there was a lower level of crp (p = 0.00) in children compared with adults. there were 8 (57.1%) asymptomatic cases and 6 (42.9%) symptomatic cases among the 14 children cases. the age of asymptomatic patients was younger than that of symptomatic patients (p = 0.03). even among asymptomatic patients, 5 (62.5%) cases had lung injuries including 3 (60%) cases with bilateral involvement, which was not different compared with that of symptomatic cases (p = 0.58, p = 0.74). conclusions: the clinical symptoms of children are mild, there is substantial lung injury even among children, but that there is less clinical disease, perhaps because of a less pronounced inflammatory response, and that the occurrence of this pattern appears to inversely correlate with age. since the first atypical pneumonia case was reported in wuhan, china on december 31, 2019 [1] , the pathogen was soon identified [2] and tentatively named 2019-ncov by the world health organization (who) [3] . on 11 feb 2020, the world health organization officially named it sars-cov-2 (severe acute respiratory syndrome coronavirus-2) and the sars-cov-2 infection was named as the covid-19 (coronavirus disease 2019) [4] . the infection quickly spread in china, and internationally [5, 6] and is still on-going. previous studies reported the main clinical characteristics of covid-19. fever, cough, shortness of breath, muscle ache, confusion, and headache were described as clinical manifestations. laboratory tests and chest computed tomographic (ct) scans were also evaluated and the results suggested that decreased lymphocyte counts and bilateral pneumonia were common clinical features, especially in severe cases [7, 8] . at present, there is no effective anti-virus drug and vaccine for covid-19, so early detection and isolation treatment are important to control the progression and spread of the disease. however, the current data mainly come from hubei province, especially from wuhan city. clinical features, especially in children, have not been reported outside hubei province. in this study, we analyzed and compared the epidemic characteristics and clinical features in children and adults in shandong province, china. the aims were to help health workers better understanding the clinical features of covid-19 in children and provide timely diagnosis and treatment. the present study is a retrospective descriptive clinical study. a total of 67cases including 53 adult and 14 children cases of confirmed covid-19 from the jinan infectious diseases hospital and rizhao people's hospital, which were the designated hospitals in jinan and rizhao city, between 23 jan 2020 and 15 feb 2020 were enrolled in this study. the project was approved by the ethics board of jinan infectious hospital (no. 20200203). informed consent was obtained from each patient or their guardian. all patients enrolled in this study were diagnosed according to the pneumonia diagnosis and treatment plan for the new coronavirus infection formulated by the national health commission (trial version 5) [9] . diagnostic criteria for asymptomatic cases: individuals infected by sars-cov-2 who remain asymptomatic throughout the course of the infection with or without abnormal chest ct imaging findings. diagnostic criteria for mild cases: mild clinical symptoms, no radiographic findings of pneumonia. diagnostic criteria for common cases: fever, respiratory symptoms, and radiographic manifestations of pneumonia. diagnostic criteria for severe cases: (1) respiratory distress, respiratory frequency ≥ 30 times/min; (2) hypoxemia, with resting oxygen saturation ≦ 93%; and (3) arterial partial oxygen pressure (pao 2 )/oxygen absorption concentration (fio 2 ) ≦ 300 mmhg (1 mmhg = 0.133 kpa). the diagnostic criteria for critical cases: (1) respiratory failure and mechanical ventilation is required; (2) shock; and (3) complicated with other organ failure requiring icu care. there are four types of infection: (1) imported cases defined as a history of a sojourn in hubei province within 14 days of onset of the illness. (2) family cluster defined as 3 or more than 3 people with confirmed cases in one family. (3) close contact defined as close contact with cases from hubei province. (4) unclear; the method of infection is unknown. a child is defined as less than 18 years of age. the medical records of patients were analyzed. information recorded included demographic data, exposure history, symptoms, signs, laboratory findings and chest computed tomographic (ct) scans. epidemiological, clinical, laboratory, and radiological characteristics data were obtained with data collection forms from electronic medical records. the date of disease onset was defined as the day when the symptom was noticed. throat swab specimens were collected from patients with suspected covid-19. covid-19 was confirmed by rt pcr using the same protocol. the dual-target detection kits were provided by the shanghai jienuo company. results: the cut-off value was 40, a ct value < 37 was positive, a ct value > 40 was negative, and 37-40 was a gray area (the diagnosis needs to be repeated). to characterize the effect of sars-cov-2 on the biochemical indicators and production of cytokines in the acute phase of the illness, plasma cytokines (pct, crp, and il-6) and biochemical indicators were measured using the human cytokine standard 27-plex assays panel and the bio-plex 200 system(bio-rad, hercules, ca, usa) for all patients according to the manufacturer's instructions. the normal range for each indicator is shown in table 2 . retrieved data were recorded into microsoft ® excel for mac (version 16.30) and analyzed. the statistical package for social sciences version 16.0 (spss 16.0;spss inc., chicago, il, usa) and the prism statistical software package (version 5.0; graphpad software inc. la jolla, ca, usa) were used. measurement data were described as mean ± standard deviation. background factors were compared using student's t-test (numerical data) or the χ 2 test (categorical data). differences were regarded as significant if the p value was less than 0.05 on either side. a retrospective analysis was performed on patients in jinan and rizhao, shandong province from 23 jan to15 feb 2020. there were 53 adult cases among the 67 cases, with a median age of 41.47 years (range 21-65 years), and 26 cases (49.1%) were males. most of cases were mild (8 cases, 15.1%) and conventional (44 cases, 83%), with only 1 severe case (1.9%). the most common symptoms at onset of the illness were fever (60.4%), dry cough (54.7%), phlegm (37.7%), pharyngalgia (35.8%) and fatigue (32.1%). less common symptoms were, headache (20.8%), anorexia (15.1%), myalgia (13.2%), chest distress (11.3%), nausea (5.7%), diarrhea (3.8%), dizziness (3.8%), and vomiting (1.9%) ( table 1) . there were 14 children cases among the 67 cases, with a median age of 6.2 years (range 0-16 years), and 6 cases (42.9%) were males. all the cases in children were familial clusters (p = 0.00), with 3 cases (21.4%) of the mild type and 11 cases (78.6%) of the conventional type and no severe or critical case. clinical symptoms in children were mild or even absent, only 5 (35.7%) cases showed signs of fever, 3 cases (21.4%) had dry cough, and 1 case had phlegm, other clinical signs of headache, fatigue, pharyngalgia, and myalgia were rare in children. none of the children developed anorexia, chest distress, dyspnea, nausea, vomiting, diarrhea, dizziness, or abdominal pain. however, dry cough and phlegm were not the most common symptoms in children compared with adults (p = 0.03) ( table 1) . laboratory tests of children and adults were analyzed. the results showed that the white blood cell counts of children were all normal, with decreased neutrophil counts (p = 0.00) and increased lymphocyte counts (p = 0.00) compared with adults ( table 2) . considering the physical characteristics of the children with lymphocytes making up 60% of the white blood cells before 6 years and the proportions of lymphocytes and neutrophils in white blood cells being close to that of adults after 6 years, we further analyzed the difference in blood cell counts between children over 6 years and adults. there are 5 cases over 6 years (35.7%) among 14 children cases, the neutrophil counts (1.62 ± 0.34 × 10 9 /l) were more decreased in children over 6 years than in adults (p = 0.02). however, lymphocyte counts (1.91 ± 0.93 × 10 9 /l) showed no difference (p = 0.41) between the two groups. covid-19 caused liver function damage, cardiac muscle damage, and coagulation function changes. the abnormal rate of alt, ast, ldh, ck, myo, pt, and d-dimer was 17%, 7.5%, 18.9%, 15.1%, 3.8%, 13.2%, and 35.8% in adults and 7.7%, 7.7%, 50%, 28.6%, 0%, 7.1%, and 35.7% in children, respectively ( table 2 ). the value and positive rate of ldh in children were more significantly increased than in adults (p = 0.01; p = 0.02) ( table 2 ). according to evaluation of infection indicators, the value of pct showed no difference between children and adults, however, the value and positive rate of crp were more significantly increased in adults than in children (p = 0.00, p = 0.02) ( table 2 ). of the 67 cases, 56 cases had complications of lung injuries (83.6%) including 11 children and 45 adults. a total of 6 children (64.5%) and 30 adults (66.7%) had lung injuries with bilateral involvement, with no difference between the two groups (p = 0.45). however, lung injuries in adults were more severe compared with that in children ( table 2 , fig. 1 ). there were 8 (57.1%) asymptomatic cases and 6 (42.9%) symptomatic cases among the 14 children cases, with a median age of 3.98 years, and 5 cases (62.5%) were males in the asymptomatic cases and with a median age of 9.17 years, and 1 case (16.7%) was male in the symptomatic cases. the age of asymptomatic patients was younger than that of symptomatic patients (p = 0.03) ( table 3) . the white blood cell and lymphocyte counts were more decreased in symptomatic children cases than that in asymptomatic children cases (p = 0.04, p = 0.04). although there were statistically significant differences in levels of cr and ast between asymptomatic and symptomatic children cases, these differences were within the normal range and had no practical clinical significance. even among asymptomatic patients, 5 (62.5%) cases had lung injuries including 3 (60%) cases with bilateral involvement, with no difference compared with that of symptomatic cases (p = 0.58, p = 0.74) ( table 3 ). the severe acute respiratory syndrome corona virus 2(sars-cov-2) infection outbreak has spread in china and around the world [10, 11] . who defined it as a public health emergency of international concern (pheic). coronaviridae (covs) are the largest known single stranded rna viruses [12] . they have been categorized in three groups, alpha-covs, beta-covs, and gamma-covs according to phylogenetic analyses and antigenic criteria [11] . the human severe acute respiratory syndrome (sars) virus, the middle eastern respiratory syndrome (mers) virus, and the sars-cov-2 all belong to beta-covs [13] . the bat coronavirus (bcov) and the sars-cov-2 share 96.2% sequence identity. bats were once thought to be the source of sars-cov-2. however, sufficient evidence is lacking. a total of 67 cases including 53 adult and 14 children cases, of which only 1 case was the severe type and no one was in critical condition, is far lower than reported in wuhan [14, 15] . there were no deaths reported because of few cases of the severe or critical type. the reason for this is considered as follows: (1) there is no major epidemic in the regions except hubei province for strong prevention and control and (2) the virulence and pathogenicity of the virus decrease in the 2nd and 3rd generation of transmission [16] . all cases of children belong to the mild or conventional type of covid-19, and all children are part of family clusters. previous studies found no cases in children, who were once thought to be less susceptible [17] . according to the current trend, all people including children are susceptible to sars-cov-2, and person-to-person transmission develops familial clusters [18] . the reason why all children are in family a retrospective analysis on clinical features of children of covid-19 compared with that of adults from two research centers was made. the findings showed that clinical symptoms were mild in children with fever and dry cough being the most common symptoms, and other symptoms were rare. however, dry cough and phlegm are not the most common symptoms in children compared with adults. this is because a lower inflammatory response to lung injuries causes milder clinical symptoms in children compared with adults. previous reports indicated that decreased lymphocytes counts were common clinical features, especially in severe cases [7, 8] . as a result of viral infection, white blood cell and lymphocyte counts can be reduced by consumption. however, similar results did not appear in our study, with only a total of 6 cases including 1 case of a child with decreased lymphocytes counts, and the value of lymphocytes counts showed no significant differences in children over 6 years compared with adults. on the other hand, neutrophil counts decreased in children compared with adults (p = 0.02). so, lymphocyte decline is not an important indicator for the diagnosis of childhood cases and the neutrophil count decline should be focused on. these may be due to a lower inflammatory response in children. sars-cov-2 infection may induced lung injuries, liver function damage, cardiac muscle damage, kidney damage, and coagulation function changes [7, 8] . among these laboratory tests, elevated ldh is more common in children than in adults (p = 0.02). an elevated ldh is commonly seen in cardiopulmonary disease and inflammation. the elevated ldh suggests that the sars-cov-2 infection appears to be more likely to cause cardiopulmonary injury and inflammation in children, although lung inflammation in children is less severe than in adults. of course, since the level of ldh is not high in children, it may not have practical clinical significance. both crp and il-6 are major indicators of inflammation. an elevated level of crp and il-6 was shown in adults, but not in children, which suggests that inflammation caused by viral infection, especially in the lungs, is less severe in children than in adults. in addition, lung injuries are not uncommon in children and are characterized by bilateral involvement, which is similar to that of adults. however, lung injuries in adults were more severe compared with children (fig. 1) . of the 14 children cases, 8 cases were asymptomatic cases. the age of asymptomatic patients was younger than that of symptomatic patients. the white blood cell and lymphocyte counts were more decreased in symptomatic children cases than that in asymptomatic children cases, which was due to a lower inflammatory response in asymptomatic children cases. even among asymptomatic patients, 62.5% of cases had lung injuries. therefore, lung injuries caused by sars-cov-2 is still relatively obvious. the ct findings may further provide the reason why elevated levels of crp were shown in adults, but not in children and elevated levels of ldh are common in children. the spike-protein (s) of sars-cov that mediates entrance to human respiratory epithelial cells by interacting with cell surface receptor angiotensin-converting enzyme 2 (ace2) is the most important means of pathogenesis [19, 20] . the number of ace2 receptors are significantly lower in children than in adults and more in the lungs than in other organs [20] , which may be the primary reason why children have fewer clinical symptoms and organ dysfunction than adults and lung injuries even among asymptomatic children cases. in addition, the weak immune response triggered by viral infection because of children's weakened immune function maybe responsible for this state. in summary, most cases in children were mild and conventional cases, with mild clinical signs and symptoms, our data suggest that there is substantial lung injuries even among children, but that there is less clinical disease, perhaps because of a lower pronounced inflammatory response, and that the occurrence of this pattern appears to inversely correlate with age. acknowledgement thanks to dr. edward c. mignot, shandong university, for linguistic advice. author contributions yjh takes responsibility for the integrity of the data and the accuracy of the data analysis. yjh, wh, zxg, and zsw had full access to all data in the study. dwj had the idea of and designed the study. lq contributed to the review and zzf approved the final version. the authors have no conflicts of interest to disclose. ethical approval the project was approved by the ethics board of jinan infectious hospital (no. 20200203). pneumonia of unknown cause-china, emergencies preparedness, response, disease outbreak news, world health organization (who) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia chinese center for disease control and prevention. coronavirus disease (covid-19) situation reports chinese center for disease control and prevention. coronavirus disease (covid-19) situation reports epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan interpretation of "guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 5 a novel coronavirus from patients with pneumonia in china the continuing 2019-ncov epidemic threat of novel coronaviruses to global health: the latest 2019 novel coronavirus outbreak in wuhan, china origin and evolution of pathogenic coronaviruses genomic variance of the 2019-ncov coronavirus who. novel coronavirus (2019-ncov) situation reports. situation report-23 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china coronaviruses post-sars: update on replication and pathogenesis early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster coronavirus spike proteins in viral entry and pathogenesis comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations key: cord-269457-i02brfzr authors: kabba, mustapha s.; forde, martha y.; beckley, kevin s.; johnny, bernadette; jah-kabba, ann-marie b. m.; seisay, samuel b.; dawoh, alusine m.; ogundiran, temidayo title: gossypiboma with perforation of the umbilicus mimicking a complicated urachal cyst: a case report date: 2020-10-17 journal: bmc surg doi: 10.1186/s12893-020-00904-7 sha: doc_id: 269457 cord_uid: i02brfzr background: a retained surgical sponge, also known as a gossypiboma, is a rare cause of serious postoperative complications. diverse retained surgical materials including instruments such as clamps and sutures have been reported, but surgical sponges are the most common material. we report an unusual case of a gossypiboma mimicking a complicated urachal cyst that led to perforation of the umbilicus. case presentation: a 38-year-old female patient presented in our facility with a palpable periumbilical mass and discharge of pus from the umbilicus for 7 months after an open appendectomy. since the onset of symptoms, the patient had been treated conservatively in a peripheral hospital where she had been operated on. as no improvement was seen, an ultrasound scan was performed that suggested an intraperitoneal abscess adjacent to the umbilicus. consequently, the patient was referred to our specialist outpatient department for surgical intervention. suspecting a complicated urachal cyst, an exploratory laparotomy was performed but revealed a retained surgical sponge as the underlying cause. the gossypiboma was resected, and the postoperative period was unremarkable. conclusion: this case demonstrates that gossypibomas, even though rare, continue to occur. they may clinically and radiologically mimic other pathologies, especially abscesses and tumors. preventive measures as well as the inclusion of gossypibomas in the differential diagnosis of intraabdominal masses or fistulation detected in patients with a history of surgery are of utmost importance to minimize morbidity, mortality, and potential medicolegal implications. a retained surgical sponge, also known as a gossypiboma, is a rare but serious postoperative complication that is a nightmare for every surgeon. a range of retained surgical materials including instruments such as clamps and sutures have been reported, but the surgical sponge, possibly because of its amorphous composition and frequent usage, is the most prevalent material [1, 2] . for reasons such as fear of litigation, the exact incidence of gossypiboma is difficult to ascertain [1] . silva and sousa estimated its incidence to be 1:1000-1:1500 laparotomies [3] , whereas bani-hani et al. estimated it to be much lower (1:5027) [4] . gossypiboma may lead to chronic abdominal pain, an abdominal mass, bowel obstruction, occult infections, fistulae and transmural migration of the foreign body into the small and large bowel or stomach [1] [2] [3] . we report a case of a gossypiboma with spontaneous perforation at the umbilicus mimicking a complicated urachal cyst, which, to the best of our knowledge, has not been reported before. a 38-year-old female patient presented to our facility in october 2019 with chronic abdominal pain, swelling in the umbilical region, and pus discharge from the umbilicus for 7 months. the swelling started 3 months after an open appendectomy was performed in a peripheral private hospital using a right paramedian incision. one week after the appearance of swelling, the umbilicus started discharging thick offensive fluid, which was associated with abdominal pain. there was no fever, nausea, or loss of appetite. the patient reported a weight loss of 5 kg in 7 months. conservative treatment yielded no improvement. as a result of an abdominal ultrasound scan, which suggested an intraperitoneal abscess ( fig. 1) , the patient was referred to our specialist outpatient department. upon presentation, the patient was depressed, dehydrated, and pale. the abdominal examination revealed an everted umbilicus with pus discharge and granulation tissue. in the umbilical region, there was a palpable intraabdominal mass that measured 20 × 16 cm, was moderately tender, and was fixed to the umbilicus but was otherwise mobile (fig. 2a ). there were no signs of peritonitis, and the abdomen was soft with moderate periumbilical distension. the right paramedian appendectomy scar had healed completely and was unremarkable. a routine blood examination showed a hemoglobin concentration of 9.6 g/dl and a white blood cell count of 3.0 × 10 9 /l. further diagnostic tests and imaging could not be performed due to financial constraints. based on the history and physical findings, a provisional diagnosis of an infected urachal cyst or postoperative intraabdominal abscess was made, and the patient was prepared for an exploratory laparotomy. intraoperatively, the abdominal cavity was clean. a well-circumscribed 18 × 16 cm adhesive mass was seen around the ileocecal junction. it consisted of an encapsulated lesion at its center, and an ileal segment, the transverse colon, and the umbilicus were all adhered to the mass (fig. 2b ). during adhesiolysis, the wall of the central lesion was accidentally opened, thus surprisingly exposing a surgical sponge and thick offensive pus. the bowel loops were further mobilized, resulting in a large mesenteric defect. impaired blood supply to the dependent bowel loop necessitated the resection of a 10 cm long ileal loop, and an end-to-end ileo-ileal anastomosis was created. adhesiolysis and mobilization of the lesion from the transverse colon led to a 3 cm iatrogenic defect, which was sutured. finally, the encapsulated lesion, including the umbilicus, was (fig. 2c) . the resectate measured 10 × 8 cm in size and contained a retained surgical sponge (fig. 2d) within a confined collection of pus. the postoperative period was uneventful. bacteriological examinations were not carried out due to financial constraints. consequently, empiric antibiotic therapy with intravenous ceftriaxone and metronidazole was administered for 5 days and was then switched to oral antibiotics, which were given for an additional 2 days. early postoperative enteral feeding was well tolerated with normal bowel activity on the third postoperative day. the laparotomy wound healed by primary intention, and the patient was discharged on postoperative day 9. follow-up visits in our sop clinic have also been unremarkable. gossypiboma is described as a retained surgical sponge or towel after a surgical procedure [1, 4] . other terminologies, such as textiloma, gauzeoma, cottonoid, muslinoma, and cottonballoma, are used to refer to this condition. the most common site of presentation is the abdominal cavity. however, other locations, including the pleural and pericardial cavities, the extremities, the central nervous system, and the breast, have been documented [5] . to the best of our knowledge, a gossypiboma with perforation at the umbilicus has not been reported before. a retained surgical sponge is a preventable postoperative complication that usually occurs in emergency surgeries, unexpected surgical procedures, prolonged operations, and surgeries on unstable and obese patients. furthermore, it is associated with inexperienced staff, poor organization in the operating theatre, and rapid or failed sponge counting during an operation [2, 6] . the possible predisposition to the occurrence of the retained foreign body in our patient at her initial surgery could have been an emergency procedure in a poor organizational setting with inadequately trained staff. essentially, there are two types of pathological reactions provoked by a retained foreign body: an exudative inflammatory process with the formation of an abscess or an aseptic fibrotic reaction that encapsulates the corpus alienum [6, 7] . in our patient, the gossypiboma led to the formation of an abscess and discharge of its liquid content at one of the weakest points of the abdominal wall, which in this case proved to be the closely related umbilicus-a process equivalent to the commonly observed spontaneous drainage of abscesses via fistula formation. intraabdominal gossypibomas are usually reported with signs of an intraabdominal mass, bowel obstruction, intestinal hemorrhage, or fistulation. rarely, the retained surgical sponge undergoes transmural bowel wall migration without leaving a defect [1, 2, 8] . the diagnosis of gossypiboma is often challenging, as there is generally a wide range of nonspecific manifestations, and most times, the actual diagnosis is not even considered. in line with this phenomenon, neither the peripheral hospital nor our facility included gossypiboma in the differential diagnosis. the presentation of our patient with a periumbilical mass and discharge of pus at the umbilicus prompted the potential differential diagnosis of an infected urachal cyst. incomplete urachal obliteration during fetal development gives rise to distinct malformations of the median umbilical ligament, such as a urachal cyst. most anomalies are asymptomatic and resolve during early infancy, but some go unrecognized until adulthood. these rare cases can present with acute abdominal symptomatology secondary to infection of the urachal remnant [9] . the clinical presentation of urachal cysts is nonspecific. urachal cysts are mostly asymptomatic until infection occurs. however, a triad of symptoms consisting of a tender midline infraumbilical mass, umbilical discharge, and sepsis should arouse suspicion of an infected urachal remnant, such as a urachal cyst. without surgical excision, a urachal cyst may slowly increase in size and drain through the umbilicus into the bladder or both, resulting in an alternating sinus [10] . in our patient, a high index of suspicion for gossypiboma should have been raised by the history of surgery associated with persistent abdominal pain, signs of infection, and a palpable mass. a preoperative diagnosis of gossypiboma using the clinical manifestation and recommended imaging modalities was observed by rajiv et al. in only 75% of cases, highlighting the fact that a significant number of gossypibomas are diagnosed only intraoperatively [11, 12] . in the case of radiopaque impregnation of a foreign body, diagnosis may be facilitated by radiographic and ct images. the sponge we evacuated showed no discernible impregnation, which may have been due to the primary absence of impregnation, as especially seen in resource-poor settings or disintegration [2, 10] of the radiopaque material. in such cases, the foreign object may not be readily identified by imaging. removal of the retained foreign body by either open or laparoscopic surgery is the mainstay of treatment. however, if the retained foreign body migrates into the stomach, it could be endoscopically removed [6] . prevention of gossypibomas is essential and can be achieved by "simply" ensuring and insisting on a thorough pack count before closure of the operating cavity. a routine brief but careful postoperative wound and cavity exploration prior to wound closure should be performed by every surgeon. the use of sponges with radiopaque markers is now strictly recommended. moreover, newer technologies such as radiofrequency chip identification and barcode scanners are helpful for decreasing the incidence of gossypiboma [13] . however, radiopaque chip identification and barcode scanners are only available in highly developed centers. theatre staff should be regularly sensitized and trained to avoid this preventable condition. this case shows that the diagnosis of gossypiboma can be complicated by its nonspecific presentation, which clinically and radiologically imitates other common pathologies, such as abscesses and tumors, and rare conditions, such as urachal cysts. moreover, gossypiboma is associated with significant morbidity, mortality, and potential medicolegal implications. consequently, prevention of its occurrence and the inclusion of gossypiboma in the repertoire of actively thought differential diagnoses in patients with intraabdominal masses or fistulation concurrent with a history of surgery are of utmost importance. complete transmural migration of a retained surgical sponge: an atypical case in image mimicking intussusception: a case report gossypiboma: complete transmural migration of retained surgical sponge causing small bowel obstruction gossypiboma after abdominal surgery is a challenging clinical problem and a serious medicolegal issue retained surgical (gossypiboma) gossypibomas, a surgeon's nightmare -patient demographics, risk factors, imaging and how we can prevent it transgastric migration of gossypiboma remedied with endoscopic removal: a case report preventableerrors in the operating room: retained foreign bodies after surgery a transmural migration of a gossypiboma in the right colon responsible for a mass which mimicked an abscessed colonic tumor: a case report surgical management for an infected urachal cyst in an adult: case report and literature review infected urachal cyst in an adult: a case and review of the literature gossypiboma posing as a diagnostic dilemma: a case report and review of the literature a study of abdominal gossypiboma initial clinical evaluation of a handheld device for detecting retained surgical gauze sponges using radiofrequency identification technology not applicable. 1 none. not applicable. ethical approval was given by the medical ethics committee of connaught hospital, university of sierra leone teaching hospitals complex, freetown, sierra leone. for publication of this case report, including the images contained within, informed written consent was obtained from the patient. a copy of the consent form is available for review by the editor of this journal. the authors declare that they have no competing interests. key: cord-010027-r0tl01kq authors: nan title: dublin pathology 2015. 8th joint meeting of the british division of the international academy of pathology and the pathological society of great britain & ireland date: 2015-09-15 journal: j pathol doi: 10.1002/path.4631 sha: doc_id: 10027 cord_uid: r0tl01kq nan the christie nhs foundation trust, manchester, uk one controversy in preparing the rcpath "dataset for tumours of the urinary collecting system [2nd edition])", april 2013 was which who grading scheme (1973 or 2004) to use for urothelial carcinoma. since there is a split within grade 2 (1973) between low grade & high grade, use of both schemes in parallel was recommended. this has the advantage of better indicating where a particular patient lies in the grading continuum & minimises the consequences of 'grading error' for cases close to the threshold between low & high grade in the 2004 scheme, (a critical distinction for management if 2004 who grading is used in isolation). the 2015 nice guidance on bladder cancer (http://www.nice.org.uk/guidance/ ng2) incorporates risk stratification tables for ta/t1 bladder cancer utilizing this parallel who grading recommendation in a multiparameter formula that also considers tumour size, pt classification and the presence/absence of cis or aggressive subtype(s). amongst the more aggressive bladder cancer subtypes is invasive micropapillary urothelial carcinoma and the nested variant. a bladder origin can be difficult to recognise at metastatic sites, especially for micropapillary and discohesive/plasmacytoid subtypes. uroplakin ii has recently become available, is more sensitive than uroplakin iii & may assist within a panel. spindle cell lesions of the bladder are often difficult & loss of cytokeratin expression is common in sarcomatoid carcinoma. there is potential to mistake inflammatory myofibroblastic tumour for a malignant tumour. two molecular pathways for bladder cancer are recognised. fgfr3 at 4p16 is the most frequently mutated oncogene in bladder cancer & is prevalent in low grade papillary tumours. p53 loss of function mutations and loss of rb1 are prevalent in cis. no prognostic molecular test is currently validated for clinical use in bladder cancer, though some (e.g. fish) are in use as an adjunct in diagnosis. indiana university school of medicine, indianapolis, usa some dogmas in testicular pathology do not hold up to scrutiny. the belief that all pure, postpubertal teratomas are malignant is invalid. within this group there is a small subset that is benign; these may be divided into dermoid and non-dermoid types that, however, share features that distinguish them from the usual teratoma of adults. these include absence of: atypia, regressive parenchymal changes, intratubular germ cell neoplasia, and i(12p). also they usually show organoid arrangements, and prominence of ciliated epithelium, squamous cysts and smooth muscle. patients do not need further intervention beyond excision sufficient to establish the diagnosis. cases often interpreted as isolated testicular polyarteritis nodosa because of the presence of fibrinoid vascular necrosis are mostly attributable to chronic, intermittent torsion. they usually present as pain-associated, palpable or ultrasound-detected masses, with the "mass" corresponding to infarct and/or hemorrhage. there are associated chronic vascular changes, with frequent marked intimal hyperplasia of arteries, mural fibrosis of veins, dilated venules and arteriolar hyalinization, consistent with torsion-induced venous outflow obstruction and secondary arterial hypertension. these patients do not develop systemic vasculitis on follow-up. many "sarcomas" in patients with germ cell tumours, especially after chemotherapy, are more correctly regarded as sarcomatoid yolk sac tumours. they are reactive for cytokeratin and glypican 3. they often show characteristic features: nodular growth, myxoid and fibrous stroma, spindled and epithelioid cells, abrupt changes in cellularity, and tumor "ringlets." most are high grade and aggressive. most regressed germ cell tumours can be recognized through a combination of findings, although the only diagnostic ones are a scar with coarse intratubular calcifications or with intratubular germ cell neoplasia. alcohol consumption has doubled in ireland and the uk in the last 60 years. the causes of this increase include increased affordability of alcohol and its widespread availability. as a consequence, the health harms associated with alcohol have dramatically increased. binge drinking and alcohol consumption among by women have risen especially dramatically. for example, the mortality from cirrhosis has doubled in the last 20 years in both men and women. in response to this, the medical profession on both islands have led informal and later formal campaigns to encourage policy change regarding alcohol at a national level. in ireland, this was driven by rcpi. the involvement of the medical profession has had a powerful influence, as doctors do not have a conflict of interest in this matter, in contrast to the alcohol industry. the policy changes advocated include particularly minimum unit pricing (mup), which has been shown to be effective in reducing alcohol consumption, alcohol-related admission to hospital and crime in canada. modelling of the data suggests it would have similar benefits in uk and ireland. this is regarded as the single most important first step. other steps which will help include actions around alcohol labelling, availability and breaking the link between alcohol and sports and leisure promotion. turning off the tap of cheap alcohol will hopefully soon reduce alcohol health harms in uk and ireland  barrett's oesophagus: an evolving challenge for the gastroenterologist p dot o'toole cost-effective surveillance programmes for barrett's oesophagus (bo) needs to be focused at risk groups. it is not only a question of identifying more individuals with bo (initial screening) but screening and subsequent surveillance has to identify at-risk individuals with bo who can benefit most from surveillance or therapy. advances in endoscopic imaging (high resolution endoscopy ([hre] with dye-based chromoendoscopy, electronic chromoendoscopy, and autofluorescence,...) certainly prove beneficial in better detecting dysplasia within known bo and can guide sampling and subsequent therapy. dysplasia can be patchy and easily missed during routine biopsy sampling of bo and adequate training with high resolution instruments is needed to increase detection rates. once dysplasia is detected, endoscopic ablation is recommended. until recently, the standard treatment for hgd was oesophagectomy but endoscopic resection and ablation techniques are now available to eradicate dysplasia and mucosal adenocarcinomas. resecting visible lesions (using endoscopic mucosal resection [emr] or endoscopic submucosal dissection techniques) allows full pathological t staging. when invasive cancer is eliminated at multidisciplinary review (i.e., purely mucosal neoplasia confirmed with a nodal risk <2%), further endotherapy to ablate residual metaplasia in bo can be performed using radiofrequency ablation (raf). in a tertiary centre use of staging emr is frequently necessary (>60%) in patients referred for endotherapy and expert endoscopy is required to ensure safe and complete oncological resection. conversely pt1 submucosal cancers detected following emr are confidently triaged for oesophagectomy. combination of emr and rfa in expert groups exceeds >95% for eradication of neoplasia and metaplasia. adverse events are quite low (stricture form healing, 4%; self-limited haemorrhage, very rare perforations ...) and recurrence of bo is also low (<10% at 5 years). careful follow-up endoscopies is necessary at 3 to 6 months initially; intervals theerafter probably yearly.  the pathologist's role in the diagnosis and management of neoplasia in barrett's oesophagus p c muldoon st. james's hospital, dublin, ireland the last decade has seen a revolution in the management of neoplasia in barrett's oesophagus. more detailed biopsy protocols, along with the advent of sophisticated local resection and ablation techniques, have radically altered the management of this expanding cohort of patients. these new treatment modalities, coupled with a massive increase in the numbers of cases of barrett's being diagnosed, have significantly altered the demands placed upon pathologists involved in this area. these changes have presented pathologists with an opportunity to challenge our existing practices, to improve the reproducibility of our analysis and to increase the clinical relevance of the way in which we report neoplasia in this setting, where the pathologist plays a critical role in the multidisciplinary management approach. this talk aims to outline a practical approach to the handling of these specimens and to provide clear guidelines as to how to report them in the most clinically useful way.  modern management in ibd p mwr vieth 1 ; h neumann 2 1 klinikum bayreuth, pathology, bayreuth, germany; 2 university hospital erlangen, medical clinic i, erlangen, germany ulcerative colitis and crohn's make a distinct histological picture. around 80% of an ibd diagnosis is the clinical information and about 20% derives from histology, only. for routine purposes it is recommendable in case of a first manifestation of an ibd to make a diagnosis such as: "picture of ulcerative colitis or picture of crohn's disease" and to recommend a follow-up endoscopy with biopsies not prior to 8 weeks after the first endoscopy and than confirm the diagnosis later to exclude mimickers of ibd. in crohn's disease it is helpful to take biopsies from the upper gi-tract to get further hints of crohn's disease. in case of neoplasia, the guidelines leave some room for local endoscopic treatment of low grade dysplasia whereas high grade dysplasia is still seen as an indication for operation since there is a high probability of detecting a carcinoma in the operation specimen afterwards. operation means on normal complete proctocolectomy. this has been indivdually questioned in the last time. there are exceptions for discussing the indication of an operation in ibd: cases with numerous pseudopolyps that cannot be searched for neoplasia, low grade lesions that cannot be completely removed, multiple neoplastic lesions and unresponsiveness to medical treatment.operation and endoscopic specimen in ibd need a subtile search for neoplastic lesions. a microscope with reverse light may help to identify suspicous lesions and may help to decide where exactely to cut a specimen. in conclusion a tight cooperation between clinical and histopathological partners is recommended to reach a high standard for patient care. second opinions may help to achieve and fuel the own learning process esp. in an institution with a lower number of ibd patients during the year. colorectal or bowel cancer screening (bcs) is commonplace and organised national screening programmes have been developed in many countries, most notably in western europe. traditionally, faecal occult blood (fob) detection has been the screening method of choice, those testing positive being selected for subsequent colonoscopy, but this is changing, with alternative or additional screening tests gaining favour. most of the problems in bcs pathology practice are particularly related to fob-based screening programmes, as these are enriched for large, bleeding sigmoid adenomas, in comparison to programmes utilising endoscopy as the primary screening modality. experience within the closely related uk bcs programmes to date has yielded several recurring problems: the diagnosis of stage pt1 or 'polyp' cancers, in particular distinguishing common epithelial misplacement from 'true' invasion; the management of stage pt1 cancers, in relation to indications for surgical intervention after such a diagnosis; and the minimum criteria for a biopsy diagnosis of colorectal adenocarcinoma. these issues will be discussed with illustrative examples, along with the somewhat more mundane but highly important practical issue of measuring various parameters related to bcs pathology. the importance of quality assurance measures to ensure high standards within bcs pathology is emphasised. with the introduction of colorectal cancer screening in various countries of the eu there is a sharp increase in the incidence of early colorectal cancer. a significant part of these early tumours presents in a pedunculated polyp. in most cases, these carcinomas are already completely removed by polypectomy. classic risk factors that suggest a high risk for lymph node metastases include haggitt level 4, positive resection margins, poor differentiation and lymphatic or vascular invasion. however, the evidence is rather thin. most pt1 studies are performed on sessile polyps. risk factors are more firmly established and include differentiation grade, lymphatic invasion, kikuchi level sm3 or the presence of budding. however, for a clinical useful decision model, we will need an integrated approach, and both specificity and sensitivity of the various factors should be taken into account. radical surgery seems overtreatment for a large number of polyp cancers. leeds university, leeds, uk minimum datasets have changed cancer reporting. this talk will explain the decision making processes behind the latest datasets both for cancer reporting and bowel cancer screening. it will also look at where we may be going in the future for staging and molecular reporting. the intestines play host to a broad spectrum of infective organisms ranging from viruses, through bacteria , fungi and unicellular parasites to worms. the spectrum of infections seen varies with geographical location, due to socioeconomic factors and due to changes in human behaviour. immunocompromisation due to infections (in particular hiv), malignancy (especially haematological tumours) and the use of immunosuppressive drugs also has an important role in determining the infections commonly seen in the gi tract. the typical pathological features of intestinal infections will be discussed together with suggestions on how to optimise the diagnosis of such pathologies. erasme university hospital, brussels, belgium pathologists are confronted with different types of colitis, most commonly infectious colitis and inflammatory bowel disease (ibd) followed by microscopic colitis and ischaemic colitis. several other forms of colitis, however, exist and might be underrecognised; these diseases include segmental colitis associated with diverticulosis, diversion colitis, eosinophilic colitis and behcet's colitis. clinical presentations of these rare types of colitis vary, and laboratory data are often non-specific; mucosal biopsy is essential in establishing the diagnosis. segmental colitis associated with diverticulosis (scad) is mainly characterised by the involvement of the sigmoid colon with sparing of the rectum and proximal colon. scad often mimics ibd at endoscopic and histological examination; since scad has a self-limited course that resolves without further recurrence or need for treatment, the implications of an inaccurate diagnosis are obvious. diversion colitis is a non-specific colonic inflammation following surgical diversion of the faecal stream. is is characterised by a chronic lymphoplasmacytic infiltrate, and the existence of lymphoid follicular hyperplasia is considered to be a hallmark feature. the development of diversion colitis is attributed to a lack of short chain fatty acids. eosinophilic colitis is etiologically obscure and can be associated with involvement of other sections of the gastrointestinal tract. an infiltrate of eosinophilic granulocytes is found to varying degrees in all wall layers. a history of food intolerance or allergy is present in most of the patients, and peripheral eosinophilia is present in 80% of cases. gastrointestinal involvement has been reported in up to 25% of patients with behcet's disease. in cases with ileocolonic involvement, it is often difficult to distinguish behcet's disease from other inflammatory bowel diseases. the diagnosis, therefore, often depends on clinical manifestations and intestinal ulcerative lesions. s·8  how to write a paper and get it published p cs herrington 1 ; p dm berney 2 1 university of edinburgh, edinburgh, uk; 2 barts health nhs trust, london, uk scientific papers have a predetermined structure, and writing in this way requires practice. most journals accept only a small fraction of submitted papers and it is important that any paper has something specific to say; and says it in a clear and concise way that can be understood by editors, reviewers and readers, all of whom play a role in assessment of its contribution. editors look for novelty and significance in the context of the aims and scope of their journal; and scientific rigour, which expert reviewers help them to assess. writing a paper and having it assessed by a journal is an iterative process. during the writing phase, the scientific rigour of the argument can be refined; and following submission and peer review, reviewers and editors often make constructive comments that help to improve it still further. the peer review process therefore acts not only as a quality filter but also as a mechanism for quality improvement. writing papers and submitting them for publication is therefore generally a positive experience, particularly if one remembers that the process is iterative and (inevitably) not all papers will be accepted for publication by the first journal that they are sent to.  large-scale routine diagnostics using whole-slide imaging in sweden -the linköping experience p c lundström cmiv, linköping university, linköping, sweden this presentation will describe the large-scale routine usage of wsi at linköping university hospital, sweden. since 2011 all histology slides are scanned, amounting to more than half a million slides to date. to a significant extent the digital images are used for primary review. the initial implementation led to several of the benefits foreseen with digital pathology, but it could also be concluded that further development was needed to unlock the full potential, in particular within the it solutions. therefore, a consortium led by cmiv, linköping university was formed in 2012 to create innovations for a new generation of digital pathology. this triple helix consortium also includes industry and more than half of sweden's health care providers, an engagement that reflects the dominating view in swedish pathology that large-scale adoption of wsi practice is possible and desirable. this talk covers the experiences made during the initial digitization, including laboratory process adjustments, and the later additions to the digital pathology toolbox accomplished by the ongoing innovation project. apart from obvious targets such as the pathologists' workstation, the developments also touch upon other areas including grossing and enterprise image management.  digital pathology -are we there yet? p sm hewitt national cancer institute, bethesda, maryland, usa the implementation of whole slide imaging for diagnostic histopathology is far more complex than connecting an instrument to a server, and placing a computer on a pathologist's desk. the technology is additive to the histology workflow, with additional cost beyond the current practice of review with a microscope. the adoption of digital pathology for histomorphologic diagnosis requires the restructuring of the workflow, additional technology advances beyond the imaging instrument, and development of new tools to assist the pathologist. the end goal is to improve pathologist's productivity and provide additional diagnostic information. digital pathology, to succeed must become a value-added proposition. the adoption of digital pathology requires: 1)improvements in scanner performance as measured by defined quality metrics. 2)advancement in server and networks to distribute images to the desktop efficiently. 3)software to facilitate review and diagnosis, beyond presenting only and image of the slide. evolution of the current technologies is required to provide an economic impetus for widespread adoption and use of digital pathology in the diagnostic setting. to a significant extent, the distinction between melanocytic naevi and malignant melanomas is based on tissue architecture. amongst the best known architectural features pointing to malignancy are absence of lesional symmetry and maturation, and presence of melanocyte ascent. however, each of these three features has significant pitfalls. as a rule, naevi are 'roughly symmetrical' and melanomas are not, but there are asymmetrical naevi (traumatized naevi, most larger congenital naevi; some combined naevi; some large acral and genital naevi) and symmetrical melanomas (including many small melanomas, especially small nodular melanomas; some spitzoid melanomas). in addition, it is not always clear whether a lesion should be considered 'roughly symmetrical' or not. i suspect that not uncommonly, a diagnosis is reached first, and the verdict regarding symmetry is adjusted according to that diagnosis. similar caveats relate to absence of maturation as an indicator of malignancy. it is seen in blue naevi and all its variants; deep penetrating naevi; some bap1 naevi. melanomas not uncommonly feature smaller cells in their deeper parts, or there may be an underlying naevus remnant with smaller cells. naevi with ascent include many spitz naevi; reed naevi; some naevi in early infancy; traumatized naevi; naevi of acral skin. over-interpretation of ascent may result from inexperience with melan-a and some other immune stains. melanomas devoid of ascending melanoma cells comprise a wide variety of subtypes including, desmoplastic melanomas and, vexingly, some spitzoid melanomas. these architectural features must, therefore, be evaluated in the context of all other findings, and with a 'splitter's' mind set, taking into account the individual characteristics of the specific naevus and melanoma variants that are of relevance to the case under study. most melanomas are fairly easy to diagnose on histological grounds. however, melanoma is a tumour that can histologically mimic almost any other tumour including epithelial and mesenchymal neoplasms. pathologists need to familiarize with the wide histological appearances of melanoma to avoid serious misdiagnoses. of crucial importance is the knowledge that a number of melanomas can closely mimic benign naevi. some variants of melanoma represent distinctive clinicopathological entities and these include desmoplastic melanoma, "malignant" blue naevus, pigment synthethizing melanoma, naevoid melanoma, spitzoid melanoma and epidermotropic metastatic melanoma. tumoral melanosis refers to complete regression of a melanoma, a diagnosis that it is often missed because of the absence of tumour cells within the regressed area. a small percentage of melanomas display focal or extensive histological changes that closely mimic other neoplasms and often a combination of histological features with immunohistochemistry is necessary to arrive to the correct diagnosis. microscopic variants of melanoma include adenoid (pseudoglandular), angiotropic and angiomatoid, signet ring cell, balloon cell, clear cell, rhabdoid and follicular (with exclusive involvement of hair follicles). some melanomas display heterologous differentiation also known as transdifferentiation. the latter should not be confused with the so-called collision tumour in which a melanoma co-exists with a neoplasm of different lineage. a wide variety of heterologous differentiation has been described in melanoma including osteosarcomatous and chondrosarcomatous (mainly seen in acral melanomas), meiomysarcomatous, rhabdomyosarcomatous, neuroendocrine, ganglioneuromatous and even epithelial. except for desmoplastic and pigment synthetizing melanoma, all other variants of the tumour have the same behaviour as ordinary melanomas. melanocytic tumours with spitzoid features represent one of the most challenging and controversial areas in dermatopathology. what is currently known as spitz naevus was initially reported as "juvenile melanoma" by sophie spitz on 1948. she recognized the relatively indolent but somewhat unpredictable behaviour of these distinctive melanocytic lesions that are particularly common in young children. over the years, the histological spectrum of these tumours was expanded, and it has become clear that classical spitz naevi follow an entirely indolent disease course. the prognosis of tumours with atypical histological features remains somewhat unpredictable. this presentation will give an overview of the morphological spectrum of spitzoid melanocytic tumours, their behaviour and recent advances of their molecular characteristics. optimisation is a core tenet in radiography and involves the radiographer ensuring that images of diagnostic quality are produced with minimum radiation dose burden to patient and staff [1, 2] . in paediatric practice this is particularly important due to the more radiosensitive nature of the child [1] . in alignment with the isrrt 2013 world radiography day theme 'radiographers optimise dose', radiography students in an institution submitted clinical case study coursework that focused on paediatric radiation dose optimisation. the purpose of the current study was to analyse these case studies as examples of prevailing radiographic practice and to compare students' perception of optimisation with the evidence within each case. the evidence of optimisation was established through independent and objective image analysis along with thematic analysis of the case commentaries. the case study evidence demonstrated that optimised techniques were generally well implemented. the exception was collimation, which was sub-optimal in 84% (n=31) of the examinations, and on average irradiating an area 27% larger than necessary. students were generally able to correctly identify techniques as optimal or not. however, when appraising exposure, positioning and collimation, between 9% and 33% of students were inaccurate in their assessment of what is optimal. overall the study reflects positively on current irish paediatric radiography with regard to dose optimisation, although more accurate collimation needs to be practised. similarly student perceptions show good understanding of optimal techniques, although appreciation of exposure, positioning and collimation errors could be improved. the aim of this project was to design and prototype immobilisation devices for children who are unable to independently maintain upright sitting posture during radiographic investigations. while current market devices exist, they are seldom used by radiographers -particularly in europe as their methods of restraint have been deemed 'culturally unacceptable' with some claiming that they are in violation of the human rights of the child [1] . the design challenge was to create devices that were functional (fit for purpose [2], radio-lucent, compliant with infection control and easy to use) while minimising discomfort and intimidation. a search of the literature, prior art, patent landscape and current market devices was performed in order to identify product requirements. triz methodologies -a problem solving, analysis and forecasting tool derived from the study of patterns of invention in the global patent literature were used to identify the physical contradictions underlying the design challenge and generate potential solutions. eight unique concept designs were identified from these methods. these were then evaluated using pugh criteria -a ranking system of the relative merits of each concept based on design requirements identified. four of the eight concepts were chosen to be prototyped: a 3-d printed seat, a swing based template, an acrylic-based support and an adaptable wheelchair. the prototypes were made in collaboration with the ucd school of engineering and tested using paediatric phantoms in ucd radiography department. the final prototypes will be trialled in crumlin children's hospital with a view to future use and development. thrombotic microangiopathy (tma) is a pathology that results in thrombosis of capillaries and arterioles due to endothelial injury. it is usually characterized by an atypical haemolytic syndrome (ahus) or thrombotic thrombocytopaenic purpura (ttp). tma is considered to be caused by infections, drugs, autoimmunity, tumours, pregnancy, transplants and inherited abnormalities involving the alternate complement pathway. this presentation describes the pathology of tma. it includes a retrospective 15 year study (1999 -2013) of all renal biopsies reported by one pathologist. all renal biopsy request forms and reports, where cases included light (lm), fluorescence(fm) and electron microscopy(em), were reviewed. cases without all 3 modalities (lm, fm, and em) were excluded. 6639 biopsies were reported in the study period (328 in 1999 (328 in to 629 in 2013 . 2105 were transplant biopsies. 284 biopsies were insufficient (lm, fm and em all not possible. this resulted in 4250 native renal biopsies as the study group. following review of the reports 641 cases were reported as tma. 66 were associated with thin membrane nephropathy, 41 with minimal change disease and 24 with plasma cell dyscrasia/ b cell malignancy. this resulted in 510 cases with tma as the only pathology reported which represents 12% of all adequate native medical renal biopsies. clinical indications included proteinuria in 73%, nephrotic syndrome in 15%, increased creatinine in 43%, increased blood pressure in 55% and haematuria in 43% of the cases. acute renal failure was described in 10% and hus in just 1% of the cases. pathological changes were predominently arteriolar sclerosis and glomerular double contours on lm with chronic subendothelial injury on em. the conclusions of this presentation are 1. tma is overwhelmingly a chronic lesion as seen in renal biopsy pathology. 2. it is a very common pattern of injury. 3. it is not usually associated with clinical hus or ttp features at presentation. the era of targeted cancer therapeutics has brought forth new challenges for molecular diagnostic laboratories. the list of genes, and indeed specific mutations, that predict drug responses keeps growing, and with it grows the demand for molecular sub-classification of tumors. in colorectal carcinoma, for example, recent studies support expanding testing beyond kras to include nras and braf in predicting resistance to egfr-targeted therapies. similarly, in non-small cell lung carcinoma standard screening for egfr mutations and alk gene fusions may be insufficient when actionable alterations involving ros1, ret, her2, met, braf and other genes are being targeted (successfully) in ongoing clinical trials. fortunately, the introduction of next-generation sequencing (ngs) into the clinical laboratory is meeting the demand. due to its quantitative output, ngs not only provides precise mutant allele ratios, but it can also be used to detect gene gains and losses. furthermore, when applied to rna, ngs supports the detection of gene fusions and serves in assessing gene expression levels. while ngs is a powerful tool for molecularly characterizing solid tumors, the quality of the results in large part rests on the selection of appropriate input material; therefore, review by a pathologist prior to testing remains a cornerstone to success. other growing uses of ngs include monitoring minimal residual disease in the setting of hematologic malignancies, and in the detection of targetable mutations in cell-free dna within the plasma. the advent of next generation sequencing has ushered in an era of tremendous potential for identifying the molecular causation of simple and complex disorders, both rare and common. the successes of next generation sequencing reflect the combined interpretive skills of geneticists, bioinformaticians and clinicians working in close collaboration. in the realm of muscle disease, we have witnessed both the strengths and the weaknesses of next generation sequencing technologies. the use of exome sequencing in patients with rare muscle disease who have been carefully phenotyped has proven to be a successful strategy for identifying causative variants in new genes as well as in known genes. in fact, exome sequencing has significantly expanded both the clinical and the histological phenotypic spectra of muscle conditions associated with causative variants in known genes. in the absence of careful phenotyping or large genetic reference data sets for identifying variants of interest, causative variants may be missed, however. the use of rna sequencing-using rna extracted from muscle biopsy specimens-has proven to be a powerful tool for finding causative variants affecting gene splicing or expression, which may be missed with next generation sequencing. muscle pathology plays an essential role in complementing next generation sequencing. the deep phenotyping of patients with muscle disease relies heavily on muscle histological and immunohistochemical findings in combination with muscle imaging, clinical history and neuromuscular examination findings. examples of how next generation sequencing coupled with careful clinical and histological phenotyping has uncovered causative variants in new genes as well as in known genes will be discussed in this talk. medicine, diagnostic pathology, technology, and diagnostic tests are evolving at an extremely rapid pace. drug developers and diagnostic developers each face unique challenges. companion diagnostic development is a key component of pharma drug development strategy. we will discuss the importance of companion diagnostics in the success of personalized medicine, the fda position on companion diagnostics, and the role, advantages and disadvantages of tissue based companion diagnostics. other technologies, such as next gen sequencing, will increasingly be utilized in a complementary fashion along with traditional slide based immunohistochemical and in situ hybridization. the scope and limitations of available technologies will be reviewed. diagnostic technologies of all types will complement each other to provide the most accurate diagnostic information for clinicians and patients. following the 2008 who classification of haematological malignancies there has been a greater emphasis on the integration of molecular information with clinical and morphological data not just for diagnostic purposes but also to help convey both prognostic and therapeutic information. this talk will concentrate on routine testing in the work-up of common haematological malignancies focusing specifically on clonality and translocation analysis in lymphoproliferations and mutational testing in bcr-abl negative myeloproliferative neoplasms. using case studies to illustrate common indications for testing this talk will also highlight some of the practical points and pitfalls in the interpretation of these tests. beaumont hospital, dublin, ireland "should i keep the brain?" is one of the most frequent questions addressed to neuropathologists by surgical pathology colleagues. fears relating to inappropriate organ retention coupled with decreasing availability of expert neuropathology opinion and the widely held belief that advances in neuroimaging have replaced the brain autopsy, have all contributed to a decline in the post mortem study of human brain tissue. leaving aside the critical relevance of neuropathology to forensic medicine, the vital role played by careful examination of the post mortem brain extends far beyond pathology and has contributed greatly to science and medicine. in general, prolonged retention of entire brains may be avoided. in hospital practice it is uncommon for a patient to die without brain imaging. access to pre-mortem brain imaging will guide the surgical pathologist in careful and appropriate sampling of calvarial, dural, meningeal, vascular and parenchymal central nervous system components. spinal cord examination requires prior experience in spinal cord removal but most post mortem technologists are expert in cord extraction. sampling and appropriate processing of nerve and muscle requires prior experience or neuropathology advice. high quality photography obtained at all phases of post mortem brain examination including the coronally sectioned individual cerebral hemispheres, with retention of blocks from each of the brain lobes together with cerebellum, brain stem, vessels and dura -meninges will ensure that in the event of a neuropathology opinion benign required -that opinion will not be compromised. specific issues which will be addressed will include the death of patients with epilepsy, dementia, stroke and undiagnosed neurological disease. the key learning objective will be to ensure that pathology trainees approach post mortem examination of the nervous system with interest and excitement. following several high profile misdiagnoses in ireland a national quality assurance (qa) programme for cellular pathology in 2009 was initiated with a vision of establishing a patientcentred pathologist-led framework that would enhance the quality of patient care with timely, accurate and complete pathological diagnoses and reporting. national qa guidelines were developed based on 17 key quality activities generating a total of 51 key quality indicators (kqi). examples of the quality activities include turnaround time, monitoring of amended reports, frozen section correlation and various elements of peer review. all 25 cellular pathology departments in the public state-funded hospitals participate in the programme in addition to 7 laboratories within privately-run hospitals. each laboratory enters codes on individual cases designed to capture the relevant kqi and the anonymised encrypted qa data is then electronically extracted from the laboratory information system to a national database. this national central database is managed by a novel information technology system, the national quality assurance intelligence system (nqais)-histopathology, that was designed to process and display the qa data so that each individual laboratory can analyse their own data and also compare their performance to the national average for each kqi. since 2013 complete national data has been inputted into the nqais system and in 2014 initial qa targets were agreed for turnaround time, frozen section correlation and rate of intra-departmental consultation (idc). in 2015 additional targets for autopsy icd, frozen section deferral rate and turnaround time have been added. to our knowledge this programme has enabled ireland to be the first country to publically report national metrics on the quality of their pathology services.  the politics of eqa: the nhs england qa review and its consequences p de hughes in 2013, a review of external quality assessment processes was carried out on behalf of nhs england. the main recommendations of this review related to the strengthening of governance of eqa, both nationally and within pathology provider organisations. recommendations specifically affecting cellular pathology were: (i) professional bodies, led by rcpath, should develop methodologies for assessing the performance of individuals in eqa schemes. (ii) all pathologists reporting pathology results and providing clinical advice should be participate in eqa schemes relevant to their practice, should achieve levels of performance determined by the professional bodies and this performance should be noted at annual appraisal. (iii) where a need to improve performance is identified, additional remedial training should be carried out, or practice in the area of concern should be stopped until appropriate retraining has been undertaken and revalidation achieved. this process should supported and resourced by the employing organisation, as should eqa scheme participation. (iv) interpretative eqa schemes are designed to assess and improve individual performance, and attempts at collusion are considered matters of professional probity. the professional response to these recommendations is expected to be led by the rcpath under the guidance of a newly-established national oversight group working on behalf of nhs england and should be more clear at the time of the pathsoc conference. a key part of this response will be to separately consider the implications of this review for technical schemes, affecting laboratories, and interpretative schemes, affecting individual practitioners. roche tissue diagnostics, tucson, arizona, usa tumour samples to guide treatment decisions have become of increasing significance. most importantly the results of companion diagnostic testing directly influences the management of individual patients as more drugs are approved for treatment of specific molecular distinct subgroups. reporting suboptimal quality test results may be harmful to the patient and cause the mismanagement of a prescribed companion drug. the consequences of unsatisfactory performance and measures for improvement are the responsibility of the laboratory. presently, there are number of eqa schemes for molecular testing available in europe however, their results clearly indicate the need for eqa since 10%-15% of laboratories do not carry out according to the standard set by the eqa provider or utilize standardized procedures. continual improvement programs, internal quality control and validation program assist laboratories however; by using standardized quality practices such as iso 15189 and the use of external quality assurance schemes can provide essential feedback to the laboratory to assure accurate molecular testing results.  how to run a histopathology eqa in the digital age p nj mayer 1 ;p jd oxley 2 1 cork university hospital, cork, ireland; 2 southmead hospital, bristol, uk interpretative eqa schemes in histopathology were first introduced in the uk in the mid-1980s, well before the advent of the internet and high resolution digital images. in this lecture we will outline key developments, as eqa schemes have evolved into the digital era, with particular emphasis on the national urological eqa scheme, which we have run since 2007. we will outline the main practical issues involved in running an eqa scheme and share our personal experience of the development and introduction of the web-based eqalite software, which is being utilised by increasing numbers of schemes. we will address the pros and cons of traditional glass slide-based circulations versus virtual circulations using scanned digital images and show how the digital archive generated from old eqa circulations has become a valuable educational and teaching resource. we will also briefly explore, from an organiser's perspective, the major issues facing eqa schemes in the future, as eqa performance becomes more embedded into revalidation and fitness to practice.  molecular pathology: the future? p cs herrington molecular pathology is already central to stratified medicine. and the ability of pathologists to understand disease phenotype is essential for interpretation of the current explosion in '-omics' data. moreover, the future of stratified medicine will require integration of information from different sources, in the context of disease phenotype, to inform patient management: pathologists are ideally placed to lead this integration. this applies not only to data derived from ex vivo cells and tissues but also to molecular imaging data, which require accurate correlation with cell and tissue phenotype for accurate interpretation. molecular pathology is key to the future of pathology; and this future extends beyond the traditional light microscope the following plenary, oral and poster abstracts have been subjected to peer review. hypothesis: suppressor of cytokine signalling (socs) family members play a vital role in the activation of the jak/stat signalling pathway via a negative feedback loop and have been implicated in the development of cancers. in breast cancer (bc), socs2 mrna has been correlated with oestrogen receptor (er) positive tumours favouring a good prognosis (bmc cancer 2007, 7:136) . this study aimed to determine whether socs2 at the protein level correlates with tumour morphology and low grade in bc. methods: differential expression analysis between tubular and grade matched nsts were undertaken in the metabric cohort. primary breast cancer tissue microarrays (n=1041) were immuno-stained for socs2 and expression patterns correlated with clinico-pathological and molecular variables including outcome. results: differential gene expression analysis on the metabric data identified socs2 as the top gene with a significant overexpression in the tubular type as compared to low grade nsts (adjusted p value=0.004). immunohistochemistry on the tenovus series showed positive nuclear socs2 expression to correlate with tumours of low grade (p<0.0001), low proliferation (ki67 p<0.0001), er/pr positive (p<0.0001) phenotype and tubular morphology (p<0.0001); as well as negative her2 status (p=0.005) and non-triple negative status (p<0.0001). survival analysis revealed significant associations with long term breast cancer specific survival (p=0.019). positive socs2 correlations were also observed with the expression of androgen receptor (ar) (p<0.0001) and stat3 (p=0.001), further indicating its role in these two signalling pathways. conclusions: results from this study suggest socs2 to be a marker of favourable prognosis: identifying low grade, er positive breast tumours with particular correlations to the tubular histological tumour type. background: ovarian cancer is the fifth leading cause of cancer in women and has poor long-term survival, in part, due to chemoresistance. tumour hypoxia is associated with chemoresistance in ovarian cancer. however, relatively little is known about the genes activated in ovarian cancer which cause chemoresistance due to hypoxia. this study aimed to firstly identify genes whose expression is associated with hypoxia-induced chemoresistance, and secondly select hypoxia-associated biomarkers and evaluate their expression in ovarian tumours. methods: cisplatin-sensitive (a2780) and cisplatin-resistant (a2780cis) ovarian cancer cell lines were exposed to combinations of hypoxia and/or cisplatin as part of a matrix designed to reflect clinically relevant scenarios. rna was extracted and interrogated on affymetrix human gene arrays. differential gene expression was analysed for cells exposed to hypoxia and/or treated with cisplatin. potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by rt-pcr. results: a wide range of genes associated with chemoresistance were differentially expressed in cells exposed to hypoxia and/or cisplatin. selected genes [angptl4, her3 and hif-1α] were chosen for further validation in a cohort of ovarian tumour samples, n=35. high expression of angptl4 trended towards reduced progression-free and overall survival. high expression of her3 trended to increased progression-free but reduced overall survival, while high expression of hif-1α trended towards reduced progression-free and increased overall survival. conclusion: this study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. we have also identified many potential biomarkers of hypoxia and platinum resistance and provide initial validation of a subset of these markers in ovarian cancer tissues. methods: analysis of affymetrix™ human exon 1.0st microarray data revealed differentially expressed genes (degs) between a bc group and a control non-bc group. ingenuity pathway analysis (ipa; bioinformatics software) was used to identify networks of the degs. the expression of a micro-network was validated using immunohistochemistry. double immunofluorescence was undertaken to identify the lineage of cells expressing components of the network. results: we identified a network of interacting genes that were upregulated in fcdiib compared to normally formed cortex or fcd without balloon cells (fcdiia). some components of this network were expressed in bcs but others were expressed in novel cell populations. double immunofluorescence identified a cell with the phenotype of a glial progenitor that was only present in fcdiib but not in normally formed cortex. conclusions: we have identified a novel population of glial progenitors found frequently adjacent to bcs in fcdiib. paracrine signaling between bcs and the novel chi3l1 positive cells is likely to be involved in the pathogenesis in fcdiib. further investigations into the role of these cells would give us a better understanding of the molecular abnormalities underlying fcd and possibly provide novel therapeutic targets. excellent anatomical knowledge of the anal sphincter complex (asc) is essential for the treatment and understanding of low rectal and anal pathology. some of the current descriptions of the asc are contradictory. in this study, the three-dimensional (3d) anatomy of the asc is described with relevance to low rectal and anal surgical pathology. six human adult cadaveric specimens (three males, three females) were obtained from the leeds gift research tissue programme. paraffin embedded mega-blocks containing the asc were serially sectioned at 250 µm intervals. sections were stained with haematoxylin & eosin, masson's trichrome and millers' elastin, from which 3d reconstructions were developed. the asc is a complex structure, varying between individuals in the size and distribution of its layers with intermingling of fibres and inconsistency of the longitudinal smooth muscle affecting the creation of the surgical intersphincteric plane. longitudinal fibres penetrate the internal and external anal sphincter to anchor in the submucosa and ischiorectal fossa. striated muscle fibres from the external sphincter were identified in the submucosa in four of six specimens. the asc is highly complex due to the degree of variation in its structure and intermingling of smooth and striated muscle fibres and their penetration of major structures. this creates potential tissue planes for the spread of infection, fistula extension and tumour spread. the complex anatomy of the asc also impacts on the staging of low rectal cancers in this region, which requires further investigation. p h thorpe; a asiri; m akhlaq; d jackson; m ilyas cten is upregulated in a number of tumour types and in colorectal cancer expression is associated with advanced dukes stage, poor prognosis and distant metastasis. cten is localised at focal adhesions and regulates cell motility but knowledge of underlying signalling mechanisms is sparse. epithelial to mesenchymal transition (emt) is a process whereby cells acquire an invasive phenotype to aid cell migration and is found to occur in a number of biological processes including cancer metastasis. we investigated whether cten increases cell migration through emt pathways in colorectal cancer. cten was forcibly expressed in colorectal cell lines and snail expression determined by qpcr and western blot. the cycloheximide pulse chase assay was used to assess any changes in snail protein stability. further to this, the transwell migration assay was performed to investigate changes in cell motility. forced expression of cten was shown to increase snail protein expression in hct116 and caco2 cell lines. there was no change in the level of snail mrna suggesting that cten regulates snail at a post transcriptional level. inhibition of protein synthesis confirmed this and showed that cten regulates the stability of snail protein. simultaneous forced expression of cten and knockdown of snail demonstrated that this relationship was functionally active. forced expression of cten increased cell migration (p<0.05) which was subsequently lost when snail was knocked down (p<0.001). we are the first to identify snail as a downstream target of cten signalling. this finding advances the understanding of cancer cell motility regulatory networks and further highlights cten as a potential therapeutic target in colorectal cancer. work supported by a pathological society grant. treatment strategies for patients with advanced colorectal cancer p sd richman 1 ; gj hemmings 1 ; p chambers 1 ; m taylor 1 ; hm wood 1 ; e tinkler-hundal 1 ; k southward 1 ; jm foster 2 ; a ouime 2 ; kg spink 2 ; p quirke 1 1 leeds institute of cancer and pathology, leeds, uk; 2 affymetrix, high wycombe, uk treatment for advanced colorectal cancer is moving to combination therapies, targeting multiple signalling pathways. indeed, mrc focus4 has been designed to assess this. we determined pten protein expression, and assessed this in relation to other biomarkers associated with signalling downstream of the epidermal growth factor receptor. tissue microarrays were constructed from 2 advanced colorectal cancer (acrc) clinical trials (focus and piccolo) for immunohistochemistry (ihc). mutation status of kras, nras, pik3ca and braf was assessed by pyrosequencing. copy number variation was assessed on oncoscan® ffpe assay kit (affymetrix inc.). pten protein expression was correlated with mutation status, mmr status, primary tumour location and copy number. pten protein expression for 1288 patients showed complete loss of expression in 85/787 (10.8%) -focus and 64/501 (12.8%) -piccolo. braf mutation status was significantly different between the pten negative and pten positive populations (p<0.0001), with significantly more pten negative tumours having the braf v600e mutation. loss of pten expression correlated with genomic deletions involving the pten gene. 20/30 (66%) of pten negative tumours exhibited loss of the pten region (10q), half of which were focal deletions. only 54/202 (26.7%) pten positive tumours showed deletions of this region, and none were focal events. there was no significant difference in either primary tumour site or mmr status (p=0.1765) between the pten negative and pten positive populations. signalling pathways do not stand in isolation; they are interlinked in a complex signalling network. current treatment interventions must target the correct pathway combinations if patients are to benefit from targeted therapy. our data suggests a subset of patients may require dual akt and mek pathway inhibition, in addition to anti-egfr monoclonal antibody therapy and inhibition of braf. zonal differences in pd1 expression in centre of tumour versus periphery in microsatellite stable and unstable colorectal cancer p gm o'kane 1 ; m lynch 2 ; j aird 3 ; s hooper 3 ; c muldoon 1 ; n mulligan 3 ; c loscher 2 ; dj gallagher 3 1 st. james's hospital, dublin, ireland; 2 dublin city university, dublin, ireland; 3 mater misericordiae university hospital, dublin, ireland colorectal cancers (crc) that show evidence of microsatellite instability (msi-h) are marked by a high tumour infiltrating lymphocyte (til) population which is thought to be prognostic. programmed cell death 1(pd-1) is a negative regulator of the immune system and targeting the interaction with its ligand pd-l1 offers a potential therapeutic target. we aimed to characterize cd8 and pd-1 expression in both the tumour centre (ct) and tumour periphery (pt) of microsatellite stable (mss) and unstable crc. methods: paraffin-embedded tumour blocks were cut at 5um, prepared and stained using specific antibodies for cd8 and pd-1. the pt was defined as the area within a 400x high power field (hpf) from the outline of the tumor. the ct was defined as the area at least one 400x hpf apart from the tumor outline toward centre of the tumor. images were taken at 40x, 100x, 200x and 400x. positive cells were averaged across 3 high power fields and classified as high or low positivity. results: forty-two specimens have been analysed to date including 28 msi-h and 13 mss tumours. sixty-eight percent of msi-h were stage ii and 69% of mss were stage iii. in the msi-h group, a high cd8 count in the ct and pt correlated with and earlier tumour size and stage. pd-1 positivity was seen in 61% of msi-h ct compared to 0% positivity in the ct of mss tumours. the periphery of both mss and msi-h specimens showed significant pd-1 expression with 71% and 85% of samples showing positivity respectively. there was no association between high or low densities of staining and stage. conclusions: zonal differences exist in the expression of cd8 and pd-1 in microsatellite stable and unstable tumours. a high proportion of msi-h tumours show pd-1 activity in the centre of the tumour despite an improved prognosis. further profiling of other t cell populations may help to further understand this expression which may act as a biomarker or provide a therapeutic target biomarkers that are able to distinguish stage ii and iii colon cancer patients at high risk of developing disease recurrence, who may benefit from adjuvant chemotherapy, are still lacking. genome-wide profiling of somatic aberrations, including gene point mutations, dna copy number aberrations (cna) and structural variants (sv), is expected to provide better insight into the molecular pathology of tumour progression and clinical outcome. genome-wide analysis of cnas was performed using high-resolution comparative genomic hybridization for microsatellite stable (mss) stage ii and iii primary colon cancer samples (n=114). in addition, the prevalence of genes suffering from cna-associated chromosomal breaks, indicative for svs, was determined. the mutation status of commonly affected apc, tp53, kras, pik3ca, fbxw7, smad4, braf and nras genes was examined for 60 samples using targeted massive parallel sequencing. associations of genomic aberrations with disease-free survival (dfs) rates were explored by log-rank tests using 10,000 permutations. disease recurrence and dfs rates differed significantly for several cna-regions (p<0.05). a total of 267 genes were recurrently affected by cna-associated chromosomal breaks (fdr<0.1), among which 168 genes (66%) that were also identified in a previously analysed cohort of 352 metastatic colorectal cancers. gene point mutation frequencies were in concordance with literature. in a univariate analysis, none of the individual mutated genes appeared to be significantly associated with dfs. in summary, several associations are found between highly prevalent genomic cnas and disease recurrence in this cohort of mss stage ii and iii colon cancers. further in-depth analysis is required to unravel underlying biology that contributes to disease recurrence. km sutton 1 ; d bottomley 1 ; d morton 2 ; p quirke 1 ; p np west 1 accurate and reliable methods for assessing the molecular profile of clinical tumour samples are important for the delivery of personalised medicine. when adopting a targeted amplicon sequencing method in combination with next generation sequencing (ngs), it is ideal to call mutations against a control sample to enable artefacts to be removed from the analysis. blood is considered the gold standard control but may not always be available. we compared the use of histologically normal mucosa to blood as a control in colon cancer. we examined mutations in 40 colon cancers from the ncri foxtrot trial using the fluidigm access array for ngs library preparation. we assessed the use of both blood and normal colonic mucosa as a control for assessing mutations in 11 genes. all samples were tested in duplicate. the work was partly funded by a pathsoc career development fellowship and is presented on behalf of the foxtrot collaborative group. mutation calls made using normal mucosa as a control compared to blood were in good agreement; a mathew's correlation coefficient above 0.7 was seen for all of the genes where agreement could be assessed. we found that false positive mutations were due to poorer amplification of the normal mucosa samples and false negatives were due to mutation calls in the normal mucosa. overall we found that when assessing mutations in hotspot oncogenes, testing in duplicate and the use of a normal control tissue is not required to make mutation calls. however, where a normal control is required, normal mucosa from the resection margin is a suitable alternative to blood where it is not available. we report a series of four unusual ovarian or extraovarian neoplasms composed of an admixture of adenosarcoma and a predominant component comprising a sex cord tumour. the neoplasms occurred in women aged 50 to 69. three cases arose within the ovary and one was extraovarian (pelvis and abdomen) in location. in all four cases, there were minor areas with morphological features of adenosarcoma with a phyllodes-like architecture and periglandular increased cellularity with mitotic figures. in two cases, the stromal component was morphologically in keeping with a juvenile granulosa cell tumour. in one case, the stromal component had some features of both adult granulosa cell tumour and sertoli cell tumour within a fibromatous background. the fourth case morphologically could not be categorised as any of the usual types of ovarian sex cord tumour and was categorised as an unclassifiable sex cord tumour. in all four cases, there was immunohistochemical evidence of sex cord differentiation. in each case, we propose that the sex cord tumour arose from a pre-existing adenosarcoma thus representing an unusual form of sarcomatous overgrowth of sex cord elements which can occur within adenosarcomas. this phenomenon is not well described in the literature. background: human epididymis protein 4 (he4) is a secreted protein that is overexpressed in some cancers. he4 is emerging as a useful biomarker in diagnosis and follow-up of endometrial cancers. the aim of this study was to evaluate the potential role of serum he4 in the diagnosis and management of endometrial cancer. methods: patients undergoing surgery for endometrial disease were recruited into this study and had pre-operative serum samples taken, n=157. demographic, clinical, radiological and laboratory data were reviewed. he4 and ca125 serum levels were analysed using the fujirebio diagnostic elisa kits and results correlated with clinicopathological details. standard cut-off points of 70 pmol/l for he4 and 35 u/ml for ca125 were used. results: he4 showed a sensitivity of 64% and specificity of 97.50% for detection of endometrial cancer. ca125 had a very low sensitivity of 14% for endometrial cancer diagnosis. he4 was elevated in all stages of endometrial cancer and demonstrated the ability to distinguish between benign and malignant groups. he4 also provided information about myometrial space invasion. conclusion: he4 has a role in endometrial cancer diagnosis and prognosis and has the potential to be used in a screening setting or as a triage marker in the primary care setting. for women diagnosed with endometrial cancer, he4 has the potential to stratify them into treatment regimens where the most appropriate treatment can be delivered resulting in improved quality of life and outcome for endometrial cancer patients. platelets drive metastatic changes in ovarian cancer cells p cd spillane 1 ; nm cooke 2 ; s o'toole 3 ; d kenny 2 ; o sheils 1 ; jj o'leary 1 1 histopathology department, trinity college dublin, dublin, ireland; 2 department of molecular and cellular therapeutics, rcsi, dublin, ireland; 3 department of obstetrics and gynaecology, trinity college dublin, dublin, ireland background: ovarian cancer is the 5th leading cause of cancer related deaths in women. previously we described a dynamic interaction between ovarian cancer cells and platelets in vitro, involving platelet adhesion, activation and induction of pro-survival and pro-angiogenic signals in the cancer cells. this study looked to further investigate this phenomenon in ovarian cancer cells by assessing the molecular changes it induced. methods: cell lines 59m and skov3 were used as in vitro models of metastatic ovarian cancer. platelet cloaking of cells was quantified by flow cytometry. cells co-cultured with/ without platelets for 24hrs were examined by rt-pcr for emt related changes and by affymetrix gene2.0st arrays for whole transcriptome changes. results: significantly more platelets adhered to skov3 cells than 59m cells. while there were different rates of adhesion, the platelets induced similar changes in emt related genes in both. there was a significant loss in expression of epithelial genes and an increase in mesenchymal genes, indicating the induction of emt. whole transcriptome analysis showed that there were a greater number of gene expression changes occurring in skov3 cells compared to 59m cells, correlating with the adhesion data. a 32 gene panel of commonly affected genes in both cell lines was identified, many of which form part of an interlinking pathway that is regulated by tgfβ1 and associated with cell adhesion/ecm remodelling. though only 32 genes overlapped, the biological processes affected in both cell lines were very similar, with 103 of the 148 processes enriched in the 59m data set also seen in the skov3 data set. conclusion: this study shows that platelets can enhance the metastatic potential of ovarian cancer cells through the induction of emt and ecm changes. in addition, it has identified a set of 32 genes that hold potential to be in vivo markers of this interaction. background: during the metastatic cascade, circulating tumour cells rapidly and efficiently adopt a platelet cloak. platelet cloaking of tumour cells promotes metastatic disease by promoting cellular proliferation, angiogenesis and emt while inhibiting autophagy and apoptosis. the aim of this study is to examine whether the platelet cloak contributes to tumour cell evasion of nk cell mediated immune surveillance. methods: freshly isolated pbmcs were harvested from healthy donors and stimulated for 18 hours with il-2 (500u/ml). pbmcs were co-incubated with ovarian (59m and skov3), melanoma (sk-mel-28) and cml (k562) cell lines that were either uncloaked, or cloaked with washed platelets from healthy donors. the nk-tumour cell receptor ligand systems, nkg2d-mica/micb and cd96/cd226-cd155 were examined using nk cell cd107a expression and interferon-gamma production to quantify nk cell mediated recognition and 'killing' of cancer cells. results: we first demonstrated that ovarian and melanoma cancer cell lines when cloaked with washed platelets strongly inhibited nk cell antitumor reactivity. platelet cloaking induced down-regulation of the stress ligands mica and micb on the tumour cell coupled with their release into the microenvironment, a known nk cell immune decoy strategy. in addition, platelets significantly down-regulated both cd96 (nk cell) and cd155 (tumour cell), inhibiting nk cell activity. both mechanisms occur in tandem to comprehensively incapacitate nk cells and promote tumour immune evasion. conclusions: ovarian and melanoma tumour cells are efficiently cloaked by platelets, which facilitates immune evasion by actively suppressing nk cell cytotoxicity and cytokine production. purpose of the study: glioblastomas (gbm) are the most common and most aggressive primary malignant brain tumours in adults. one of their histopathological hallmarks is the microvascular proliferation; these tumours are among the most angiogenic of malignancies by displaying the highest degree of microvascular proliferation. igfiir/man-6-p is a receptor that belongs to the insulin-like growth factor (igf) system. the involvement of igf-iir/man-6-p in the process of angiogenesis has been postulated in rare earlier studies. to our knowledge, the role of igf-iir/man-6-p in the neovascularisation of human gbm has never been studied. methods: igf-iir/man-6-p expression was evaluated in the vascular compartment from 322 human gbm and from 10 normal adult brain samples by means of quantitative immunohistochemistry on tissue microarray sections. in vitro cell line experiments were carried out in order to characterise the igfiir/man-6-p role in angiogenesis. summary of results: igf-iir/man-6-p was strongly expressed in the cytoplasm of endothelial cells in hyperplastic vessels and exhibited a dot-staining pattern. we found a higher expression of igf-iir/man-6-p in gbm vessels compared to normal brain vessels (p=0.05). furthermore, preliminary in vitro experiments suggest a role of igf-iir/man-6-p in tube formation but not in growth of the ea.hy926 endothelial cell line. conclusions: this work shows a possible role of igfiir/man-6-p in the process of neovascularisation of gbm angiogenesis. additional investigations are required to confirm the role of this receptor as a direct actor of angiogenesis in gbm. purpose of the study: vasa vasorum (vv) are microvessels which supply vessels that cannot be nourished by diffusion from their own lumina. vv are believed to be a key element in the pathogenesis of vascular diseases. a number of different imaging methods have been used to study the vv but there is still no definitive consensus on their structure. the aim was to describe the normal microvessel anatomy of temporal arteries. methods: human temporal artery, obtained following routine biopsy with ethical approval and patient consent. samples were embedded into paraffin blocks and serially sectioned at 5 micron intervals. alternate sections were stained with h&e and scanned to create virtual slides. the slides were aligned, vv were segmented (annotated) and iso-surfaced to generate 3d reconstructions. summary of results: the reconstruction shows the structural arrangement of the vv as a complex plexus. no connection to the vascular lumen was visualised. in this segment a hierarchical branching structure was not observed. vv were almost exclusively restricted to the adventitia of the vessel wall. mean ± sd area of the vv (n = 5283) is 2287.23µm2 (±4956.03). the mean ± sd number of vessels per slide is 60.76 (±15.37). these metrics are based on one arterial specimen. conclusion: this method allows us to study the three-dimensional spatial relationships of microvessels within arterial specimens. furthermore, metric data generated in the process can support the 3d images to study the microvasculature. this method will be applied to diseased arteries in future to generate novel hypotheses about the inflammatory process. acknowledgements: this research was supported by a pathsoc intercalated studentship. purpose of the study: it is controversial whether mesothelioma can be diagnosed with confidence in effusion cytology and therefore an ancillary marker of malignant mesothelial cells would be clinically valuable. brca-1 associated protein (bap1) is a tumour suppressor gene which shows biallelic inactivation in approximately half of all mesotheliomas. bap1 expression is commonly lost in mesothelioma. we investigated whether loss of bap1 expression can be used to support a diagnosis of mesothelioma in effusion cytology. methods: immunohistochemistry (ihc) for bap1 was performed on cell blocks from effusions associated with confirmed mesothelioma cases, effusions containing mesothelial cell atypia, benign effusions, and effusions from patients with lung adenocarcinoma. results: ihc for bap1 was performed on 75 cases of confirmed mesothelioma. 43 (57.3%) showed negative staining in the presence of an internal positive control. in 57 effusions considered to have atypical mesothelial cells in the absence of definitive diagnosis of mesothelioma, 8 cases demonstrated negative staining for bap1. on follow up, 6 of these patients received a definitive diagnosis of mesothelioma in the subsequent 14 months (2 were lost to follow up immediately). only 5 of 100 consecutive benign effusions were interpreted as bap1 negative. 47 patients with confirmed adenocarcinoma demonstrated positive staining for bap1. conclusion: we conclude that loss of bap1 expression in effusion cytology is quite specific for mesothelioma. whilst it is not definitive, it can be used to support the diagnosis of mesothelioma in atypical effusions. we caution that interpretation of bap1 ihc on cell block may be difficult and that convincing positive staining in non-neoplastic cells is required before atypical cells are considered negative. we also note that bap1 loss is not a sensitive test and cannot be used to exclude mesothelioma. the south-east of scotland experience on the molecular detection of egfr, kras and alk mutations in lung adenocarcinomas p y kheng 1 ; l williams 2 ; k walsh 1 ; j fairley 1 ; s camus 1 ; l gilroy 1 ; k gilmour 1 ; d stirling 1 ; w wallace 1 ; d harrison 1 ; a oniscu 1 1 royal infirmary of edinburgh, edinburgh, uk; 2 the university of edinburgh, edinburgh, uk the approval of novel targeted treatments for egfr-positive and alk-positive non-small cell lung cancer (nsclc) has led to the increased requirement for mutation testing services in south east of scotland. egfr mutations are typically found in females, asians and never smokers whereas kras mutations are associated with smoking. alk rearrangements are commonly found in younger patients and never smokers. this study aimed to determine the prevalence of egfr, kras and alk mutations in south east of scotland and to evaluate our experience in testing of alk with ihc and fish. data of all patients tested were collected retrospectively from clinical records. from january 2011 to may 2014, we reported mutation rates of egfr, kras and alk to be 10.4% (67/643), 35.8% (86/240) and 2.3% (7/304) respectively. in our cohort, an increase in one pack years of smoking resulted in a decrease in the odds ratio of egfr-positivity (or 0.94, 95% ci 0.92 -0.96, p<0.001). kras-positivity was associated with a history of smoking, with rates in both former (or 6.26, 95% ci 2.00-19.56, p=0.002) and current smokers (or 6.82, 95% ci 2.18-21.35, p=0.001) significantly higher than in non-smokers. the number of smoking pack years had no influence on the rates of kras-positivity. alk-rearrangements were found to be associated with never smokers (p<0.001) and younger patients (≤50 years old) (p<0.001). to date, no false positives were reported for parallel testing of alk with ihc and fish. we observed 100% sensitivity (7 ihc+/7 fish+) and 96.6% specificity (113 ihc-/117 fish-) when comparing ihc with fish. in conclusion, the prevalence of egfr mutation in south east of scotland has reflected mutation rates reported in west of scotland. our findings further support the use of alk-ihc as a diagnostic screening tool. purpose of the study: the molecular mechanisms of metastasis and progression of penile squamous cell carcinoma (pscc) are unclear. nobody, to our knowledge, has investigated the expression of cell-cycle proteins in advanced or metastatic pscc. we aimed to determine the extent of hpv infection in patients with advanced pscc and its effect on the expression of the key cell-cycle proteins p53, p16ink4a and retinoblastoma (rb). methods: archival paraffin embedded blocks were obtained from 27 primary penile cancers, all patients having developed locally-advanced or metastatic disease. all patients were treated in the phase ii trial of docetaxel, cisplatin & 5-fluorouracil (tpf) chemotherapy cruk/09/001 (nicholson et al. bjc 2013; 109: 2554-9) . samples were analysed immunohistochemically for p16ink4a, p53 and rb protein expression on a tissue microarray. all tumours were hpv typed using pcr. summary of results: hpv dna was detected in 8/22 (36%) with hpv 16 present in 7/8 (88%). 5 cases were not suitable for analysis. no association was found between hpv and expression of either p16ink4a (p= 0.3426), p53 (p= 0.1365) or rb (p= 1) using fisher's exact test. conclusions: hpv dna is detected in less than half of progressive pscc, suggesting either the loss of hpv in advanced disease or that non-hpv related cancers progress more commonly. the lack of correlation between hpv and these cell-cycle proteins suggests that they may undergo somatic mutation that is not driven by hpv, leading to increased growth and invasiveness. treatment strategies may be hampered by this genetic diversity, which requires further investigation. prostate cancer is the second most common form of cancer in males, and the incidence of this disease is predicted to double globally by 2030. more than 1.1 million new cases of prostate cancer are diagnosed each year and two thirds of these patients are from the western world. current diagnostic tests for prostate cancer are limited in both sensitivity and accuracy, and a method for accurate prognosis in these patients is yet to be developed; therefore, there is a need for a sensitive and specific prostate cancer test to implement early and appropriate therapy. the recent discovery of altered endosomal-lysosomal biogenesis in prostate cancer cells has identified a fundamental change in the cell biology of this cancer that holds great promise for the identification of novel biomarkers that can predict disease outcomes. investigation of the endosome compartment and endosome biogenesis revealed elevated gene and expression of critical machinery components that are required for endosome biogenesis and endocytosis. here we demonstrate significantly altered expression of endosomal and lysosomal genes in mrna microarrays of prostate cancer tissue compared to non-malignant tissue, and that specific endosomal and lysosomal genes are predictive of patient outcomes. two endosomal tri-gene signatures were identified that had a significant capacity to stratify patient outcomes. changes in the expression of these genes was further ascertained by qpcr in fresh-frozen prostate tissue specimens, which further implicated altered endosome biology during disease progression, with significant changes in expression observed between aggressive prostate cancer and indolent disease or normal prostate tissue. these findings support the initiation of a retrospective trial to determine if these new biomarkers can accurately predict clinical progression in prostate cancer patients. m craze; c joseph; c nolan; a green; ea rakha; io ellis; p a mukherjee university of nottingham, nottingham, uk introduction: lymphovascular invasion (lvi) is an important step in the metastatic cascade. identification of a molecular signature for the lvi positive phenotype will help identify relevant drivers and pathways. this study aimed to investigate determinants of lvi from a biomarker database. methods: biomarkers (n >200) from a well annotated series (n=1929) were analysed for correlations with lvi [clinical/ihc (d2-40) supplemented]. proteins with significant associations with lvi were interrogated for pathway enrichment analysis [corrected for false discovery rate (fdr)], using the string 9.1 platform incorporating gene ontology (go), kegg and nci. results: biomarker analysis related to both clinical/ihc determined lvi identified 35 positively associated markers, 14 in both clinical and ihc categories (e.g. ada3, cd8, foxp3, kpna2). a further 21 markers were negatively associated, 8 in both categories (e.g. bcl2, brca1, mage3 and sox10). significant pathways (p<0.001) unifying the positively associated proteins include metabolism, immune responses (t-cell regulation and differentiation), cell activation and transcription [go] ; t-cell receptor signalling pathways and pathways in cancer and haematopoietic cell lineages [kegg] . for negatively associated proteins, the following were significant: ubiquitination processes, regulation of the mitosis [go]; p53 pathways [kegg] and apoptotic and cell cycle pathways [go & kegg] . on cross-validating a subset included in the metabric cohort, there were overlapping enrichments for immune response regulation (go) and haematopoietic cell lineages (kegg). conclusions: these preliminary findings are the first to unify biomarkers for lvi pathway analysis in bc, using protein based data. within the constraints of selection bias, data mining from immunohistochemistry of multiple biomarkers in relation to biological processes hold promise. *am supported by the nihr and the academy of medical sciences abstracts s·17 assessment of her2 status on needle core biopsy of breast cancer: impact of histopathological concordance p m pigera; ahs lee; io ellis; ea rakha; z hodi nottingham city hospital, nottingham, uk one of the key recommendations introduced in the asco/cap update guideline recommendation on her2 testing is the novel concept of "histopathological concordance." it is proposed that certain tumour morphological features such as histologic type and grade should trigger repeating a molecular test in cases of "discordance". in this study we have we have reviewed 3104 breast cancer cases consecutively reported in routine practice in nottingham in the last 4 years. data on her2 status was collected and cases with her2 assessed on resection specimens (rs) were analysed in details. results: of all cases, 98 patients (3%) had her2 status assessed on core biopsy and the corresponding tumour rs. the main reasons for a repeat were tumour multifocality and morphologically different or heterogeneous tumours. a few cases were repeated because of borderline negative fish results or neoadjuvant therapy. 18 cases were repeated due to insufficient tumour in the core biopsy. in this study the her2 status of the index tumour was changed in 2 cases and both were in the borderline result category. her2 testing of different tumour foci of multifocal or morphological heterogeneous tumours was consistent with that of the index tumour assessed on the core biopsy apart from two cases; one positive and one negative. 17 tumours were upgraded from grade 2 on core to grade 3 on excision and her2 status did not change. no contribution of hormone receptor or tumour type was identified. conclusion: there is excellent agreement between her2 assessed in core biopsy and rs. histopathological discordance seems to play a minor role which does not justify test repeat in routine practice. tamoxifen prevents breast cancer in a sub-set of high-risk women in a mechanism that appears to be dependent on reduction of md. animal model studies suggest that tamoxifen remodels the mammary stroma to a tumour-inhibitory phenotype. this study aims to analyse the effect of tamoxifen on breast fibroblast function and identify potential protumourigenic pathways contributing to density-associated risk. methods: primary human breast fibroblasts were treated with hydroxytamoxifen (100nm-5µm). fibroblast function was analysed by measuring: proliferation; expression of stromal proteins fibronectin (fn), lox and collagen 1; effects on tgf-β signalling via smad phosphorylation and upregulation of the myofibroblast marker sma. genome wide analysis was performed using rna-seq. summary of results: fibroblasts from 25 patients were treated with tamoxifen. all patients showed reduced proliferation with treatment. in 62% of patients tamoxifen treatment resulted in reduced expression of fn. tgf-β-mediated upregulation of sma and fn were consistently inhibited by tamoxifen, as was fibroblast contraction of collagen gels. rna-seq analysis revealed modulation of a number of metabolic pathways by tamoxifen, including significant upregulation of dhcr7, part of the microsomal antioestrogen binding site (aebs). conclusions:these data indicate that tamoxifen can directly remodel the stromal microenvironment, generating a less 'reactive' stroma. modulation of aebs activity has been proposed to be anti-tumourigenic, and also is implicated as a suppressor of hedgehog signalling. thus, tamoxifen impacts on multiple pathways to create a tumour inhibitory phenotype. this work was supported by the pathsoc small grant scheme. purpose of the study: the phenotypic features of basal like (bl) breast cancer (bc) resemble those occurring in brca1-germline mutation carriers. several lines of evidence suggesting the overall tendency of basal-like/triple negative bc to spread through vascular rather than lymphatic routes. the latter has recently been attributed to the activation of cadherin switch, an emt-like phenomenon, in blbc. this study aims at studying the cadherin switch expression profile tgfb1, a key emt-trigger, expression in brca1 mutated compared to sporadic bc. the expression of e-cadherin, n-cadherin and tgfb1 were studied in a subset of germline brca1 mutated bc (n= 47) compared to non-selected cohorts of non-lobular sporadic invasive blbc (n= 422) and non-basal bc (n=1190) using ihc and tma. summary of results: compared to sporadic bc, brca1 mutated cases were of younger age, more grade 3, with more medullary-like tumours, and more lvi positive. e-cad was significantly less expressed in brca1 cases than in the sporadic non-basal and in the blbc. however, n-cad was not significantly expressed in brca1, non-basal, and blbc. tgfb1 was significantly less expressed in sporadic bc, both non-basal blbc than brca1 mutated bc. e-cad/n-cad combinatorial expression phenotypes were significantly different between brca1 mutated and non-basal and blbc. higher proportions e-cad-/n-cad+ were significantly observed blbc than non-basal bc. brca1 mutated cases displayed the least e-cad+ expression and the highest e-cad-/n-cad+ in the studied series. conclusions: despite the known similarities between brca mutated and blbc, results of this study demonstrate the more occurrence of cadherin switch in brca1 mutated breast cancer. e-cad repression appears to contribute more than n-cad gain in blbc than non-basal bc. exploring molecular mechanisms underlying lymphovascular invasion in breast cancer p sn sonbul 1 ; a mukherjee 1 ; r russell 2 ; om rueda 2 ; m aleskandarany 1 ; ar green 1 ; e provenzano 3 ; c caldas 3 ; io ellis 1 ; ea rakha 1 the development of tamoxifen resistance (tr) in oestrogen-dependent breast cancer (bc) is a therapeutic challenge. insulin-like growth factor binding proteins (igfbps) may play a role in this process. we have investigated the role of igfbp proteins in tr bc. igf axis genes were evaluated in mcf-7 (wt) cells and tamoxifen-resistant (tamr) variants using qrt-pcr and confirmed by elisa, western, and ligand blotting. igfbp-2 & -5 were knocked down by shrna transfection, and subsequent sensitivity to 4-hydroxytamoxifen (4-ht) was determined via wst-1. cell migration was investigated by using the incucyte system. igfbp-2 expression was evaluated in 424 bc cases by tma immunohistochemistry. five out of 10 genes of the igf axis (igf-ir, igf-2r, igfbp-2, -4 and -5) had the highest expression levels by both parental wt and tamr cells. igfbp-5 was down-regulated by ~7-fold while igfbp-2 was up-regulated by ~2-fold in tamr versus wt cells (mrna and protein levels). significantly, a knockdown of igfbp-2 in tamr cells restored sensitivity to (4-ht), reduced erα expression to 45 ± 11.9% and enhanced cell migration. expression of igfbp-2 was significantly (p< 0.001) associated with survival advantage in tr patients. igfbp-2 and igfpb-5 are reciprocally regulated in the acquisition of tr by mcf-7 cells. igfbp-2 may play a role in the development of tr in vitro and its high levels in clinical samples may predict tr. purpose of the study: several lines of evidence are currently suggesting that the morphologic heterogeneity of breast cancer is mirrored at the genetic level. understanding the molecular genetic evolution of bc would contribute further insights into the molecular derangements driving disease progression. moreover, varied clinical outcome and response to similar therapeutic regimen is attributed, at least in-part to intratumoural heterogeneity. ngs can reliably study the genetic events using miniscule amounts of genomic dna. methods: gdna was extracted from ffpe tissue sections from a case of invasive duct carcinoma (3 primary tumour samples and 3 samples from positive axillary lymph node metastases). sample preparation and exome enrichment was performed using nextera rapid capture exome kits (illumina, fc-140-1000 ). exome sequencing was performed using illumina miseq with 15x depth of coverage (following adapter/barcode trimming). exploratory analyses and data mining were executed regarding variant (s) concordance/ discordance between primary tumour samples and their respective metastatic variants. summary of results: initial findings revealed 37 candidate indels common to all three axillary lymph node samples yet absent from the three primary tumour samples. several genes have been identified as having frameshift mutations caused by indels. molecular players previously linked to anti-angiogenesis are amongst the genes affected by indel mutations in their coding sequences that may lead to potential abrogation of protein function. conclusions: these initial findings provide the framework for detailed molecular analyses for assessing molecular evolutionary events in primary breast cancer and their corresponding metastases. c-myc is amplified in approximately 15% of breast cancers (bc) and is associated with poor outcome. c-myc protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in bc. we hypothesised that the functional role of c-myc differs between molecular subtypes of bc. we therefore investigated the correlation between c-myc protein expression and other proteins involved in cell cycle control, proliferation, apoptosis and dna damage together with clinicopathological parameters, outcome and treatments in early invasive primary bc (n=1,106) using immunuohistochemistry. the metabric bc cohort (n=1,980) was evaluated for c-myc mrna expression. in whole series, there was significant association between c-myc protein expression with higher tumour grade, lymph node(ln) positivity and medullary-like tumours. c-myc showed differential association with other proteins in the molecular classes. in luminal a tumours, c-myc was associated with atm (p=0.005), cyclin b1 (p=0.002), pik3ca (p=0.009) and ki67 (p<0.001). in contrast, in basal-like tumours, c-myc showed positive associated with cyclin e (p=0.003) and p16 (p=0.042) expression. c-myc was an independent predictor of a shorter distant metastases free survival in luminal a ln+ tumours treated with endocrine therapy (et; p=0.013). c-myc expression did not predict patient outcome in the other molecular subtypes with respect to adjuvant treatment. high c-myc mrna expression was associated with higher grade and basal phenotype (p<0.001). in luminal tumours treated with et, c-myc mrna expression was associated with bc specific survival (p=0.001). c-myc function is associated with specific molecular subtypes of bc and confers resistance to et. the diverse mechanisms of c-myc function, particularly in luminal a bc, warrants further investigation. metasin axillary predictive score (maps): a measure of axillary nodal disease prediction to provide an informed choice for breast cancer patients and surgeons p pp gopinath 1 ; d george 1 ; p sai-giridhar 2 ; s jader 1 ; e arkoumani 1 ; s holt 2 ; g francis 3 ; c yiangou 3 ; s al ramadhani 4 ; s el sheikh 5 ; n agrawal 3 ; v sundaresan 1 purpose of study: intra-operative sentinel lymph node sampling and molecular analysis empowers the surgeon to carry out axillary clearance as a one-step process. we have recently completed the clinical validation of metasin, an intraoperative molecular assay for sentinel lymph node analysis in breast cancer patients (1836 cases). method: the assay uses 2 positive predictive markers and is quantitative, enabling the prediction of tumour volume using 2 markers ck19 and mammaglobin. in this study group, 439 patients had positive sentinel nodes and 444 cases underwent axillary clearance. of the axillary clearance cases, 26% contained positive lymph nodes. 84% were sentinel node (snb) macrometastases, 5% were snb micrometastases and 11% were snb negative or contained isolated tumour cells. informative data was available for sentinel nodes from 125 positive cases. results: using the qpcr values (from metasin assays using standardised pre-mixes) and clinical axillary clearance data, the cases have been stratified on the basis of the involvement of other axillary nodes. we have shown a three-tiered predictive grouping exists: group a includes low tumour volume disease with a nodal positivity of 25% within the axilla (n=16): group b with a 44% positivity of other nodal involvement (n=80) and group c with positivity of 73% of axillary clearances (n=29). the clustering of the metasin data is dependent on the qpcr results and shows that the cases can be sub-grouped to provide a probability basis for prediction of axillary nodal involvement; dependent on the qpcr cut offs. this gives the patient and surgeon a statistical basis for determining the likelihood of other axillary nodal disease. sequencing of the brca1 and brca2 genes has long been used in genetics laboratories to identify cases of familial breast and ovarian cancer. however, the advent of chemotherapy for ovarian cancer based on parp inhibitors, which requires the presence of a brca1 or brca2 mutation, is turning this specialist test into a commonly-applied companion diagnostic. at the same time, the introduction of new dna sequencing technologies is posing challenges even for experienced genetics laboratories. emqn has been providing eqa of brca1 and brca2 gene sequencing world-wide for 15 years. the rate of serious diagnostic errors has varied from year to year, but the mean has hovered stubbornly around 3%. in eqa, just 3 samples per year are sent out, and the quality and experience of participating laboratories varies greatly. we recently carried out a collaborative study to measure the quality of brca gene sequencing by traditional and new methods in 20 experienced, expert laboratories from 11 countries. ten dna samples (8 with pathogenic mutations, 2 with normal dna sequence) were sent to each laboratory. ten labs used next-generation sequencing (ngs) alone, 3 used sanger sequencing alone, and the others used combinations of sanger sequencing, ngs, mlpa and other technologies. seventeen (85%) of labs identified all clinically-significant variants on all 10 samples. four false negative results were reported by 3 labs. two were due to deficiencies in the bioinformatics pipeline of the ngs process, while 2 were attributed to a sample swap, and incorrect interpretation of a melting profile. no significant trend was identified with respect to the genotyping accuracy of the different methodologies used. the observed error rate of 2% amongst expert laboratories indicates the complex and challenging nature of this kind of testing. caution will be required when applying these technologies to sub-optimal ffpe samples in pathology laboratories. p n wolstenholme 1 ; sj patton 1 ; z deans 2 ; s abbs 3 ; j coxhead 4 ; k brugger 3 ; p westwood 5 ; k thomson 6 ; h scheffer 7 next generation sequencing (ngs) is increasingly being introduced into clinical diagnostic laboratories worldwide. the huge amount of data generated by ngs cannot be duplicated by alternative methods for laboratories to internally validate all results, therefore external assessment of data is required. the uk national external quality assessment scheme (ukneqas) for molecular genetics and the european molecular genetics quality network (emqn) have developed a joint eqa scheme for ngs, with the aims to: (a) assess and improve quality; (b) enable laboratories to benchmark their ngs service against others and against best practice; (c) work towards consistency of reporting clinical results generated by ngs; and (d) contribute towards best practice. emqn and ukneqas offer numerous disease-specific, molecular pathology and technical eqa schemes. the objectives for developing ngs eqa were to make it generic (independent of genes, diseases, platforms, and testing context (e.g,. somatic, germline etc)) and applicable all users. two pilot eqas have been run and 157 labs from 32 countries participated. these labs were sent a genomic dna sample and asked to sequence either their smallest gene panel or largest single gene which the lab tested, submit technical details, and genotypes at known snps. the results were compared against a "consensus eqa genome" established by multiple validations of the dna. 12187 different genes were tested. most labs are using small panel of 1-10 genes. 60% of all variants were detected by every lab which tested for them. a detailed summary of the key findings will be presented. both pilots have proved to be challenging to meet our objectives, however the results have enabled clinical diagnostic labs to start to address the quality of their ngs testing. tumours invade the vasculature, which transports circulating tumour cells (ctcs) to distant sites enabling growth of secondary tumours. ctcs hold the potential to monitor: therapeutic response, emergent mutations and act as a screening tool for the early detection of cancer. there are numerous methods to isolate ctcs. once isolated, epcam and/or panck positivity and cd45 negativity are used to verify ctc status. however, due to the metastasis associated process of epithelial-mesenchymal transition, epithelial markers may be ineffective at identifying all ctcs. to overcome such protein marker based limitations, we have developed a novel staining pipeline (ctc-5) that combines histochemical staining (giemsa) with immunofluorescene (dapi, epcam/panck, her3 and cd45) staining and whole slide imaging for robust identification, enumeration and characterisation of ctcs from cancer patients. ctcs are isolated from whole blood using screencell cyto devices. cyto devices are then slide mounted, giemsa stained and digitised. giemsa staining is washed out and slides are immunofluorescently stained for epcam/panck, cd45, her3 and counter stained with dapi. fluorescently stained slides are digitised. giemsa stained and four colour immunofluorescent digital slides are processed in silico generating a single z-stacked digital slide for pathological assessment. the ctc-5 staining pipeline has been experimentally validated via ctc characterisation of peripheral blood from lung, breast and ovarian cancer patients, with respect to healthy donor and spiked-in controls. the ctc-5 pipeline overcomes recognised weaknesses in ctc characterisation. histochemical staining is added to the current gold standard of epcam/panck and cd45 staining, while also preserving a fluorescent channel for assessment of biomarker status (e.g. her3, apoptosis or platelet cloaking). such advancements enable robust pathological assessment of ctcs in the clinic. thorough interrogation of diseased tissue requires the use of multiple biomarkers in order to investigate biological pathways. unless fluorescent technology is used, multiple sections are required from each tissue block as each section can only be tested for a limited number of markers. histogenic molecular mapping (hmm) is a technique which used digitized images to evaluate multiple biomarkers. although each section cut from a block is slightly different from the immediately preceding section, the similarity is sufficient to allow non-linear registration of images of successive sections. if the order is known, multiple sections can be mapped onto each other by registering each with the immediately preceding section. this allows several biomarkers to be mapped into a single "composite" section thereby giving a representation of the pathways activated/expressed in the tissue. we used hmm to investigate the mismatch repair pathway in colorectal cancer. sequential tissue sections were stained for mlh1, pms2, msh2 and msh6 and then scanned. bespoke computational algorithms were used for image registration and composite images were binned as either "mismatch repair proficient" or "mismatch repair deficient". validation of each category could be obtained by quantification of pixels in binarized images or pixel distribution using stereology. our data show that hmm can be used for interrogating biological pathways in tissue sections and, ultimately, automated diagnosis of disease states. personalising whilst pre-operative radiotherapy is the standard of care in locally advanced rectal cancer (larc), only half of patients respond. individualised treatment based on a predictive test could avoid unnecessary radiation exposure in poor responders. macrophages in the tumour microenvironment with tumoricidal m1 and tumour protective m2 phenotypes could be modulating this response. this study investigated the possible predictive value of m1 and m2 subpopulations in identifying the response to short-course radiotherapy (scrt). pre-treatment biopsies and post-treatment resection samples were taken from 29 patients with larc given scrt. dual-staining immunohistochemistry was performed with cd68, hla-dr (m1 marker), and cd163 (m2 marker). samples were scored for hot-and-random spots by nuance software (version 3.0.2) and compared with tumour response measured by reduction in tumour-cell density. the work was partly funded by a pathsoc career development fellowship. samples showing a low score for hla-dr positive m1 macrophages exhibited a better response to scrt with a median 80% reduction in tumour cell density (iqr 47 to 85). those with a high score exhibited a poor response with only a 20% reduction (iqr 0 to 49, p=0·017). no such trends were observed for cd163+ m2 macrophages. the ratio of hla-dr+ to cd163+ macrophages for biopsy and resection samples was significantly different showing a drop in the hla-dr positive macrophages in the resection samples (biopsy median 2·53, iqr 1.98 to 3.08; resection median 1·38, iqr 0.96 to 1.80; p=0·024). assessment of macrophage subpopulations in pre-treatment biopsies appears to predict the degree of response to scrt in larc. further investigation to validate these findings is now required prior to developing a predictive test for use in routine clinical practice. patients with a poor predicted response could avoid toxic and costly radiotherapy and undergo alternative strategies including chemotherapy. next-generation sequencing technologies (e.g. 16s profiling) are increasingly used to investigate complex bacterial communities. they have advantages over classical methods, as a significant proportion of bacteria are 'non-culturable'. however, they do not distinguish 'viable' and 'non-viable' populations, which may skew results, particularly following antibiotic exposure. here we report culture and 16s data from a clinically reflective human gut model, describing changes in the gut microbiota following exposure to multiple antibiotics. a triple-stage chemostat model was inoculated with pooled human faeces from healthy volunteers to establish gut microbiota populations. the model was sequentially exposed to clindamycin (33.9 mg/l, qds, 7days), vancomycin (125mg/l, qds, 7days) and fidaxomicin (200 mg/l, bd, 7 days). specific bacterial populations were enumerated daily on selective agars. periodically, 16s profiling of gut model samples was performed; dna was extracted on a qiaxtractor, 16s v4 pcr products were sequenced on an illumina miseq, and resulting data were analysed using qiime. both culture and 16s profiling demonstrated marked alterations in gut microbiota populations following antibiotic exposure. for many populations, notably bifidobacteria and enterobacteria, changes seen by culture correlated with 16s profiling. however, as culture describes numerical changes in populations, and 16s profiling describes proportional changes, results are not always directly comparable. 16s profiling greatly increased microbiome coverage, particularly for clostridia. population diversity (number of observed species and shannon index) decreased with sequential antibiotic exposure. use of culture and molecular methods in tandem can greatly increase understanding of changes occurring in complex microbial populations. barrett's oesophagus is the erosive replacement of the normal squamous oesophageal lining with a glandular epithelium and is the major precursor of oesophageal adenocarcinoma. barrett's patients are enrolled into active surveillance programmes in order to detect and treat oesophageal cancer at an early stage. surveillance however is costly and burdening to patients. to improve screening efficacy there is an acute need for accurate biomarkers of cancer progression risk in barrett's patients. understanding the pattern and pace of clonal evolution that occurs within the barrett's segment is a key step towards achieving this goal. opinion is divided over whether goblet cells (intestinal metaplasia) on oesophageal biopsy are required for a diagnosis of barrett's oesophagus. this is based on the unproven assumption that goblet cell differentiation marks increased cancer risk in barrett's oesophagus patients. we have investigated the clonal structure of non-dysplastic and neoplastic barrett's oesophagus by combining state-of-the-art 3d modeling and genetic lineage tracing. by tracing the clonal origin of an early oesophageal adenocarcinoma through whole-exome sequencing and mitochondrial dna sequencing, we find that this cancer developed from non-goblet columnar epithelium, whereas the adjacent goblet-bearing mucosa was free of oncogenic mutations. our results have important implications for the harmonization of the clinical diagnosis of barrett's oesophagus. long-course chemoradiotherapy (crt) is used to down-stage locally-advanced rectal cancer (larc) prior to resection. an interval period prior to surgery allows for tumour shrinkage to facilitate surgical removal. the optimal time interval remains unclear, with little high-quality evidence to guide clinical decisions about when to operate. this study explores the pathological outcomes from a pilot randomised controlled trial comparing an interval of 6 weeks versus 12 weeks between crt and surgery. thirty one patients were recruited from seven uk centres between june 2012 and may 2014. photographs were taken of the specimens and assessed by a blinded histopathologist for the quality of the mesorectal dissection. rates of pathological complete response (pcr), down-staging, and circumferential resection margin (crm) involvement were determined. response was also assessed using novel tumour cell density (tcd) assessment where the slides from the resected specimen and baseline biopsy were scanned at 400x magnification, the tumour area selected and 285 to 315 data-points analysed by a blinded expert to describe the percentage of different tissue components. the work was partly funded by a pathsoc career development fellowship and is presented on behalf of the starrcat trial investigators. twenty three patients underwent surgery (10 from the 6-week arm and 13 from the 12-week arm). the mesorectal fascial plane was intact in 7 specimens from the 6-week arm (70%) and 8 from the 12-week arm (62%). three patients at 6-weeks and two patients at 12-weeks showed a pcr. only one patient (from the 12-week arm) had an involved crm. tcd was 0.3% for the 6-week arm and 4.3% for the 12 week arm (p=0.12). in this small randomised trial, rates of mesorectal quality, crm status, pcr and tcd were similar following either a 6 or 12 week interval after crt. further studies are now needed to clarify whether a longer interval does facilitate on going down-staging. the role of tissue factor pathway inhibitor (tfpi) in liver injury p g petts 1 ; h kudo 1 ; a dorling 2 ; m thursz 1 ; r goldin 1 1 imperial college london, london, uk; 2 kings college london, london, uk introduction: studies have demonstrated that inhibition of the coagulant cascade is associated with less advanced liver fibrosis and better outcome in acute liver injury. tfpi is a serine protease inhibitor that acts as a homeostatic inhibitor of the coagulation cascade and may be a target to modify outcome in liver disease. methods: transgenic mice carrying a genetic modification that allows cells expressing a-smooth muscle actin (asma; e.g. activated hepatic stellate cells) to simultaneously express tfpi were used in models of chronic liver injury (carbon tetrachloride, ccl4) or acute liver injury (paracetamol) and culled at set time points after dosing. results:chronic liver injury: at 24 hours after the last dose of ccl4 the transgenic mice had significantly decreased asma expression and tissue inhibitor of metalloproteinase (timp) -1 gene expression but no difference in matrix metalloproteinase (mmp) -2 and -9 gene expression compared to wild types. this suggested a microenvironment that would promote fibrosis resolution. however after 24 hours this difference was lost. at all time points there was no significant difference between fibrosis in transgenic and wild type mice as demonstrated by sirius red staining, hydroxyproline assay and collagen 1a1 gene expression. acute liver injury: in paracetamol induced liver injury there was a significant difference in parenchymal necrosis in transgenic mice compared to wild types at 24 and 48 hours after dosing (24 hours: mean necrosis 6% vs. 30% respectively, mann-whitney test p=0.008. 48 hours: mean necrosis 2% vs. 20% respectively, mann-whitney test p=0.036). conclusion: these results suggest that tfpi is an unlikely therapeutic target in chronic liver injury. however in acute paracetamol induced liver injury tfpi appears to rescue the injured liver in a sustained manner from 24 hours after the initial insult and suggests a role for tfpi in managing acute liver injury. (research funded by the pathological society). analysis of adenocarcinoma and non-small cell lung cancer (nos) for egfr mutations now forms part of the royal college of pathologists' lung cancer dataset. identification of patients harbouring these mutations facilitates delivery of targeted therapies with superior efficacy. testing of these tumours for alk has also been introduced in our centre. we assessed our compliance with the college guidelines in this area for 2013 and 2014. in those tumours positive for egfr or alk mutations, we examined the original sections to assess any correlation between mutation status and morphological subtype. 96 of the 116 appropriate cases (83%) diagnosed histologically in 2013 were sent for egfr mutation analysis, increasing to 183/190 (96%) in 2014. 20 of the cases over this time (7%) were positive for an egfr mutation. of these, 12 showed an acinar growth pattern, 3 were solid, 1 lepidic, 1 papillary and 1 micropapillary. it was not possible to characterise the growth pattern in two of the cases analysed as cell blocks. the most common mutation, a missense mutation at codon 858 of exon 21, was most frequently associated with an acinar growth pattern. of the 2013 cases, 22 (19%) were sent for alk mutation analysis, compared with 152 (80%) in 2014. both of the two cases with an alk translocation (2p23 rearrangement) showed an acinar growth pattern. our compliance with college guidelines in sending appropriate lung specimens for mutation analysis is improving. the correlations between mutation status and morphological subtype add to, and are in keeping with, the current body of evidence in this area. primary synovial sarcoma of the heart -an interesting case report and review of literature p s venkatesan 1 ; p sloan 1 ; s kendall 2 ; m giles 2 1 royal victoria infirmary, newcastle, uk; 2 james cook university hospital, middlesbrough, uk seventy five percent of primary cardiac tumours are reported to be benign atrial myxomas. the remaining are malignant tumours with most of them being sarcoma, particularly angiosarcoma and malignant fibrous histiocytoma. synovial sarcoma of the heart is a very rare malignancy accounting for less than 1% of all primary cardiac tumours. most of them arise from the pericardium and the right side of the heart and is considered to be highly aggressive with reduced survival rates. diagnosis in these rare locations is also challenging. we report a 42-year-old gentleman who presented to us with productive cough, chest pain and paroxysmal nocturnal dyspnea. echocardiography revealed a calcified left atrial mass arising from the posterior leaflet of the mitral valve and radiologically was thought to be a benign atrial myxoma. excision was planned with histology showing a malignant biphasic spindle cell tumour exhibiting marked cellular atypia and numerous mitoses. on immunohistochemistry, the glandular component expressed diffuse positive staining for bcl-2 and ema with focal positive staining for pancytokeratins. the spindle cell component expressed cd99 and ema and was found to be negative for cd34, s100, desmin, melan-a and hmb-45. cytogenetic testing revealed ss18-ssx1/2 gene fusion with ss18 rearrangement confirming the diagnosis of synovial sarcoma in this rare location. a postoperative computed tomography was performed which showed no evidence of metastasis or primary lesions elsewhere. there was excellent postoperative surgical recovery and adjuvant chemotherapy was considered in the multi disciplinary meeting. primary cardiac synovial sarcoma is an extremely rare malignancy especially when arising from the left atrium posing diagnostic difficulty mimicking atrial myxoma. in contrast to the poor prognosis mentioned in the literatures, there was excellent recovery in this gentleman. swyer-james-macleod syndrome (sjmls) is a rare lung condition that manifests radiologically as unilateral hemithorax lucency as a result of post-infectious obliterative bronchiolitis, leading to small airways obstruction and secondary emphysema. the histological features of sjmls are poorly and infrequently described. we present three cases of the syndrome that underwent lobectomies in our institution from 2013 to 2015, in three women, aged, 33, 46 and 22 years, presenting with recurrent lower respiratory tract infection, shortness of breath and pleuritic chest pain. two underwent left upper lobectomies, one left lower lobectomy. the first case demonstrated hyperlucency of the affected lobe with markedly reduced blood vessel attenuation. the radiological findings of the second case were of extensive bronchiectasis, hyperlucency, mucus plugging and hypervascularity. the radiological findings of the third case were of an apical bulla and upper lobe cavitating lesion with lobar hypolucency and hypoperfusion. the main histological findings were bronchiolar changes with bronchiolectasis, mucus plugging, constrictive / obliterative bronchiolitis and various degree of peribronchiolar inflammation. emphysema was mild and diagnosed as loss of attachment of alveolar walls. in addition, case 1 had dystrophic, hypoplastic or absent branches of the pulmonary arteries. case 2 showed prominent bronchial arterioles and abnormal tortuous dilated pulmonary arteries and veins. case 3 had established bronchiolar scars in the bronchovascular bundles, pleural arteries showed medial hypertrophy and the interlobular septa contained dilated prominent veins, as well as cystically dilated inflamed peripheral bronchus. these cases highlight the importance of vascular changes in sjmls, likely secondary to the bronchiolar inflammation and destruction leading to capillary bed destruction from secondary emphysema and reactive pulmonary and arterial changes. mediastinal nodal staging with ebus is recommended for patients with resectable non-small cell lung cancer and has emerged as a safe tool to establish granulomatous pathology in suspected sarcoidosis. we conducted a retrospective analysis of the outcomes of ebus performed in a large teaching university hospital with a rapid access lung clinic over a 12 month period and correlation with endobronchial and ct guided biopsies, and surgical resections, when available, and compared the adequacy of ebus when performed with and without rapid on-site evaluation (rose background and aims: in interstitial lung disease (ild), when an aetiological factor appears absent and clinical-radiological correlation is non-contributory, histology is required. the traditional surgical lung biopsy (slb) is not without risks. cryotechnically obtained specimens contain more alveolated lung tissue and less crush artefact than conventional transbronchial biopsies and may offer an alternative to slb in selected cases. we aimed at studying the complications of cryoprobe transbronchial lung biopsy (cpbx) and the quality and pathological characteristics of the tissue obtained. methods: this is a prospective study of patients who were selected for cpbx including cases of possible/probable idiopathic pulmonary fibrosis (ipf). complications of the procedure as well as the quality and pathological characteristics of the tissue are studied. results: twenty-seven procedures were performed in 24 patients, 20 of which were radiologically ipf. a total of 77 biopsies were obtained (average 2.85 biopsies per procedure). only one was inadequate initially. fibroblast foci and features consistent with usual interstitial pneumonia (uip) pattern were present in 18 biopsies from 16 patients (66.7% of total; 80% of suspected ipf cases). granulomas were identified in 4 patients (16.7%), 3 of which were radiologically suspected ipf (15% of suspected ipf cases). two patients (8.3%) had organizing pneumonia; both were inconsistent with ipf radiologically. the findings in the remaining 3 patients were nonspecific; two of these were radiologically ipf (10% of ipf cases). seven patients (25.9% of procedures) developed pneumothorax, only 2 of them (7.4%) required chest tube drainage. five patients (18.5%) developed bleeding (moderate in 3 (11.1%) and mild in 2 (7.4%)). conclusion: cpbx was useful in this cohort at potentially identifying features not typical of ipf and displayed an acceptable complication rate. cardiomyopathy zj van der klooster 1 ; s sepehrkhouy 1 ; m harakalova 1 ; r goldschmeding 1 ; n de jonge 1 ; ajh suurmeijer 2 ; ra de weger 1 ; f asselbergs 1 ; p a vink 1 introduction: genetic dilated cardiomyopathy is a heterogenous group of diseases caused by mutations in various genes. several types of cardiomyocyte cell death have been implicated in dilated cardiomyopathy: (macro)autophagy-related cell death, apoptosis, necroptosis and oncosis. one plausible mechanism of genetic cardiomyopathy is proteotoxicity of accumulated protein aggregates. we investigated the association of such aggregates as sign of autophagy-related cardiomyocyte cell death with specific pathogenic mutations. methods: hearts from 30 patients with a genetic dilated cardiomyopathy or a combined phenotype of dilated and arrhythmogenic cardiomyopathy were included. microscopic slices from 8 regions were immunohistochemically stained for p62, a marker for aggregated proteins destined for autophagy. results: sporadic p62 positive cells were seen in control hearts (0.5% of cardiomyocytes, range 0.1-0.8%). troponin mutations (tnnt2 and tnni3; 0.7%, range 0.2-1.2%, n=3) showed hardly any increase in p62. titin (1.6%, range 0.7-2.7% ,n=5) and lamin a/c (1.7%, range 1.1-2.5%, n=5) mutations showed a threefold increase in p62 staining. a tenfold positive staining was found in desmosomal mutations (pkp2 and dsp; 3.8%, range 3.7-4.0%, n=3) and myosin mutations (myh7 and mybpc3; 4,6% range 3.3-5.7%, n=3). phospholamban mutations (8.8%, range 4.1-16%, n=8) and desminopathies (desmin and alpha-b crystallin; 17% of cardiomyocytes, range 4.0-31%, n=3) showed the highest number of p62 positive cells. conclusion: accumulation of p62 positive protein aggregates is associated with the type of mutation underlying the dilated cardiomyopathy. titin, lamin a/c and troponin mutations revealed little protein aggregation, whereas desminopathies, phospholamban, desmosomal and myosin mutations show abundant aggregates. this suggests that the type of mutation plays an important role in determining distinct mechanisms of cardiomyocyte cell death. major trauma centre status and its impact on the department of cellular and anatomical pathology in a large tertiary referral centre p ra hadden background: major trauma has been centralised into major trauma centres which act as the focus of major trauma networks. in april 2012, derriford hospital in plymouth, devon became operational as the regional major trauma centre for the south west peninsula. as a result, there was potential for an increased number of trauma-related deaths to be referred to the local coroner, as well as surgical specimens, potentially increasing the work load on pathologists. the case mix could include post-operative cases, neurosurgical cases, polytrauma cases and forensic cases. methods: on admission, all eligible trauma patients are recorded onto the trauma audit & research network (tarn) database. the tarn data was retrospectively analysed and cross referenced with the department of cellular and anatomical pathologies database to determine how many patients had died, how many had post-mortem examinations were performed and how many surgical specimens were sent, on patients from outside the region or transferred from smaller major trauma units. results: over the first two years, there was a small increase in workload from patients who, prior to trauma centre status would have gone to other centres. conclusions: in recieveing patients from elsewhere in the region, there was an increase in workload for both autopsy and non-autopsy work. this excluded some neurosurgical cases, which traditionally would have been referred (as derriford is the neurosurgical centre). there are several areas for implication including, apt time, mortuary space and non-autopsy surgical work. although the workload increase is small, at a time when services are being stretched it is important to ensure any increase in work will not be the "straw that broke the camels back" and can be dealt with accordingly. derriford hospital, plymouth, uk to attempt to streamline general pathologist's approach to the investigation of potentially asbestos-related deaths methods turnaround times, tissue sampling protocol and frequency with which samples were sent for formal fibre counts was investigated for 100 consecutive coronial autopsies at the author's institution. colleagues at other institutions were questioned about their own practice when investigating cases of potential asbestosis, lung cancer or mesothelioma. the author found no consensus in opinion on methods of sampling of the lungs in potential asbestosis, lung cancer or mesothelioma. the most common indication for samples to be sent for asbestos fibre counts was for malignant mesothelioma. sending tissue for fibre counts led to considerable delays in the authorisation of postmortem reports and to significant cost implications. the author presents a pragmatic algorithmic guide to approaching potentially asbestosis-related deaths with suggestions for sampling the lungs and tumour in all cases. in general terms, malignant mesothelioma previously confirmed premortem with histology and immunohistochemistry should not require extensive postmortem histological sampling. lung cancer and asbestosis require widespread sampling of lung tissue to determine amphibole count according to helsinki criteria in the former, and in the latter, assessment of the distribution and degree of fibrosis in addition to fibre count. one or more of these tissue blocks can be sent for formal counts in equivocal cases after following the algorithmic approach. conclusions although predominantly intended as a pragmatic approach to assist the busy practicing autopsy pathologist, the author believes that the algorithm presented will help departments streamline their approach to these cases and help the relative of the deceased gain access to compensation when appropriate in a more timely fashion. purpose of the study: this is a case report of a three year old girl who died suddenly at home. an autopsy was performed in order to determine the cause of death. method: an autopsy was conducted which showed no gross abnormalities. microscopy of the main organs and microbiological samples were taken for further assessment. results: histological assessment of the heart showed multiple small foci of lymphocytes around vessels and within the interstitum of the epicardium, myocardium and subendocardium. these lymphoid aggregates consisted of 20-30 lymphocytes up to larger numbers of 100 lymphocytes collectively. several foci were present within virtually all of the sections taken in both right and left ventricles. there was no evidence of myocyte necrosis. immunohistochemistry confirmed they were of t lymphocyte cell origin admixed with smaller numbers of macrophages. histology from the respiratory system showed a diffuse subepithelial lymphocytic infiltrate in the larynx and trachea, and the nasopharyngeal samples detected coronavirus, adenovirus and two types of parainfluenza virus. however, viral polymerase chain reaction (pcr) from the cardiac tissue was negative. conclusion: an unequivocal diagnosis of a myocarditis could not be made in this case due to the lack of myocyte necrosis and the absence of viral dna within the cardiac tissue. genetic testing was strongly advised as splenic material had been taken at autopsy and following molecular genetic techniques a mutation was detected in the sodium channel indicating an inherited ion channelopathy. further genetic counselling and testing of the remaining siblings and family members is being performed. varicose veins affect a third of the uk population. isolated case reports and small series of fatalities resulting from varicose vein haemorrhage appear in the literature infrequently. some of the earliest reports of fatality we have found appear in british medical journal (1958) and the lancet (1973), more recently they have appeared in journals of forensic pathology. our purpose is to establish and bring attention to the rarity of fatality resulting from varicose vein haemorrhage and the importance of the scene of death and autopsy findings. a literature review was undertaken, we obtained relevant office of national statistics (ons) mortality data for the years 2011-2013, and reviewed our own post-mortem records for demographic, clinical and scene of death information in cases we have encountered. our findings confirm that fatality resulting from varicose veins remains a rare cause of death. some of these deaths are preventable and in 2013 nice (national institute of health and care excellence, uk) issued guidelines in which haemorrhage from varicose veins constitute a vascular emergency. importantly emphasis on first aid is required, simply elevating the limb stops bleeding and is life saving, whereas direct pressure and tourniquets do not. pathologists should be aware of potential findings at autopsy in these cases. in particular, awareness that even obscure minor injury to a varicose vein could have resulted in significant blood loss leading to death. blood lost at the scene will not be apparent at autopsy, and details of blood loss could be variably recorded on the scene of death information provided, therefore vigilance is required. histopathologists practice in an era of ever advancing medical treatments for a wide variety of oncological, neurological, haematological and rheumatological diseases. immune modulating therapies are taking a more prominent place in clinical practice. however, with such great advances in therapy comes great risk, with the potential of life threatening opportunistic infections in our patients. we present a series of 9 immunosuppressed patients who acquired such infections and in whom the diagnoses were made by histopathological examination. the spectrum of these pathogens ranges from viral (cmv, ebv, herpes), parasitic (strongyloides) to fungal (p. jirovecii, cryptococcus), and the range of infections is diverse. our series includes 4 males and 5 females, with an age range of 32 -72 (mean age = 57 years). unsuspected infectious diagnoses were made at post mortem in 6 of the 9 cases. organs affected included lung (n = 5), brain (n = 1) and haematological system (n = 1). in one case both colon and lung were affected (n=1) and in a further case both liver and lung were affected (n=1). immunohistochemistry and/or histochemistry was invaluable in making the diagnoses and was used in all 9 cases (n=9). treatments leading to immunosuppression included chemotherapeutic agents, monoclonal antibodies, steroids and methotrexate. we believe that with the ever increasing use of immunosuppressive therapies (both new and old) for a wider number of disorders, vigilance should be paid to their potential to cause life threatening side effects. histopathologists play a pivotal role in the recognition of this risk and in the diagnosis of these diseases. audit of hospital-based adult autopsy practice in a university hospital from july 2013-2014 p d abu-sinn; f macsweeney the contribution of hospital-based autopsy practice to improvements in patient care is substantial; however, there remains a void in the processes of audit and raising quality of standards in autopsy services. we aim to assess the current autopsy practice compared to rcpath guidelines and identify areas for achieving a high quality autopsy service. all adult autopsy cases performed at a university hospital mortuary between july 2013 and 2014 were reviewed. a total of 522 adult autopsies were performed by 5 consultant histopathologists. ninety nine percent were coroners' cases. the median turnaround time was 38.5 days, with a range of 3-123 days, excluding 31 outlier cases (complex timeconsuming cases). there was considerable variation in turnaround times in complex cases and between the various reporting pathologists. eighty five percent of cases were compliant with rcpath minimum dataset for autopsy practice. the remainder were lacking clinical information only. histology and toxicology contributed to cause of death in 34.4% and 14.8% respectively. no organs were retained. further review of the cases not compliant with rcpath guidelines (15%), identified that the possible reasons were the inaccuracy, and sometimes irrelevance to the cause of death, of the information received by the pathologists. in many instances, the clinical information given to the pathologist may be controversial, and a certain degree of caution needs to be implemented to avoid including misleading information in the autopsy report. the turnaround times could be improved if a preliminary report is issued within a set time frame, to be followed by the complete report when the histology and toxicology results are available. however, this practice is not acceptable to some coroners who prefer one complete final report. variations in autopsy practice are to be expected as each autopsy involves substantial case-specific information to which a case-specific answer to the cause of death is expected. prostate cancer is the second most common form of cancer in males, and the incidence of this disease is predicted to double globally by 2030. more than 1.1 million new cases of prostate cancer are diagnosed each year and two thirds of these patients are from the western world. the current psa-based test for the diagnosis of prostate cancer lacks specificity, results in missed-diagnoses, over-diagnosis and unnecessary biopsies/treatment. there is an urgent need for a method that enables early accurate detection of prostate cancer. endosomes and lysosomes are cellular compartments that degrade and turnover macromolecules in order to maintain cellular homeostasis. these organelles are directly involved in the critical processes of energy metabolism, cell division, and intracellular signalling, which are all hallmarks of cancer pathogenesis. endosomes have a critical role in controlling the secretion of proteins into extracellular fluids, making them an ideal system to identify new biomarkers that are released from cancer cells. we have discovered that endosome biogenesis (formation and function of endosomes) is altered in prostate cancer. there were significant changes in the gene and protein expression for 19 endosomal proteins and differential distribution of endosome subsets in prostate cancer cell lines. there were also changes to the endosomal traffic and signalling of the transferrin receptor in prostate cancer cells. these fundamental changes in the cell biology of prostate cancer have allowed us to identify a specific set of endosomal proteins that have diagnostic potential. we are developing elisa's to quantify these endosomal proteins in patient samples and antibodies for immune histology applications. the objective for this project is to develop an effective method for the early and specific diagnosis of prostate cancer, which is important as this will have a major impact on patient outcome and survival. the incidence of malignant melanoma has rapidly increased in recent times and melanoma currently represents the second most common cancer diagnosed in young adults. diagnosis is based predominantly on histological assessment; however, due to the wide spectrum of morphological characteristics and lack of firm diagnostic criteria, accurate diagnosis can be challenging. some atypical melanocytic lesions do not display clear-cut morphological features to allow distinction of benign from malignant tumours, making diagnosis and treatment difficult. among these atypical melanocytic lesions blue nevi, spitz nevi and dysplastic lesions are common. from histological features alone, it can be difficult to exclude a diagnosis of melanoma and therefore aggressive surgical strategies may be employed in cases were they are unnecessary, highlighting the need for improved diagnostic techniques. both mrna and mirna profiling have been shown to be able to distinguish benign nevi and primary melanoma tumours. studying mirna expression levels is an attractive strategy as mirnas are highly resistant to degradation and can be easily analysed in ffpe samples. we have studied mirna expression levels in a cohort of benign, blue, spitz and dysplastic nevi versus primary melanoma tumours and their derived metastases. expression levels of key melanoma mirnas, including mirna 21, mirna 211, mirna 205 and mirna 200c can be used to distinguish between nevi and malignant melanomas. we propose an easy to implement, simple and robust molecular method based on mirna expression ratio that, in combination with histological assessment, allows diagnosis of difficult to classify atypical melanocytic lesions. background: diagnosis of lynch syndrome (ls) traditionally relies on clinical criteria to guide diagnostic genetic testing. mmr status of the patient's tumour can help detect lynch syndrome families as well as having other recognised applications for the patient's management including prognostic and predictive significance. as such, the 'dataset for colorectal cancer histopathology report' recommendations from the royal college of pathologists were updated in july 2014 to include screening of colorectal cancer patients under the age of 50 and molecular testing for abnormalities in the mismatch repair genes. in south-east of scotland we introduced molecular testing to identify individuals at risk of ls. to widen our screening in line with revised guidelines set by european experts, we expanded our cohort criteria to include those between the age of 50 and 60. methods: molecular analysis was carried out on 553 individuals: 446 via 'reflex testing' (newly diagnosed colorectal carcinoma ≤60 yrs, or clinical/ pathological features associated with mmr defects, such as pre-menopausal endometrial carcinoma, multiple tumours and medullary-type carcinomas) and 107 via 'request testing' (clinical criteria and referral dependent). 'molecular-positive' profiles for ls were identified for genetic pre-testing counselling/diagnostic testing. results: 41 patients with potential ls were identified, 24 (58.5%) underwent genetic counselling/testing and 13 cases were confirmed ls with germline pathogenic mutations in the mmr genes. eight of these were identified using reflex testing. conclusion: this is the first uk study to show that screening for ls in patients with colorectal cancer under the age of 60 is effective at identifying families with ls. the testing protocol is in line with the recent recommendations. the human microbiome is rich and diverse, especially in the oral cavity and gastro-intestinal tract, where it has been shown to be more stable in adults, although various factors such as diet and antibiotics mays influence its composition. this pilot study aimed to examine and compare the oral and gut microbial composition in four individuals using a culture-independent approach. methods: saliva and faecal samples were collected from volunteers within the same day on two separate occasions. the v4 region of the 16s rrna gene was amplified in all samples and pcr products sequenced on an illumina miseq. unique barcodes were used to sequence 24 multiplexed libraries together. the data were analysed using the quantitative insights into microbial ecology (qiime) software. a second series of 42 samples of faeces from 3 individuals were run to investigate consistency over time. operational taxonomic units (otus) were assessed and showed 5 major phyla represented in the saliva samples: firmicutes, proteobacteria, bacteroidetes, fusobacteria and actinobacteria. similar phyla except forfusobacteria, were found in the stool samples. the weighted unifrac pcoa analysis displayed a clear separation of the 2 sample groups, and also showed a more disperse bacterial profile for the saliva samples, based on population sizes, whereas rarefaction curves and unweighted analysis indicated higher bacterial diversity in the stool samples. each individual could be distinguished either by oral or faecal microbiome. one volunteer who had had previous radiotherapy to the mouth displayed a particularly distinct oral microbiota. the microbial community profiles of saliva and faecal samples of four individuals were found to be distinct from each other, despite sharing similar phyla. analysis of multiple samples from each volunteer clearly separated each sample by volunteer and by sample type. are current automated approaches for determining the phylogeny of multiple deposits capable of interpreting the complexity of cancer evolution? p tg palmer; hm wood; m taylor; w fateen; im carr; p quirke tumour heterogeneity is central to chemotherapy resistance and disease progression in advanced malignancy. this heterogeneity arises due to the evolution of clones within the tumour cell population; the advent of high throughput sequencing has allowed the detection of different tumour cell clones within and between primary tumours and their metastases, potentially allowing mapping of tumour evolution. several, automated bioinformatic approaches have been devised for determining tumour phylogeny from changes in genomic copy number (cn); either by the overall similarity of genomic changes between tumour deposits or by examining the occurrence of shared breakpoints. we have compared these automated approaches with a manual determination of phylogeny based upon shared breakpoints identified from four cases of metastatic colorectal cancer consisting of between 6 and 53 deposits. we illustrate several recurrent issues identified with the use of automated systems for the determination of tumour phylogeny associated with an inability to correctly identify and interpret changes in ploidy, an inability to identify heterogeneity within tumour deposits, the masking of smaller events by larger ones, overinterpretation of convergent, but unrelated events, over calling sequencing artefacts as changes in cn, and non-calling of genuine cn changes due to low tumour cell content or low sequencing depth. we conclude that manual interpretation of bioinformatics data is still required to determine the phylogeny of metastatic cancer within an individual. results: levels of agreement between each sample size and the 'gold standard' were evaluated using bland-altman plots. 7 separate pairwise comparisons were performed. some small sample sizes were shown to have small mean difference and narrow limit of agreement. the ki-67 pis were then translated into grades and similar comparisons were performed by calculating the kappa score for categorical variables. additionally, the interobserver variation between the two independent researchers were calculated. conclusion: smaller sample sizes (below 1000) tend to overestimate the ki-67 pis, possibly due to the effect of concentric counting starting from the center of the hotspot. however, the ki-67 pis do start to stabilise closer to 2000 (e.g. 1500). the interpretation of whether a lower sample size can replace the current standard would be a subjective decision, but the kappa score gives a rough idea of how much it affects the clinical grading. updated data will be presented. purpose of the study: targeting the stem cell properties of tumor-initiating cells is an avenue through which cancer treatment may be improved. before this can be achieved, so-called cancer stem cell (csc) models must be developed and characterized in specific malignancies. methods: in this study, holoclone formation assays were used to characterize stem-like molecular signatures for prostate cancer (pca) cells. summary of results: lncap and pc3 parent cells were capable of responding to stem cell differentiation morphogen retinoic acid (ra), suggesting the presence of inherent stemlike properties. lncap cells, which represent early, androgen-responsive disease, formed holoclones after twenty six days. pc3 cells, which represent advanced, metastatic, castrationresistant disease, formed holoclones after only six days. holoclones displayed decreased expression of ra-genes, suggesting a more immature, less differentiated phenotype. gene and mircorna arrays demonstrated that holoclones downregulated a number of stem cell differentiation regulators while displaying enhanced regulation of g2 to m transition and the mitotic spindle checkpoint components of the cell cycle. pc3 holoclones displayed pronounced downregulation of known regulators of osteoblast differentiation from mesenchymal stem cells and epithelial mesenchymal transition. conclusion: our results suggest that some pca cells retain the ability to transition to a more immature state in which differentiation and metastatic mechanisms are changed. the highlighting of osteoblast differentiation regulators in this mechanism is particularly notable, considering the propensity of pca to metastasize to bone. we examined by flow cytometetry the interaction in vitro between platelets and 15 human cancer cell lines of different origin and metastatic potential. the emt profile of cells 24hr post platelet exposure was assessed by morphology and gene expression analysis (rt-pcr). here we showed that platelet cloaking of cancer cells is universal, occurring across all 7 tumour types examined. however, it is heterogeneous with adhesion rates varying both across and within tumour types, from 35% (pc3-metastatic prostate cancer) to 83% (skmes1-metastatic lung cancer). changes indicative of emt were seen in all cell lines. however, again they were heterogeneous in nature; with morphology changes akin to emt observed at varying degrees across the cancer types. also, there was no consistent pattern to the emt-like gene expression changes seen, with one exception a significant increase in the expression of plasminogen activator inhibitor 1 (pai-1)was observed in 93% of the cell lines examined. in this study we describe the universal nature of platelet cloaking and that even though the interaction is not inducing precisely the same molecular changes in all the cancer cells; overall it is driving these cells into a mesenchymal phenotype. giant cell tumours of bone (gct) are primary locally aggressive bone tumours with a recurrence rate of up to ~30%. the tumour is characterised by numerous osteoclasts and neoplastic stromal cells. making a diagnosis can be challenging because the differential diagnosis incudes an array of benign osteoclast-rich tumours but also osteoclast-rich osteosarcoma. recently the occurrence of h3f3a p.gly34try (g34w) and g34l mutations was reported in 96% of gct, the latter occurring rarely. these mutations occur in less than 2% of >400 other benign and malignant bone tumours. it has been emphasised that a diagnosis of gct should be made with caution in the absence of detection of g34w substitution. given the diagnostic importance of g34w mutation in gct, we have developed a simple, quick and cost-efficient diagnostic test to detect this recurrent alteration in ffpe dna using droplet digital pcr (ddpcr). the ddpcr data from dna of >100 gct have been compared with previous 'genotype' data generated using sanger sequencing, and a number of next generation sequencing approaches (whole exome, whole genome and targeted panels). the g34w and g34l can both be detected in a single assay. we have demonstrated the sensitivity, specificity, repeatability and robustness of the test to be very high with a turnaround time of no more than 5 working days. as ~30% of gct recur locally following curettage a blood test would be valuable to monitor patients. to this end the ddpcr test also shows that the mutation can be detected in plasma. typically resistant to chemotherapy and radiotherapy, high grade disease has been treated by surgery for more than 50 years. it has recently been shown that idh1 and idh2 mutations are present ab initio in ~60% of chondrosarcoma cases and that these are retained throughout disease progression. this has opened up a number of new potential diagnostic, biomarker and therapeutic options. digital pcr is currently the most sensitive and accurate method for detecting and quantifying mutant dna molecules. the biorad qx200 digital pcr platform is also both cost effective and scalable. using the qx200 platform, we have developed assays for the 5 common idh1 mutations and the 1 common idh2 mutation. we have developed the idh1 assays both in singleplex and multiplex. we have optimised and validated all assays in tissue samples demonstrating both high sensitivity and specificity when compared to previously genotypes samples. we have demonstrated that the assays are quantitative over 4 orders of magnitude and in high quality dna we can detect idh mutations at below 1 mutant molecule in 10,000 wild type molecules. in a pilot study, we have used digital pcr to analyse circulating tumour dna levels in plasma taken pre-surgery from 14 patients whose chondrosarcoma harbour an idh1 mutation. it was possible to detect idh1 mutant molecules in plasma of all grade iii samples, 50% of grade ii and none of the grade i samples. in 4 of these cases where the ctdna was also measured post-operatively, the levels of ctdna dropped dramatically. tumour necrosis factor receptor, cd40, gene functions as an oncogene and promotes cell proliferation in colorectal cancer cell lines p haa almasmoum; h thorpe; m ilyas introduction: cd40 is a tumour necrosis factor (tnf) receptor which regulates a range of cellular responses. cd40 is activated by its ligand cd40l and may promote tumourigenesis in haematological cancers. however, cd40 functions as a tumour suppressor in solid cancers. cd40 maps to chromosome 20q13, a region which is amplified in 40-50% of colorectal cancer (crc). the functional activities of cd40 were tested in crc cell lines for cell proliferation and motility. methods: expression of cd40 was screened in crc cell lines by western blot. to define the role of cd40 in human crc, we knocked down cd40 using small interfering rna (sirna) and the knockdown was confirmed by qpcr and western blot. the prestoblue assay was used to study proliferation in colorectal cell lines, and flow cytometry to study the cell cycle. transwell migration and wound healing assays were performed to investigate the effect of cd40 on cell motility in crc. result: cd40 was expressed in crc cell lines hct116, rko, dld1 and ht29, and not expressed in sw480 and sw620 cell lines. knockdown of cd40 reduced cellular proliferation in hct116 (p=0.0158) and dld1 (p=0.0020) cell lines. knockdown of cd40 showed a higher number of cells in the sub g0 phase (dead cells) in the cell cycle analysis compared to the control. however, knockdown of cd40 in hct116 did not have an effect on cell motility in both the transwell migration (hct116= p=0.253) and wound healing assays. discussion: cd40 exhibited oncogenic activity in crc cell lines. cd40 enhanced cell proliferation but not cell motility in crc cell lines. the expression of cd45 (common leucocyte antigen) and cytokeratin is thought to be mutually exclusive with cd45 expression largely restricted to haematological malignancies and cytokeratin expression largely restricted to carcinomas. we report two clinically relevant cases. the first case is a urinary bladder biopsy showing a high grade malignant tumour with cells that had scanty cytoplasm, hyperchromatic stippled nuclei and high mitotic activity. nuclear molding was present. the tumour cells showed focal strong positivity for ck7 and diffuse positivity for cd56 and synaptophysin. focal positivity for cd45 was present and confirmed on repeat staining. the morphology and immunoprofile was consistent with a small cell carcinoma showing aberrant cd45 expression. the second case is a maxillary tumour biopsy composed of medium/large atypical lymphoid cells with hyperchromatic nuclei, small nucleoli and scanty cytoplasm. mitoses and apoptotic cells were noted. immunohistochemistry showed the atypical cells to express cd20, cd79a, bcl6 and mum1 but not cd45, cd5, cd10, cyclin d1, cd30, tdt, alk1, cd2, cd3, neuroendocrine or melanocytic markers. a high ki67 proliferation fraction was present. the appearances were consistent with a diffuse large b-cell lymphoma. the above cases highlight the possibility of aberrant expression as well as loss of expression of immunohistochemical markers by neoplastic cells in undifferentiated malignancies. attention to tumour morphology may provide diagnostic clues. interpretation of immunohistochemistry in the context of tumour morphology as well as awareness of aberrant expression/loss of expression can help avoid diagnostic error. accurate, timely diagnosis is the ultimate aim in surgical pathology. numerous histological pitfalls and lesional mimics exist, with the need to maintain an awareness of such entities vital if potentially serious misdiagnoses are to be avoided. this case report describes two distinct lesions within the same lymph node, both of which are potential mimics of each other. a 43 year old female presented with a three week history of a left breast lump. she had no known previous breast disease or any associated risk factors. a needle core biopsy of this clinically and radiologically suspicious mass yielded a diagnosis of grade 2 invasive lobular carcinoma. left axillary sentinel node biopsy was thus undertaken. two hot and blue sentinel lymph nodes were excised. one was free of neoplasia, the second contained benign naevus cell inclusions within the capsule together with a micrometastasis. immunocytochemistry confirmed the presence of two distinct cell populations; the benign naevus inclusion cells stained positively for s100 but not for ae1/3, the reverse pattern was observed in the invasive lobular carcinoma cells. heterotrophic benign inclusions within lymph nodes are an infrequent yet well recognised entity. ridolfi et al reviewed the lymph nodes from 909 axillary surgery patients and found 0.017% of lymph nodes contained benign naevus cell inclusions. small benign naevus cells within the capsule of a lymph node can resemble the 'indian file' pattern of classic invasive lobular carcinoma. this case is unusual in that both metastatic carcinoma and benign naevus inclusion cells were present within the same lymph node, enabling a clear comparison of the cytomorphology and immunoprofile of these two distinct lesions. an awareness of benign inclusions within lymph nodes helps to avoid the potential for misdiagnosis. the judicious use of immunocytochemistry can be useful in distinguishing benign inclusions from carcinoma. recently there has been increasing recognition of distinct breast cancer phenotypes. of these, basal phenotype breast cancer (bbc) has attracted particular interest since the majority are triple negative (tn); have an aggressive natural history and can be associated with brca1 germline mutation. this area is mired in difficulty, as a precise unifying definition of bbc remains elusive. several morphological features more prevalent in bbc have been identified. in our practise we noticed variable use of 'basal' in reports. given this, whilst no specific therapies to bbc currently exist, we felt it necessary to understand how accurate our designations have been and whether this is a worthwhile practise. method: the diagnostic database was searched for all malignant tn breast resections or reports containing the word 'basal' within 12-months. tn was defined as allred score er 0-2/8, pr 0-2/8 and her2 0, 1+ or 2+ negative on fish. for completeness, we considered including all breast cancers, but pragmatically this was not possible. we carried out ck5 and ck14 staining on all cases where not performed. results: of the 618 invasive breast cancers, 69 cases (11%) were identified, of which 88% were tn and 28% were designated bbc in the report. 16% were both tn and bbc. where a diagnosis of bbc was made, 37% of cases had additional markers requested. preliminary results showed 94% were ck5+ and ck14+. this is higher than other studies, implying specificity but not sensitivity in suspecting bbc amongst reporting pathologists. discussion: a limitation of this review is that it cannot identify the rare non-tn bbc not diagnosed as such. it also represents current practise in a single institute and may not reflect national practise. we identified patchy use of the designation bbc, with overall under-reporting of this subtype. we recommend that if it becomes necessary to distinguish bbc lesions, additional markers studies, such as ck5 and 14 be consistently performed. purpose: neoadjuvant chemotherapy (nact) is increasingly used for the management of large but operable, inflammatory, and locally advanced breast cancer (labc). little is known about predictors of response/survival following nact. the topoisomerase iiα (top2a ) gene, a key regulator of dna repair and modelling, is thought to be target for anthracyclin and other chemotherapeutic agents. the aim of this study is to assess the role of top2a as marker for response/resistance to nact and patient outcome. methods: patients who underwent nact, predominantly anthracyclin, for primary and operable invasive carcinoma or labc in the period between 2005 to 20013 at a single large tertiary referral breast unit were identified. comprehensive data on chemotherapy regimen, surgical treatment, pathological response and survival were collected. pre-treatment tumour samples were stained for standard predictive and prognostic markers and top2a. results were correlated with pathological response (pr) and patient survival. results: 252 patients fulfilled inclusion criteria. mean age was 48.94ys. complete pr was achieved in 15.4%. the mean expression level of top2a in pre-treatment core biopsies was 75.4%, range 0-95%. there was significantly higher expression in high grade tumours (p=0.04) and positive correlation with ki67 expression (r=0.405, p<0.001). there was no correlation with nodal status, pr or her2 expression. cases with high expression (>50%), had significantly worse overall survival (mean 38 vs 52 months, p=0.01). this was also identified in the endocrine non responsive group (er allred score≤4), mean 74 vs 33 months, p=0.04. on multivariate analysis, top2a was not an independent factor for overall survival. conclusions: top2a protein is expressed in high grade breast carcinoma with high ki67 proliferation index. its expression in pre-treatment biopsies predicted patient outcome in the neoadjuvant setting. this strong adverse effect on survival warrents further prospective investigation as a marker of outcome in nact patients. the risk of circumferential resection margin (crm) involvement is confined to tumours of the rectum with the risk of peritoneal involvement increasing the further a tumour is located above the peritoneal reflection. there is no internationally accepted definition of the upper limit of the rectum, and the term 'rectosigmoid' is frequently applied to tumours in this area leading to confusion around the risks and whether radiotherapy can be given. the photographs from 331 abdominoperineal excision specimens were available for quantitation using aperio imagescope. both fresh and fixed specimen images were included where available. the position of the anal verge, top of the sphincters, anterior peritoneal reflection, mesorectal apex (defining the limit of the mesorectum) and high vascular tie were identified and the distances between each point measured. the work was supported by a pathsoc bursary. there was wide variation in the length of the mesorectum in both fresh (median 172 mm, iqr 146 to 199 mm) and fixed (166 mm, 140 to 196 mm) specimens. the length of the anal canal also showed variation (fresh 66 mm, 49 to 78 mm; fixed 66 mm, 53 to 75 mm). the height of the anterior peritoneal reflection was lower in females compared to males (fresh 125 vs. 132 mm, p=0.288; fixed 111 vs. 126 mm, p=0.034). there is marked variability in the anatomy of the rectum between individuals and genders. this potentially affects the risk of either crm or peritoneal involvement and whether radiotherapy could be offered. a fixed definition of the upper limit of the rectum for all patients is not helpful. this should be determined for individual patients on the basis of the mri findings. the term 'rectosigmoid' should be abolished and more accurate definitions based on the position of the mesorectal apex and commencement of the sigmoid mesentery should be used to define the boundaries of the rectum and sigmoid colon and determine subsequent risks to the patient. pre-operative chemoradiotherapy (crt) with anti-egfr antibodies may change the status of egfr pathway mutations. we assessed the mutational status of a number of egfr pathway genes before and after crt in the nwcog excite trial. patients with mri-threatened surgical margins were given pelvic radiotherapy (45gy) with capecitabine, irinotecan and cetuximab followed by surgery after 8 weeks. dna was retrospectively extracted from the pre-treatment biopsy and resection specimen by macrodissecting areas of greatest residual tumour. the mutational status of kras (codons 12/13/61/146), nras (12/13/61), pik3ca (542/545/546/1047) and braf (v600e hotspot) were determined by pyrosequencing. the work is presented on behalf of the nwcog excite trial investigators and was part-funded by a pathsoc fellowship. 80 patients commenced treatment and 76 underwent surgery with pathological complete response in 14 (18%) and near-complete in 6 (8%). pre-treatment testing (n=78) detected mutations in kras (n=34), braf (n=3), nras (n=3) and pik3ca (n=10). any egfr pathway mutation was detected in 58%. following crt, cases with residual tumour able to be tested (n=54) showed mutations in 32 patients (59%). there was a discrepancy compared to pre-treatment biopsy in 18 cases (33%): from wild-type (wt) to mutant (mut) in 9, from mut to different mut in 1 and from mut to wt in 7. one patient changed in 3 codons (mut to wt in kras 146/pik3ca 545 and wt to mut in kras 12). in 12 patients (22%) this changed their overall egfr pathway status (6x wt to mut and 6x mut to wt). intratumour heterogeneity may explain some of the differences in egfr pathway mutations reported between biopsies and resections presenting a challenge to personalised medicine. however, cetuximab may also drive the growth of undetectable mutant clones to detectable levels on pyrosequencing. further assessment using more sensitive sequencing technologies is currently being employed to investigate these differences. there is a vast amount of historical ffpe material held in archives, but due to variations in fixation and processing this presents several challenges when applying newer genomic technologies to it. in this study we compared the genomic information obtained with the oncoscan® ffpe assay kit (oncoscan) and next generation sequencing (ngs). samples from 378 patients were obtained from 10 centres taking part in the mrc cr07 trial of short course radiotherapy versus selective long course chemoradiotherapy in rectal cancer. dna was prepared using agilent sureselect kits and sequenced using illumina platforms in parallel to analysis using the oncoscan assay. for both methods, quality control (qc) data was generated and the sample classified as a 'pass' if it fell within the pre-defined qc boundaries. for the oncoscan assay, copy number (cn) and somatic mutation (sm) data was further investigated. this study was part funded by a pathsoc fellowship. in total, 272 cases (72%) passed the ngs qc and 232 (61%) passed the oncoscan qc. a total of 186 (49%) passed qc on both platforms with marked variability in sample pass rates between the 10 centres for the ngs (range 0% to 100%) and oncoscan (ranges 33% to 84%). when assessed manually, the oncoscan sm data was considered acceptable for 273 cases (72%), which included 40 initially classified as 'failed' by the qc data. similarly, the oncoscan cnv data was interpretable for the majority of cases. this study has shown that whilst historical dna held in the ffpe blocks of archival clinical trials like mrc cr07 can present challenges when using new genomic technologies, a large proportion of samples can still yield valuable genomic data. marked variation exists in the quality of genomic material between centres confirming that differences in specimen handling affect dna quality. prospective trials must address this by standardising fixation and processing protocols. the plane of colon cancer resection has recently been shown to predict survival. complete mesocolic excision (cme) with central vascular ligation (cvl) produces an oncologically superior specimen and appears to be related to optimal outcomes. we aimed to assess whether a regional educational programme in cme with cvl led to an improvement in the quality of colon cancer specimens. following a regional educational programme in cme with cvl in the capital and zealand areas of denmark, 686 cases of primary colon cancer resected across six hospitals were assessed by grading the plane of surgery and undertaking tissue morphometry. these were compared to 263 specimens resected prior to the educational programme. this work was partly supported by a pathsoc undergraduate bursary. across the region, the mesocolic plane resection rate improved from 58% to 77% (p<0.0001). hillerød hospital had implemented cme with cvl as standard prior to the educational programme and continued to produce optimal specimens. three of the other hospitals showed a significant improvement in the plane of surgical resection. hillerød specimens continued to be more radical with a greater distance between the tumour and the high tie, area of mesentery and lymph node yield compared to the other five hospitals. a multidisciplinary regional educational programme in cme with cvl has improved the oncological quality of colon cancer specimens as assessed by mesocolic planes, however, there has been no significant effect on the amount of tissue resected. surgeons at hillerød continue to produce more radical specimens suggesting that such educational programmes are not alone sufficient to increase the amount of tissue resected around the tumour. hillerød have recently published their long term outcomes with survivals being 10% higher when compared to other hospitals across the region. further engagement is now necessary to ensure that optimal outcomes are achieved across the region. investigating the faecal microbiome in formalin fixed paraffin embedded (ffpe) material p itr jobling 1 ; m taylor 2 ; c young 2 ; hm wood 2 ; p quirke 2 1 university of leeds, leeds, uk; 2 leeds institute of cancer and pathology, leeds, uk purpose: research into the faecal microbiome has shown a diverse population with a high level of variability between individuals. altered faecal microbiomes are present in a range of diseases but work remains to understand their role in gastrointestinal disease. current research into the microbiome makes use of fresh or frozen faecal samples. this restricts researchers to predominantly prospective study designs. one potential method for rapidly increasing and diversifying research is the retrospective study of ffpe material. we aimed to investigate the feasibility of typing the microbiome in ffpe faecal samples using next generation sequencing (ngs) technology. methods: material from six faecal samples was divided and stored as frozen or fixed and paraffin embedded creating two matched sub-groups. to assess assay sensitivity one sample was diluted to eight different concentrations before fixing and embedding. the v4 and v6 regions of the 16s rrna gene were amplified. primer pairs created approximately 240bp and 98bp targets in e.coli respectively. pcr products were multiplexed and sequenced on an illumina miseq. qiime software was used for analysis. results: analysis of alpha (within sample) diversity showed a significant difference between sub-groups when targeting v4 (p=0.05) but not v6. analysis of beta (between sample) diversity showed a significant difference between sub-groups when targeting v4 (p=0.01) while the v6 region showed a reduced, but still significant (p=0.02) difference. the sensitivity assay showed comparable results down to 0.5% concentration levels. conclusion: to our knowledge this is the first feasibility study generating ngs data on the microbiome from ffpe faecal material. variation between matched frozen and ffpe faecal material was less when targeting v6 compared to v4. we hypothesise this may be due to the shorter amplicon undergoing less dna fragmentation in ffpe material. there are several platforms available for dna mutation detection in formalin-fixed paraffin-embedded (ffpe) material, all with their relative strengths and weaknesses. we investigated the oncoscan® ffpe assay kit (oncoscan) in comparison to pyrosequencing in patients with operable colon cancer recruited to the phase ii component of the ncri foxtrot trial of pre-operative vs. post-operative chemotherapy. ffpe samples of tumour from the resection specimens of 132 cases were tested for kras 12/13/61 and braf v600e mutations using pyrosequencing. the oncoscan assay allows for the interrogation of 74 mutations across nine genes. pre-extracted dna was analysed on the oncoscan assay and quality control (qc) scores generated, indicating confidence in mutation calling results. the mutational status of all samples was automatically assessed in the affymetrix sm viewer, and then manually confirmed. this work is presented on behalf of the foxtrot collaborative and was part funded by a pathsoc fellowship. out of 132 samples, 22 failed oncoscan qc thresholds, however, only 10 of these were deemed inconclusive by manual interrogation. 130 samples were interpretable by pyrosequencing. of the 120 samples that produced conclusive results on both platforms, the concordance rate was very high at 95.8% when calling a mutated versus non-mutated kras/braf status. mutations were 'missed' by pyrosequencing in only 1 case (0.8%) and by oncoscan in 4 cases (3.4%). in addition, the oncoscan assay provides mutational data in additional genes along with copy number (cn) and loss of heterozygosity (loh) information. in patients with colon cancer recruited to the ncri foxtrot trial, the oncoscan ffpe assay shows good correlation with pyrosequencing when determining the mutational status of kras/braf. although pyrosequencing has a slightly lower failure rate, the oncoscan has the added advantage of targeting more mutations, producing genome wide cn, and loh information in one assay. excellent anatomical knowledge of the rectum and surrounding structures is essential for total mesorectal excision (tme). denonvilliers' fascia (dvf) has been frequently studied, though the optimal anterior plane in tme is still disputed. the relationship of the lateral edge of dvf to the autonomic nerves is also unclear. we studied whole-mount microscopic sections of en-bloc cadaveric pelvic exenteration specimens and describe implications for tme. four human adult cadaveric specimens (two males, two females) were obtained from the leeds gift research tissue programme. paraffin-embedded mega-blocks were produced and serially sectioned at 50 and 250 µm intervals. sections were stained with haematoxylin & eosin, masson's trichrome and millers' elastin. additionally, a developmental series of eleven human fetal pelvic specimens (embryonic age of 9-20 weeks) were studied. dvf consisted of multiple fascial condensations of collagen and smooth muscle fibres and was indistinguishable from the anterior mesorectal fascia and the capsule of the prostate or posterior vaginal wall. the lateral edges of dvf appeared fan-shaped, and the most posterior part was continuous with the mesorectal fascia. peri-rectal fasciae were not identified in fetal specimens. dvf is adherent to and continuous with the mesorectal fascia. optimal surgical dissection during tme should be carried out anterior to dvf to ensure radical removal, particularly for anterior tumours. autonomic nerves are at risk, but can be preserved by following the mesorectal fascia along the anterolateral mesorectum. the lack of evident fasciae in fetal specimens suggests that these might be formed in later developmental stages. the perineal body (pb) is poorly understood. in abdominoperineal excision (ape), there is no natural dissection plane through the pb. knowledge of the pb is essential to avoid straying in to incorrect planes leading to tumour perforation and unnecessary urogenital and anorectal injuries. this study describes the anatomy of the pb and the implications for ape. six human adult cadaveric specimens (three males, three females) were obtained from the leeds gift research tissue programme. paraffin-embedded mega-blocks containing the pb were produced and serially sectioned at 50 and 250 µm intervals. sections were stained to reveal collagen and elastin, and with an antibody against α-smooth muscle actin. the pb is formed of a fibromuscular mass, which was thicker and wider in female specimens compared to males, extending from the external anal sphincter to the rectogenital septum. muscles from the urogenital diaphragm and anterior rectal wall anchored into the pb. the longitudinal muscle (lm) of the rectal muscularis propria extended in anterolateral directions and intertwined with the somatic pelvic floor muscles to create strong fixation of the anorectum. the lm plays a dominant role in the formation of the pb. surgeons should be aware of the complex course of the lm through the pb to prevent injuries to the urogenital organs and perforation of the anterior rectal wall. the perineal phase of an ape starts with excellent exposure followed by proper tension on the pb to allow safe dissection through the densely-packed fibromuscular mass. introduction: ki67 is a proliferation marker that is exclusively present in dividing cells and absent in resting cells. its expression has already been studied in different cancers and used to understand the cellular organisation of barrett's epithelium (lavery 2014). however, very little is known about the cellular organisation based on ki67 expression patterns in upper gi sequence. this study aims to examine the cellular organisation as defined by ki67 expression patterns in upper gi cancer sequence. methods: ki67 expression within barrett's crypts was assessed in 40 cases (nbde 11, lgd 15, hgd 14) . the barrett's crypts were divided into three equal regions: crypt base (bottom third), middle region and the surface (upper third), respectively. ki67 was scored using the allred system and analysed using one-way anova with bonferroni post-hoc analysis. results: one-way anova showed significant difference across the three groups (p < 0.0001). bonferroni post-hoc analysis showed significant difference in the surface architecture between nbde and hgd (p < 0.0001) and lgd and hgd (p < 0.0001). for the middle region, although there was no statistical significance between the groups, nbde and lgd and lgd and hgd showed statistical trends (p = 0.079 and p = 0.089 respectively). for the basal compartment there was significant difference between nbde and lgd (p = 0.035). this study showed for the first time a significant difference in the ki67 expression between nbde, lgd and hgd in the basal and surface regions. middle compartments showed trends but additional ndbe, lgd and hgd groups need to be analysed to increase the statistical power. the results warrant further molecular analysis between the various groups and show a clear role for proliferation in the maintenance of the cellular architecture and organisation across the upper gi groups which might help in the understanding of the origin and development of oac. • frequency of serosal involvement in rectal cancers (suggested contributing factors for this include effect of pre-operative therapy, tumour regression and recent changes in surgical practice). • turnaround times: suggested contributing factors include increased departmental workload, retirements and reduced reporting capacity. the following action plan was implemented to improve compliance with standards: • ensure all pathologists are aware of results via presentation/dissemination of audit report • identify issues affecting turn-around times and improvement strategies • support recruitment to increase reporting capacity. • maintain awareness of the need to recognise serosal involvement in rectal excisions. • re-audit in 1 year. the design and maintenance of a pilot online digital archive of archetype colorectal polyps and gastrointestinal (gi) teaching cases for the national bowl screening programme (bowelscreen) in the republic of ireland. methods: suitable internal and referral cases were identified by bowelscreen consultants at saint vincent's university hospital. these cases were subject to both internal and external review, by the mater misericordiae university hospital, and represented typical examples of lesions seen in a national bowel cancer screening programmes (e.g adenomas, ssls, adenomas with misplacement, tsas). representative slides, including immunohistochemistry, were anonymised and digitised using the hamamatsu nanozoomer digital pathology (ndp) whole slide scanner platform and associated software packages (ndp scan and view). whole slide images (wsi) were uploaded to secure cloud storage using a generic file transfer protocol program. wsi were collated into 18 cases and accessible via the pathxl gateway (pathxl.co.uk) by approved users via an online case referral and reporting system. users were notified of pending cases via email and the viewing of wsi occurred within the user's web browser utilising an online version of ndp view program and did not require local use of propriety software. each case was referred across the two participating sites and scored in four areas; diagnosis concordance, quality of wsi, web interface and the online referral and reporting system. conclusion: with the maturation of technology involved in digital microscopy a digital archive program is now a feasible approach to the standardisation of diagnosis and a useful adjunct to traditional optical microscopy in education within the national bowel screening programme. conclusions: a committed and conscientious bms can learn how to report histopathology cases. however, if this is to be achieved, the department in which he or she works must also be committed and supportive. carcinoid tumour of the appendix: a case report p aae shalaby; p aae shalaby a case of a 24 years male operated on for acute appendicitis and an incidental finding of a carcinoid tumour at the tip is reported. the tumour was less than 2 cm in greatest dimension but it infiltrates through the wall of the appendix into the surrounding fat. it stains positive for the neuroendocrine markers. carcinoid tumour of the appendix is unusual, but it has to be looked for during examination of appendectomy specimens done for appendicitis (0.5%). women are more frequently affected than men (3:1) and the tumour is usually small less than 1 cm in diameter and frequently located at the tip. it is usually diagnosed incidentally after an operation for acute appendicitis and sometimes during other procedures (colectomy, cholecystectomy and others). the tumour rarely metastasis to the liver and this is usually related to the tumour diameter) and can cause a "carcinoid syndrome": flush, diarrhea bronchoconstriction, cardiac valve disease. diagnosis is made by the pathologist and staging by conventional radiologic procedures (tac, us), dosage of neuroendocrine mediators such as 24 hours urinary 5-hiaa. simple appendectomy is adequate treatment for appendicular carcinoids less than 1 cm in diameter. adequate treatment for tumours greater than 2 cm is right hemicolectomy. the mangement of tumours 1 to 2 cm range is controverisal, but generally, appendectomy alone is sufficient except when meso-appendix is invaded. carcinoid tumour of the appendix has a good prognosis with a 5-year-survival rate, of 85-100%. the prevalence of epithelial changes in helicobacter pylori-associated gastritis in oman: a retrospective study p aae shalaby; a al saadi there is strong association between h. pylori gastric infection and epithelial changes and progression to cancer. it has been shown that h pylori infection is strongly associated with high proliferative activity and it could be a risk of initial step of gastric carcinogenesis. the aim of this study was to examine the association between epithelial changes in the gastric mucosa and gastric h pylori infection in oman by retrospective examination of the gastric biopsies for patients presented to sultan qaboos university hospital (squh) in 2013. a total of 697 biopsies were studied with a prevalence of h pylori infection in 34% with about 13% showing epithelial changes, mainly intestinal metaplasia in 10% out of the h pylori positive cases, a few cases with low grade dysplasia and reactive atypia. in cocnlusion intestinal metaplasia was the main epithelial change that was related to h pylori infection. further studues are required to investigate the relation between h pylori infection and the progression to gastric carcinoma. purpose of the study: low rectal carcinoma may require abdomino-perineal excision of the rectum (aper), which has been associated with higher rates of tumour perforation and circumferential margin (crm) involvement than anterior resection. this increases the risk of local recurrence and may necessitate adjuvant treatment. the extralevator abdominoperineal excision of rectum (elape) in the prone position has been found to improve these outcomes and has been encouraged by the low rectal cancer national development programme (lorec). we aimed to assess the effect of increasing the practice of elape on the histological and oncological outcomes in these cases in the mid-yorkshire nhs trust, a large district general hospital. in 2011 the number of surgeons routinely performing aper was reduced and all those performing the procedure had been trained in the cylindrical resection technique. joint operating and laparoscopic procedures were encouraged. a retrospective review of case notes and histological reports between 2009 and 2012 was performed (before and after sub-specialisation). patient demographics, histological findings and complications including local recurrence were recorded. summary of results: between 2009 and 2011, 39 apers were performed, with tumour perforation in 5 (13%) and crm involvement in 8 (21%) of cases. after sub-specialisation, 28 were performed. none were perforated and 2 cases (7%) showed margin involvement. local recurrence occurred in two cases before specialisation and none after 2011 at the time of follow-up. joint operating and subspecialisation increased the number of cases performed by each surgeon, and the number performed laparoscopically. conclusions: elape in conjunction with departmental restructuring significantly improves immediate oncological outcomes in a dgh setting, with no effect on 30 day mortality. the technique may reduce local recurrence, although longer follow-up would be required. background: systems biology uses computational and simulation approaches to interrogate gene expression datasets and explore biological pathways. by employing systems biology and data mining tools we can identify new biomarkers. our objective was to ascertain the utility of a novel panel of systems biology derived biomarkers in cervical pre-cancer for more accurate grading and stratification of cin disease. methods: this project is conducted within the framework of an fp7 funded programme "systemcerv". gene pathways were analysed using matlab and sirene. along with accessing keggs online database for gene prediction and david for gene functional classification, we identified a novel panel of biomarkers. gephi software was used to visualise communities of genes related to cervical pre-cancer and cancer progression. clinical validation was performed by immunohistochemistry on a range of cervical lletz specimens (normal, cin1, cin2 and cin3). all patients gave written informed consent. in parallel, p16 ihc was performed on all specimens as a benchmark stain. the mortality associated with cervical cancer can be reduced if the disease is detected at the early stages of development or at the pre-malignant stage. the pap smear is the current screening method, but is highly subjective and can often exhibit low specificity and sensitivity. for this reason, either a replacement or supportive technique is necessary to improve the quality of cervical cancer screening. raman spectroscopy is a powerful tool that can generate a biochemical fingerprint of a sample in a rapid and non-destructive manner. in this study, raman spectroscopy has been applied to the investigation of cervical cells from preservcyt specimens. raman measurements were taken from the nuclei of cervical cells from normal, cin1, and cin3 samples. these spectra were processed, analysed and used to define a spectral signature for each grade of cervical disease. principal component analysis (pca) was used to discriminate between the two data sets. distinct raman spectral differences were detected between normal, cin1 and cin3 cells. notably, it was possible to observe spectral peak shifts representing fluctuations in guanine (dna/rna), ch deformation in proteins and carbohydrates, carbon-carbon double bonds in phenylalanine, tyrosine and tryptophan, and amide i. the pca showed an excellent discrimination between the data sets. this study has shown that raman spectroscopy can detect subtle changes between cervical cells, and may be a powerful tool for improved diagnosis of cervical dysplasia. background: myd88 and mad2 are two potential prognostic biomarkers that have been investigated in ovarian cancer. high myd88 and low mad2 ihc staining is associated with reduced pfs, both markers are also linked to paclitaxel chemoresistance. the main objective of this study was to assess the in vitro relationship between mad2 and myd88, through alteration of mad2, myd88 or its receptor tlr4 in two ovarian cancer cell lines using sirna targeting mad2, tlr4 or myd88 and a myd88 overexpression plasmid vector. following overexpression/sirna knockdown procedures, myd88, tlr4 and mad2 expression was assessed through qpcr and western blot analysis. mir-433, mir-21 and mir-146a gene expression was also assessed by qpcr. furthermore the effect of tlr4/ myd88 knockdown on chemoresponse was assessed in skov-3 cells using the cck-8 assay. results/discussion: it was found that knockdown or overexpression of myd88 in skov-3 or a2780 cells respectively or knockdown of tlr4 in skov-3 cells had no effect on mad2 expression or the expression of mir-21, mir-433 and mir-146a. interestingly however knockdown of mad2 in both cell lines induced a 3 fold increase in tlr4 expression, furthermore knockdown of tlr4 in skov-3 cells was shown to restore chemosensitivity to paclitaxel. the results demonstrate a potential in vitro link between tlr4 and mad2 and support a role for tlr4 in paclitaxel chemoresistance. background: the prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. recent evidence points to the toll-like receptor-4 (tlr4), and particularly its adaptor protein myd88, as one potential mediator of this resistance. downregulation of mad2, a key component of the spindle assembly checkpoint complex, has also been linked with paclitaxel resistance . both markers have individually been shown to be associated with poor outcome in ovarian cancer. high myd88 and low mad2 immunohistochemical staining is associated with reduced progression free survival. the main objective of this study was to assess the combined utility of mad2 and myd88 in predicting patient prognosis. methods: two tissue microarrays composed of cores from 51 high grade serous epithelial ovarian cancers patients were constructed and stained for mad2 and myd88. staining was scored based on previously derived scoring schemes for myd88 or mad2 staining. the mean overall score from triplicate cores was then used to classify patients into high and low staining categories. results: a trend towards reduced progression free and overall survival was observed in patients with high myd88 and low mad2 expression. the results demonstrate the combined utility of mad2 and myd88 as predictors of prognosis in ovarian cancer. purpose: ovarian cancer is characterised by high rates of terminal, chemoresistant recurrence. although chemoresistance is known to be a property of cancer stem cells (cscs), the mechanism is poorly understood. we have previously identified a novel four-member csc stem-progenitor cell hierarchy for ovarian cancer. the aim of this study was to characterise the contribution of each member of the ovarian csc hierarchy to chemoresistance. methods: the csc hierarchy was assessed for tolerance to chemotherapy drug cisplatin (mtt assay) both as components of the parent population (a2780 cell line) and as isolated cell types. the hierarchy was additionally assessed in the long-term cisplatin-adapted 'a2780cis' cell line. cell types were analysed and isolated via flow cytometry and assessed for stem cell characteristic via single-cell asymmetric division and murine xenograft tumourigenicity assays, and molecularly characterised (whole transcriptome arrays). results: cisplain dose-response assays from a2780-derived csc sub-populations indicated that only one of the four populations within the hierarchy had a high cisplatin-tolerance (ic50=10um) compared to the other populations (ic50=4um). this was notable as the relative cisplatin ic50s for the a2780 and a2780cis parent cell lines are 4um and 11um respectively. treatment of the parent a2780 cell line with the ic50 (48 hours) resulted in a proportional 50% loss in each of the four cell types, suggesting that this specific csc subpopulation adapts to cisplatin over a longer period of time. conclusion: although cscs are known to be chemoresistant, the mechanism though which this is achieved is poorly understood. our data indicates that only some members of a csc hierarchy are responsible for chemoresistance. notably, this sub-population appears to possess inherent chemoresistance in pre-treatmentcells. as such, it should be possible to target these cscs in the primary malignancy to prevent chemoresistant recurrence. mixed sex cord-stromal tumours of the ovary are very rare. we report a case of mixed sex cord -stromal tumour (also referred to as gynandroblastoma) containing both sertoli-leydig cell tumour and adult granulosa cell tumour in a female 61 years old who presented with postmenopausal bleeding. on histology, the majority of the tumour represented an unsual form of well differentiated sertoli-leydig cell tumour with a pseudoendometrioid appearance. minor foci of classic adult granulosa cell tumour were present. on immunohistochemistry, the tumour was diffusely positive for inhibin and sf1 and focally for calretinin, er and cd56. ema, pax8 and ck7 were negative. as far as we are aware, this is the first report of an ovarian mixed sex cord-stromal tumour containing a component of pseudoendometrioid sertoli-leydig cell tumour. a 37 year old female patient presented with bilateral painful warty lesions on the labia majora. the patient had had hiv for a long time and was on highly active antiretroviral therapy. she also suffered chronic renal failure requiring haemodialysis three times weekly. clinically, the lesions were highly suspicious of vulval cancer. the lesions increased significantly in size over a short period of time (2 months) requiring surgical resection under general anaesthesia. histological examinations revealed polypoid lesions with prominent pseudoepitheliomatous hyperplasia and dense inflammatory infiltrate, composed mainly of lymphocytes and plasma cells, extending to the hypodermis. numerous abscesses with large numbers of eosinophils were present withinin the hyperplastic epithelium. the typical intranuclear inclusions of herpes simplex virus (hsv) were identified. hsv immunohistochemistry was positive. this is a rare case of vulval hsv warts mimicking cancer. oral acyclovir was administered following surgery and resulted in good control. literature review shows only 6 previously described cases of verrucous hsv, types 1 and 2, simulating neoplasia in patients with aids on antiretroviral therapy. primary mucinous eccrine adenocarcinoma of the skin is a rare adnexal neoplasm, typically involving the head and neck region in the elderly population. here we present a case of primary mucinous eccrine adenocarcinoma of the vulva; occurrence at this site is extremely rare, with only five cases published in english literature. a 62 year old female presented with a 10mm vulval lesion, clinically suspected to be an inclusion cyst. the lesion was removed and sent for histopathological assessment. histological examination revealed a well circumscribed, partly encapsulated tumour composed of rounded and irregular nests of polygonal epithelial cells with scattered lumina, suspended in pools of extracellular mucin. the epithelial cell nuclei displayed a uniform chromatin pattern with small distinct nucleoli. the mucin pools stained positive for alcian blue and dpas. immunohistochemical staining demonstrated positivity for cea, ck7, gcdfp, oestrogen receptor, progesterone receptor, synaptophysin and chromogranin. immunostaining was negative for ck20, cdx2, ca-125, ttf-1, ch5/6, ck14, her-2, wt-1, cd56 and s100. ki-67 proliferation fraction was approximately 5%. overall, the findings were those of a mucinous eccrine adenocarcinoma with neuroendocrine differentiation. following multidisciplinary discussion, and negative imaging of the breasts and gastrointestinal tract, a diagnosis of primary mucinous eccrine adenocarcinoma of the vulva was reached. only a handful of cases of primary mucinous eccrine adenocarcinoma of the vulva have been reported. metastatic disease, particularly from breast and colon, must be excluded. follow up data from patients with primary mucinous eccrine adenocarcinoma of the skin suggests a high local recurrence rate (29.4%), necessitating close follow-up. however, risk of metastasis is low (9.6%). royal shrewsbury hospital, shrewsbury, uk introduction: primary extraskeletal myxoid chondrosaroma (emc) of the vulva is a rare mesenchymal neoplasm. the myxoid tumour differential diagnosis on a core biopsy can be quite challinging. to date, few cases have been reported in the literature. case report: a 42-year old woman noticed a swelling on the right side of the labia, thought to be a bartholin's cyst in 2011. she was managed conservatively. she had drainage and marsupialization under general anaesthesia. this resulted in extreme bruising of the vulva. this was managed with antibiotics and non-steroidal anti-inflammatory medication, and it resolved after 3 weeks. six months later, the patient presented again with a persistent vulval mass. a biopsy was obtained under general anaesthesia, and it showed a myxoid tumour with differential diagnosis of low grade chondroid tumour. an mri was performed to assess the extent of the disease. the tumour was excised. at surgery, a 7 x 5 cm lobulated, extremely vascular vulval tumour was found. the tumour was inseparable from the inferior pubic ramus of the pelvic bone. a complete macroscopic resection was obtained. histology confirmed low grade myxoid chondrosarcoma. conculsion: vulval lesions with unusual characteristics or insidious evolution in the labia majora or bartholin's glands area should be carefully and promptly investigated. differential diagnosis of myxoid tumours in the vulva should include myxoid chondrosarcoma amongst other diagnoses. (fish) showed the presence of a bcl2 rearrangement in a proportion of cells. therefore this case is best regarded as a composite lymphoma of diffuse large b cell lymphoma with hairy cell leukemia rather than blastic transformation of hairy cell leukemia. to the best of our knowledge, simultaneous occurrence of diffuse large b cell lymphoma and hairy cell leukemia in a lymph node has not yet been reported in the literature. bone marrow examination by aspirate and trephine biopsy is an important haematological investigation. ideally, aspirate findings should inform examination of the trephine biopsy, but if the two modalities are separate the aspirate report can be delayed and histopathologists may assess the trephine biopsy without being aware of the aspirate findings. we audited the availability of aspirate results to the histopathologist examining trephine biopsies, over the period in which our department implemented an integrated haematopathology reporting system. the effects on diagnostic concordance, turnaround times and immunohistochemistry requesting were also assessed. the setting was a regional specialist haematopathology centre. prior to integration, a prospective audit of 101 consecutive trephine biopsies received by a senior haematopathologist was carried out, against standards set by the international committee for standardisation in haematology. data were collected from hospital computer systems. the move to integrated reporting involved the installation of new software (hilis) to specifically handle integrated haematopathology data. ten months later, a retrospective analysis of a further 100 cases was performed using hilis data. prior to integration, 35% of aspirates were reported within 3 days, and access to the aspirate report was available at time of examination for 61% of trephine biopsies. after integration, 96% of aspirates were reported within 3 days, and reports were available at time of examination for 100% of trephine biopsies. diagnostic concordance was 72% initially, and 100% after integration. the mean number of immunostains requested per case was unchanged (5.9 vs 5.7). our findings show integrated reporting has markedly increased the availability of aspirate reports to the histopathologist, and improved diagnostic concordance. this new model benefits the haematologist, histopathologist and patient. introduction: clonality studies are carried out when the diagnosis of lymphoma is particularly challenging. the detection of clonality in lymphoproliferative lesions suspicious for lymphoma can be a valuable supplementary tool as it has a high positive predictive value. clonal studies can also help to distinguish recurrent or residual disease from reactive inflammation. however false positive and negatives are common and can be attributable to several factors, including poor dna quality. methods: 206 cases reported over a six month period (jun-nov 2014) were retrospectively reviewed, 62% of which were referral cases. we investigated various aspects of clonality studies; including dna quality, fixation method, clinical information provided and correlation between the morphological/immunophenotypical findings and clonality results using euroclonality/biomed-2 primers (igh, igk, tcr-β and gamma-delta). results: 166 (81%) had adequate dna quality, 29 (14%) poor dna and 11 (5%) had inadequate dna quality. external cases had better dna quality in the majority of cases. clinical information was provided in 75% of local cases and 53% of external cases. in 56% of cases clonality results supported the initial histological report. 9 cases showed clonal expansion despite a benign process on histology. 21 suspected cases lymphoma (13 b-nhl and 8 t-nhl) showed no clonality, 8 of which yielded poor dna quality. skin cases although had good dna quality, usually had low number of neoplastic cells resulting in poor pcr products. conclusions: dna quality is very variable and clinical information is often not provided precluding adequate assessment of clonality findings. dna was worse locally (decalcified marrow trephines using formic acid and peloris system with high temperatures that can cause dna degradation). standardisation of fixation methods and interpretation of peaks/ bands in the clinical context of the patient is essential for clonality to be informative. kikuchi-fujimoto disease: a novel diagnosis by transbronchial biopsy of mediastinal lymphadenopathy p pm ellery; n archard; a ramsay ucl hospitals nhs foundation trust, london, uk objectives: kikuchi-fujimoto disease (kfd) is a rare, self-limiting form of necrotising lymphadenitis that most commonly affects young asian women, and classically presents with fever, malaise and lymphadenopathy. the cervical lymph nodes are involved in around 85% of cases, with other sites rarely involved. we report an unusual case in which an unexpected diagnosis of kfd was made via transbronchial biopsy of mediastinal lymph nodes. a 15 year old boy of pakistani origin presented with a 4 month history of lethargy, neck stiffness and weight loss, with fever (up to 40°c) and night sweats. chest x-ray, mantoux test, blood cultures and viral pcr were negative. lumbar puncture was normal, with no acid fast-bacilli. ct showed enlargement of the deep cervical lymph nodes (petpositive on further imaging), and mediastinal lymphadenopathy. he was transferred to our hospital for further management, with a differential diagnosis of tb, lymphoma, rare infection or autoimmune disease. he underwent transbronchial biopsy of the mediastinal lymph nodes. results: his biopsy showed blood clot and cores of lymph node, with focal collections of crescentic macrophages, admixed lymphocytes and prominent apoptotic debris. immunohistochemistry demonstrated a population of cd123-positive plasmacytoid dendritic cells and granular mpo positivity in macrophage cytoplasm. the background lymphocytes were mainly cd8-positive t-cells. the features were those of kfd. conclusion: involvement of deep lymph nodes is unusual in kfd, and to our knowledge, this is the first case diagnosed via transbronchial biopsy. such biopsies often produce scanty diagnostic material, and here the detection of the characteristic immunoprofile of kfd helped confirm the diagnosis. this case highlights that kfd should be considered at sites other than the cervical lymph nodes, and demonstrates the value of immunohistochemistry in reaching a definitive diagnosis. we describe a benign intravascular proliferation of atypical polytypic cd30 positive t cells, co-expressing follicular t helper cell lineage markers coincidental to local sepsis of the buttock. a 43 year old female presented with a 5x5cm buttock abscess at the site of a longstanding palpable lump. peripheral blood showed only a neutrophilic leucocytosis. immunohistological examination showed large aggregates of atypical cd30 positive, alk negative lymphoid cells expressing a pan t helper phenotype with cd7 partially downregulated. podoplanin proved that the atypical t cells were primarily, but not exclusively, localised within lymphatic channels. a t cell receptor clone was not detected using pcr. to find intravascular concentrations of atypical lymphoid cells is uncommon in skin biopsies and raises the possibility of leukaemia or intravascular lymphoma. intravascular lymphoma is a rare variant of non hodgkin lymphoma with a minority possessing t or nk cell lineage but frequently involving skin. cd30 is a transmembrane glycoprotein and a member of the tnf superfamily involved in regulating proliferation. it is considered a reliable marker of lymphoma. primary cutaneous cd30 positive tlpds encompass a spectrum of biological aggressiveness and include primary cutaneous anaplastic large-cell lymphoma and lymphomatoid papulosis (lyp). cd30 can also be up regulated in activated b and t cells and it has been proposed that cd30 positive ivtlpds are equivalent to an intravascular form of lyp. intravascular proliferations of atypical cd30 positive t cells have been linked with chronic inflammation and abscess formation. furthermore atypical cd30 positive tlpd expressing a cd4 positive t helper phenotype and exhibiting an indolent clinical course have been reported in the arm, trunk, neck and prepuce. ultimately ivtlpd may require follow up based upon clinical features and natural progression due to overlapping diagnostic features. p g laing; s craig; l moss; c crichton; p johnston the investigation of lymphoid neoplasia requires multiple sections, in our practice consisting of twelve antibodies and thirteen single stained slides. by selecting particular antibodies for double staining, spatial relationships between cell types and overall tissue organisation can be more easily visualised. this pilot study aimed to optimise the staining intensity and specificity to provide accurate diagnostic information, reduce slide number, material and consumables costs, preparation time in the laboratory and storage space to enhance costbenefit. twelve antibodies were chosen and paired: kappa/lambda, cd3/cd20, mum-1/cd10, cyclin d1/cd30, bcl-6/cd5 and cd5/pax-5. firstly, these combinations were applied to normal tissue and then known tumours to optimise technique. once the staining protocols were finalised they were run on eleven consecutive cases with conventionally stained non-hodgkin lymphoma (nhl) panel requests. the slides were then reviewed by the lymphoma team for quality and diagnostic accuracy compared to the standard single stained slides. the results demonstrate that double staining is possible in the diagnosis of nhl. the combinations chosen have proved successful and have provided interpretable results; for example, the relationship of light chain staining in plasma cells, mum-1 and cd10 positive cells in diffuse large b-cell lymphoma proves positive. we feel time will be saved cutting sections to improve efficiency in lymphoma investigation and reduce panel storage space by around 30%. in conclusion the outcomes from this pilot study have been positive for medical and scientific staff, has shown that double staining in the diagnosis of nhl is possible and that optimising this protocol with a view to live diagnosis is worthwhile. in an age in which novel therapies are not necessarily defined by their ability to kill malignant cells, understanding the biology of malignant cells after treatment is extremely important. this is particularly true of ibrutinib (pcl-32765) therapy in chronic lymphocytic leukaemia which is characterised by lymphocytosis. imagej/fiji image analysis software could therefore be used to analyse cell shape and grouping characteristics. we used a novel assay in which chronic lymphocytic leukaemia cells, cultured for 5 days, were seeded onto fibronectin coated glass coverslips and then had their b cell receptors ligated with goat anti human igm. they were compared with cells simultaneously inhibited with ibrutinib. we tested multiple staining techniques and found that using either rose bengal or texas red phalloidin staining produced the most reproducibly analysable data when using imagej/ fiji. we demonstrated, using the assumption that the outline of interacting cells would be larger than cells which were alone that homotypic interactions were increased after b cell receptor cross linking (54 groups larger than 2 cells against 35 group larger than 2 cells per 500nm x 250nm field (p=0.019)). nuclei were also significantly larger when cross linked. (mean 33.23 (+-0.43) pixels without cross linking and 44.15(+-0.57) (p=≤0.0001)) suggesting increased nuclear spreading. these effects were reversed by the addition of ibrutinib with 18 groups larger than 2 cells per the same field(p=0.0252 compared with cross linked sells) and nuclear size mean being 33.83 (p≤0.0001 compared with cross linked cells). this study demonstrates a novel, easily reproducible assay to assess a variety of cellular responses to ibrutinib therapy and suggests a method of quantifying activity both when stimulated and inhibited. this technique could easily be scaled up to further investigate cellular behaviour following ibrutinib therapy. purpose: gliomas represent 43% of all solid intracranial tumours and are associated with a poor prognosis. recent studies indicated that the human cytosolic branched chain aminotransferase protein (hbcatc), which metabolises the branched chain amino acids (bcaa), was significantly upregulated in idh1/2 wild type (wt) glioblastomas, correlated with methylation patterns in the bcat1 promoter and is associated with a worse prognosis compared with idh mutant gliomas. the diagnostic and prognostic significance of markers of bcaa metabolism is currently under investigation. methods: 64 glioma tumour samples were compared for hbcatc, hbcatm and bckdc expression using western blotting and immunohistochemistry. dna was extracted from fresh frozen tissue. sanger sequencing of the p.arg132 region of idh1 and p.arg172 region of idh2 was undertaken using a 3730 dna analyser (applied bio-systems). summary: in idh wt tumours, like hbcatc (p=0.007), the expression of the mitochondrial isoform (hbcatm) is significantly (p=0.036) expressed relative to idh mutant gliomas. hbcatm additionally shows a more significant correlation with patient survival than hbcatc on kaplan-meier analysis. in idh wt tumours, low hbcatm expression is a positive prognostic factor (p = 0.003). hbcatm expression additionally correlated with who grade. although previous reports indicate that increased hbcatc occurs exclusively in idh-wt tumours, our studies demonstrate that 30% of idh mutant tumours express comparable levels of hbcatc. although hbcatc alone has been suggested as a putative therapeutic target, it is important to evaluate the expression of hbcatm in glioblastomas as its expression may impact the efficacy of new treatments targeting hbcatc. conclusions: idh wt high grade gliomas traditionally have a poor prognosis. however we demonstrate for the first time that relatively low hbcatm may select for a better performing clinical cohort and may be a possible candidate target for drug therapy. a 38 year old female presented with an occipital mass, presumed to be a lymph node and underwent fine needle aspiration of the lesion. fna yielded two air dried slides, upon which a diagnosis of mesenchymal neoplasm was made. the patient underwent a subsequent incisional biopsy allowing a formal histological diagnosis of myxoinflammatory fibroblastic sarcoma to be made. myxoinflammatory fibroblastic sarcoma is a low-grade neoplasm usually occuring on the distal extremities and only rarely presents as a head and neck neoplasm. fna is a useful tool in the diagnosis and subsequent management of head and neck neoplasia and we describe here the cytological features and subsequent histological diagnosis of myxoinflammatory fibroblastic sarcoma occuring in the occipital scalp. objective: presentation of giant cell fibroblastoma (gcf). because of it has a dilemma of microscopic appearances; it is mandatory to differentiating it from atypical dermatofibroma (adf), fibrous hamartoma of infancy (fhi) and vascular lesions. methods: eighteen month-male egyptian child presented with painless slowly expanding subcutaneous back swelling at the left scapular area. results: histologically, the lesion is poorly circumscribed and range from cellular to myxoid in a dense to loose collagenous stroma. the tumours composed of mixture of spindle shaped or stellate cells admixed with multinucleated giant cells with occasional pleomorphism and very low mitotic index (<1 per 50 high-power fields). these cells infiltrate around adnexal structures and through subcutaneous fat. a distinctive finding is cracking artifact of the stroma simulating angiectoid spaces. these pseudovascular spaces are lacking a true endothelial lining and lined by discontinuous layer of enlarged multinucleated giant cells. immunostains, including factor viii, cd31, cd1a, sma, s100 and cd68 were negative. ki 67 labeling index is very low. all the cellular components show positive immunoreactivity for cd34. conclusions: first case of gcf reported in egypt. we recommend a wide scaled study to categorize this tumour with molecularly similar lesions. the royal county sussex hospital, brighton, uk a 60 year old man being investigated for obstructive hydronephrosis was incidentally found to have a 3.2cm splenic mass on computed tomography (ct). no lymphadenopathy was present and the mass remained stable on sequential ct and ultrasound scans. on positron emission tomography (pet) the lesion had a low signal with moderate uptake. all haematological investigations were within normal limits including a negative epstein barr virus (ebv) test. his past medical history included previous immunosuppressive therapy for inflammatory bowel disease. a core biopsy under ct guidance was performed. the cores showed a paucicellular spindle cell lesion with bland, blunt ended nuclei, no cytological atypia and a sparse chronic inflammatory infiltrate. there was no necrosis. the spindle cells stained positive for smooth muscle actin (sma) and h-caldesmon indicating this to be a splenic leiomyoma. splenic lesions are uncommon and within their differential include, lymphoma, inflammatory pseudotumour, harmatomas and leiomyomas. (1) a splenic leiomyoma is an unusual and rare benign smooth muscle tumour with an unknown pathogenesis. they are thought to arise from the capsule and blood vessel walls of organs. (2) they have been documented in immunosuppressed states (constitutional or acquired), in those with ebv infection and in children with ataxia-telangiectasia. (2) leiomyomas within the spleen are rarely reported in the literature, especially in those patients over the age of eighteen. in this case there was historical immunosuppression, however leiomyomas should be considered in the differential diagnosis of well-defined solitary splenic lesions purpose of the study: the zucker diabetic fatty (zdf) rat is extensively used as a model of diabetic kidney disease (dkd) associated with obesity and progressive insulin resistance ('diabesity'). this study aimed to validate qualitative ultrastructural parameters of glomerular injury in the zdf animal model and apply these criteria to an interventional study investigating the effects of roux-en-y gastric bypass (rygb) on dkd. methods: superficial renal cortices were immersion-fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, processed and embedded in epoxy resin prior viewing under a technai 12 transmission electron microscope. glomerular basement membrane (gbm) thickness, podocyte foot process diameter (pfpd) and podocyte foot process frequency (pfpf) per unit length of gbm were determined for each group (sham and rygb operated zdf fa/fa diabetic animals vs non-operated non-diabetic zdf fa/+ lean controls). statistical analysis was performed using a mann whitney u test and an unpaired t-test where appropriate. summary of results: selected tem parameters (gbm thickness, pfpd and pfpf) demonstrated significant differences between specified sham-operated zdf fa/fa vs fa/+ samples, p=0.017. analysis of rygb interventional study samples still in progress. early post-operative glucose measurements showed a significant improvement in glucose homeostasis in the rygb group (rygb vs sham, p=0.0001) occurring independently of weight loss. urinary albumin:creatinine ratios were lower in the rygb group vs sham operated positive controls (p=0.0079) and were comparable with age-matched lean control fa/+ samples. conclusions: preliminary findings support a beneficial role for rygb in an animal model of 'diabesity'. validated ultrastructural parameters should assist in elucidating changes in podocyte activation and differentiation as mediators of the observed remission of albuminuria following rygb surgery. nottingham university hospital, nottingham, uk spitz naevus is a benign melanocytic lesion that shares many histological features with malignant melanoma. although the morphological criteria differentiating the two entities are well established however, some cases can be challenging. many isolated markers have been proposed to help in differentiating spitz naevus from melanoma, albeit none has been shown to be definitive. aim: this is a preliminary study looking at the immunohistochemical expression of 3 markers that are known to have important role in cell cycle regulation, proliferation and melanocytic differentiation (p16, ki67, and hmb45). the aim is provide to a combination of proteins that can help in differentiating spitz naevus from malignant melanoma. the study included 12 cases of spitz naevi, 6 benign compound naevi and 6 cases of malignant melanoma. immunohistochemical expression of p16, ki67, and hmb45 has been accessed and compared with the morphological features of these lesions. results: it is noted the mean p16 expression is higher in compound and spitz naevi than melanoma (83, 91, and 36 respectively). proliferation activity as measured by ki67 index is higher in melanoma in comparison with compound and spitz naevi (30.8, 2.8, and 1.6 respectively). hmb45 shows only junctional positivity in 9 out of 11 cases of spitz naevi while in the other two it shows week dermal component. hmb45 is constantly positive at the deep dermal component of melanoma, albeit the staining intensity is variable. the immunoprofile of spitz naevus is different from that of a malignant melanoma. a combination of biological markers as (p16, ki67, and hmb45), can provide a potential tool to differentiate between the two entities. nevertheless, expanding the biomarker repertoire on a large number of cases is necessary to further establish a reliable panel to differentiate among difficult cases. direct immunofluorescence in a tertiary referral centre: an audit of local guidelines and usage p lj lumsden; l motta; r green salford royal hospital, salford, uk direct immunofluorescence (dif) forms an important and costly adjunct to conventional haematoxylin and eosin (h&e) histology in dermatopathology, particularly in bullous diseases and other immune-mediated diseases. we aim to assess the usage and diagnostic yield of dif in our dermatopathology department. 134 requests for dif on skin biopsies received over a 5 month period met the inclusion criteria. each individual report was assessed with regard to the indication for dif, whether dif was deemed to be indicated or not indicated on assessment of the clinical history supplied on the request card, the results of dif and whether dif was contributory to the final diagnosis. we also collected data on the usage of dif over the last 2 years to assess changes in practice. all 134 requests for dif were granted in line with current departmental policy. the indication categories were divided as follows: bullous 41, alopecia 4, lupus 32, vasculitis 23, dermatitis herpetiformis (dh) 15 and 'other' 19. all requests for dif were deemed to be indicated in both the bullous and dh categories by our panel, but indicated requests varied from 8.7% to 50% in the remaining categories. in 45.5% (61 out of 134) of cases dif was deemed to be contributory to the final diagnosis. our analysis also showed that usage of dif in our department is escalating, with a 23.2% increase in requests from 2013 to 2014. our departmental policy with regard to dif is inclusive and operates solely on the basis of clinician request. with the increasing usage of dif, established departmental guidelines and/or a protocol for dif usage should be mutually agreed with dermatology colleagues in order to ensure effective use of this expensive test. overview of merkel cell carcinoma in an irish population p a cooper 1 ; j thorne 2 1 royal college of surgeons in ireland, dublin, ireland; 2 beaumont hospital, dublin, ireland purpose of study: merkel cell carcinoma (mcc) is an uncommon but highly aggressive primary cutaneous malignancy of neuroendocrine cells with a propensity for regional and distal metastases. due to it's rarity, information relating to it's epidemiology in an irish population is limited, mainly owing to difficulty in gathering large patient series. our aim was to identify all cases of mcc in our institution in a defined 10 year period and review the patient demographics compared to internationally available data. a search was carried out on the hospital laboratory system to identify all cases of mcc from 01/01/2005 to 21/12/2014. all histology reports were reviewed and any information pertaining to patient demographics was recorded in an excel spreadsheet. a literature review was performed relating to the patient profile of mcc internationally and the results were compared. results: a total of 33 reports pertaining to 25 individual patients were recovered. all patients were of caucasian irish ethnicity. the incidence of mcc was higher in men (56% of cases, n=14) than women. the median age at diagnosis was 80 years (range 57-90). men presented at an earlier age (median 78 years) than women (median 83 years). regarding the anatomic site of the tumours, 64% (n=16) were on the head or face, 20% (n=5) were on the lower limb and 12% (n=3) were on the upper limb. all were on sun-exposed sites. of note, the majority of tumours in the male population were on the head (78.6%, n=11), while the female population showed an equal distribution between the head and the lower limbs (45%, n=5 for each sub-site). the subset of patients we identified show demographics consistent with published literature for us, australian and other european cohorts. merkel cell carcinoma is a disease of the elderly affecting sun-exposed sites. we note some variation in the dominant anatomic sites between genders and conclude this is due to differing environmental exposure. background: a novel cell-dispensing instrument referred to as a single cell manipulator (scm) device was developed with the following features: i) rapid optical and fluorescent detection of single cells ii) generation of picoliter sized droplets encapsulating the isolated single cell and iii) printing of the single cell in an "ink-jet" like manner onto a chosen substrate. this technology was used to isolate cells of interest from i) heterogeneous mixed populations of cells, ii) co-cultures of cells and iii) clinical patient samples for subsequent downstream biological analysis. methods: cells were injected into a reusable silicon dispenser chip that was coupled to a live cell camera for image capture and display of cells approaching the chip's exit nozzle. an optical detection mechanism determined the presence of single, fluorescent cells within the selected region of interest close to the chip exit nozzle. a sorting algorithm ensured that only droplets containing the single cells of interest were selected for printing to the prescribed location and user-chosen substrate. results: fluorescently labelled hpv16 caski cervical cells were spiked into a cervical liquid based cytology sample and printed onto a glass slide using the scm. undifferentiated ntera2 human embryonal cancer stem cells were isolated from a mixture of differentiated and undifferentiated cells based on fluorescent tagging of the cell surface receptor, stagespecific embryonal antigen 4 (ssea4). the thyroid stimulating hormone receptor (tshr) was expressed in anaplastic v600e mutated thyroid cancer cell lines that were treated with the mek inhibitor pd0325901. treated cells were isolated using the scm. the scm pasca technology allows isolation of single cells from heterogeneous populations of cells and clinical samples for downstream analysis at a single cell level. this study aims at quantifying immunohistochemistry (ihc) stained human cell lines for protein biomarkers by manual pathologist review. staining analyses are used to calibrate tissue microarrays of tumour cores, against quantitative protein concentration allowing a systems-based data analysis. as a proof of concept, ffpe human cell line pellets (n=13) were ihc stained for smac protein and analysed using aperio image analysis software. staining quantification manually performed by pathologists provided parameters including average staining intensity, percent total cell positivity and h-score. these data were enriched by qualitative parameters pointing out possible histological artefacts. a calibration curve was plotted using h-score data and protein concentrations, previously determined by western blotting. the panel of cell lines provided a range of strong and weak/absent ihc staining using a highly specific smac antibody. the calibration curve showed a strong correlation between absolute protein concentrations and manual h-scores. expression amounts in cell lines correlate with ihc staining intensities determined by pathologist review. the linear correlation between manual h-score and absolute protein values provides an avenue to indirectly determine protein expression. further analysis will be performed on additional antigens and analysis outcomes will be then compared to digital results. this data will provide the basis for deterministic systems-biological data analysis approaches. purpose of the study: 'lean' is a management framework for maximising value and minimising waste. it originated in the automotive manufacturing industry and has been utilised successfully in non-manufacturing processes. one such application in our department was the 'leaning' of the molecular test requesting process using a smart-phone app. this study will look at the potential utility of this application within the national health service (nhs), wherein approximately twenty different molecular test request forms are currently in use. a mobile application to facilitate molecular test requesting was developed using xcode and the objective c programming language. the application was built around an email based system. patient anonymity was paramount in the design; nhs numbers are used as identifiers. the application generates a molecular test request form and can also generate a national cancer drugs fund application form for each request. administrative staff use colour coded flags to represent the progress of each email request through the workflow process to facilitate tracking. the app reduced the administrative staff workload by reducing the number of steps and paperwork involved in the molecular test requesting process. a threefold reduction in time taken by clinicians to request molecular tests was noted. a survey of staff involved with molecular test requesting revealed a 90% reduction in 'lost requests' after the introduction of the application. we present a 'lean' method for requesting molecular tests using a smart-phone app. this application can be used to standardise molecular test request forms within the nhs along with automatic generation of a national cancer drugs fund application form for each request. purpose of study: automated approaches for quantitative digital image analysis (dia) of tissues are becoming increasingly popular in pathology due to advances in whole slide scanning hardware and digital imaging technology. it is essential that dia is standardised to ensure accuracy and reproducibility of results. very limited published data exists on the effect of scanner hardware variations on the accuracy and reproducibility of dia results.the aim of this study was to test the following variables: variation in light source intensity during the day; presnap calibration & white balance of scanned images; variation in dia due to debris on peripheral parts of the section or coverslip edges. methods: immunohistochemistry stained sections from 3 patient samples were scanned on the same aperio cs scanner, hourly, for 2 consecutive days to generate 15 scanned images of each sample, representing 45 images in total. all scanned images were run through aperio software using a macro to quantify positive cell counts. for a subset of images, a region of interest was drawn around the tissue to exclude any debris/coverslip edges from peripheral parts of the slide in the subsequent dia. statistical analysis was performed to calculate the coefficient of variation between dia results from scans on different days. results: variation in light source intensity accounted for 1.2% to 2.1% variation in cell counts between repeat scans of the same slide. exclusion of debris/coverslip edges accounted for 1-2% variation in cell counts between repeat scans of the same slide. subjective analysis revealed no significant difference in appearance of different scans of the same slide. conclusion: variation in light source intensity, presnap calibration and overall white balance, plus debris in peripheral areas of the section account for minimal variation in resultant dia results. technical advances in scanner hardware have reduced variability in scanning operations. further investigations are ongoing. the complexity of ovarian cancer resistance mechanisms: a novel, clinically relevant, in-vitro investigation p s busschots 1 g blackshields 1 ; bt hennessy 3 novel carboplatin and taxol resistant cell lines were developed from upn251 oc cells in a clinically relevant selection strategy to better understand resistant mechanisms in oc. upn251-7c models carboplatin resistance and upn251-7t models taxol resistance. upn251-6calt and upn251-6talt were exposed to alternating treatments of both agents during development. affymetrix arrays were used to characterise gene/mirna signatures linked with the development of chemoresistance in oc cell lines upn251-7c and upn251-7t. bioconductor software, david v6.7 and mirna-target interactions (mtis) analysis was carried out to identify de-regulated genes/mirnas, gene pathways and gene/mirna interactions involved in resistance. upn251 sublines developed using taxol were significantly resistant to taxol, vinblastine and olaparib (p-gp substrates), and reversible with elacridar (p-gp inhibitor) treatment. significant up-regulation abcb1 was seen in upn251-7t which was reflected at the protein level. srpx2 was highly up-regulated in upn251-7t. gli3 and ccl20 were up/down-regulated respectively in upn251-7c. gli3 had a validated interaction with mir-205 down-regulated in upn251-7c. lin28b was highly deregulated in upn251-7c and upn251-7t and had a validated interaction with let-7i, down-regulated in upn251-7c. p-gp over-expression is a dominant mechanism for taxol resistance in our cell lines. mechanisms for carboplatin resistance are more complicated. the top deregulated genes are involved in numerous pathways including apoptosis, cellular transformation, signal transduction, and cell migration cervical glandular neoplasia: the influence of excision procedure on margin status although cold knife cones (ckc) have been traditionally advocated for treatment of adenocarcinoma in situ (ais), large loop excisions of the transformation zone (lletz) are increasingly used. we analysed excisions from 111 patients where ais was confirmed prior to procedure over 5 years to assess the influence of excision procedure on final margin status. we tabulated whether margins were involved, close (lesional tissue less than 5mm from margin) or excised. results: lletzs were performed in 70% (78 of 111 patients), ckc excision in 30% (33 of 111). women who had lletzs were younger than those having ckc excision (32.8 years versus 35.4 years). positive margins were present in 21% (16 of 78) and close margins in 27% (21 of 78) lletz cases. for ckc, margins were positive in 9% of cases (3 of 33) and close in 6% (2 of 33) conclusion: although complete excision is more frequently observed when ckc is performed, compared to lletz, for the treatment of ais 0%) patients, discordant low-grade lymphoma was identified in the bone marrow during staging investigations. 4 were classified as follicular lymphoma, 2 marginal zone lymphoma, 2 chronic lymphocytic lymphoma, 1 lymphoplasmacytic lymphoma, 1 non cll -like monoclonal b-cell lymphocytosis. five had an accompanying a low-grade component that could not be classified. none of the transformed high-grade lymphoma cases were ebv positive. in our cohort, we did not observe statistically significant difference in survival between the transformed and non-transformed cases. an equal proportion of cases transformed from follicular lymphoma and marginal zone lymphoma. 19.1% of transformed patients had a previous history of another cancer, compared to 12.1% of non-transformed cases. age, gender and a history of autoimmune disease were not associated with transformation. transformed chronic lymphocytic lymphoma an investigation into the presentation and nature of diffuse large b cell lymphoma within a large patient cohort p hr freer 1 t cell/histoiocyte-rich large b cell lymphoma (4.2%) was the next most common subtype, followed by primary dlbcl of the cns (3.0%). a modified r-ipi (the patient performance score was unknown) was used to stratify the patients into four risk groups and was found to be predictive of patient outcome. the average ldh level was 733.5 iu/l, well above 480, the upper limit of the normal range. of the cohort of patients, 52.8% achieved remission, 15.5% were alive with disease at the end of the study and 31.7% are now deceased. the majority of patients that did die did so within a year of diagnosis. in addition, 27 bcl-2 negative patients were identified, with a mean age at diagnosis of 66.8 years. 11 of these patients were female and 16 male. the mean ldh level was 747 some researchers included additional clinical features that were common when there was malignancy in the asymmetrical tonsils. in this study we aim to evaluate the cancer detection rate in this setting including those who possess high risk clinical features such as age (> 45), pain and history of smoking. methodology: in total, 119 consecutive tonsillectomy cases, clinically labelled as asymmetrical tonsils were analysed. out of these, 28 cases (group 1) were additionally labelled with investigation for unknown primary, obvious lesion or suspicious ulcer seen. the remaining 91 cases with a sole clinical indication of asymmetry were stratified as group 2. results: in the first group, 7 cases had a histological confirmation of tonsillar primary (25%). while, in the second group, the histological analysis showed 78 cases with benign reactive pathology (89%) while, one case was diagnosed as malignant lymphoma (1%), 6 cases with mild dysplasia (7%) conclusion: our results do correlate with the considerable agreement amongst otolaryngologists that the appearance of an asymmetrically enlarged tonsil in the presence of associated risk factors is considered an indication for tonsillectomy and histological examination given the significant rate of tonsillar malignancies in this group. anatomical difference in the depth of tonsillar fossa and asymmetry of the anterior tonsillar pillar may give a false impression of a clinically asymmetrical or unilateral enlarged tonsil metastatic adenoid cystic carcinoma to the lung and kidney: a single case report p rm doyle the tumour has three prognostically significant subtypes which form the basis for tumour grading; cribriform, most frequent, tubular and solid, associated with a more aggressive clinical course and metastasis. in the majority of cases (74%) the tumour has an insidious onset and patients have locally invasive disease at first presentation, which coupled with adjacent, important anatomical structures means a complete primary surgical resection is often not feasible. distant metastases are frequent and predominately involve the lung and bone with renal metastasis a rare occurrence. an optimal treatment regime for acc has yet to be established and while local management with combination surgery and adjuvant radiotherapy is currently favoured there is conflicting evidence regarding the use of radiotherapy and no formal surveillance guidance for local and regional recurrence or distant metastasis exists. we report a single case of a 52 year old male who presented in 2004 with an asymptomatic right neck mass and underwent right radical neck dissection and adjuvant radiotherapy for acc. the patient represented in 2014 with aspiration pneumonia on a seven month history of dysphagia and dyspnea. xray and subsequent computed tomography (ct) imaging identified large, multiple right sided lung lesions, maximum 3cm, and a 5.6cm left renal upper pole mass. ct guided biopsy of the right lung lesion and ultrasound guided biopsy of the renal mass were reported as metastatic adenoid cystic carcinoma regan 3 ; cm martin 2 ; cv timon 3 cytokeratin 7 (ck7) is a junctional biomarker with a seqika fragment which stabilises hpv-16 e7 transcripts. we assessed the expression pattern of ck7 protein in tumour specimens from patients diagnosed with oropharyngeal squamous cell carcinoma (scc) presenting at two major irish head and neck centres, within the last 10 years. methods: archived tumour specimens together with epidemiological data were collected from patients presenting with new primary oropharyngeal scc at two main head and neck centres in ireland, within the last 10 years. briefly, dna was extracted from tissue blocks and hpv testing carried out using spf10 hpv pcr immunohistochemical staining for ck7 [clone sp52, ventana] was performed on tissue blocks following optimisation on the ventana benchmark ultra immunostainer. slides were analysed by light microscopy and scored using the h scoring system with 95% of these identified as hpv-16 subtype. ck7 expression was observed in the tonsillar crypt epithelium of both normal tonsils and tumour specimens. 56% of cases were positive for ck7, with 38% of cases demonstrating h score of >60. ck7 expression in the tumour cells was significantly linked to hpv status and our results suggest that the expression of ck7 in normal tonsillar crypt epithelial cells provides a selective advantage to hpv-related carcinogenesis at this site, possible due to the unique propensity of ck7 to bind and stabilise hpv-16 e7 transcripts regan 3 ; cm martin 2 ; cv timon 3 methods: archived hpv-positive tumour specimens and epidemiological data were collected from patients presenting with new primary oropharyngeal scc at two head and neck centres in ireland over a one year period. briefly, dna was extracted from tissue blocks and hpv testing carried out using spf10 hpv pcr. the inno-lipa hpv genotyping extra test [fujirebio] was used to determine genotype. immunohistochemical staining for ck7, gda, mmp-7, agr-2, pd-1 and pd-l1 was performed following optimisation. slides were analysed by light microscopy and scored using the h scoring system (junctional biomarkers) frozen section reporting of necrotising granuloma of the liver following percutaneous instrumentation of the biliary tree: a case series eh hadjimichael; p df fielding; mi ilyas; az zaitoun; dl lobo; pk kaye nottingham university hospital, nottingham, uk percutaneous transhepatic cholangiography (ptc) is an interventional radiological technique for both diagnostic imaging and therapeutic decompression of the proximal biliary tract in malignant distal obstruction when retrograde techniques fail. recognised complications of ptc include sepsis, haemorrhage and pneumothorax. we describe four cases where necrotising granuloma, apparently secondary to previous ptc, has resulted in frozen section examination at the time of subsequent planned cancer resection, to exclude tumour metastasis. four cases of necrotising granuloma in the liver have been identified between january 2013 and february 2015. all cases were planned whipple's procedures for pancreatic cancer where initial intraoperative evaluation revealed solitary subcapsular liver lesions. biopsy and intraoperative frozen section examination were performed to exclude metastatic disease. all frozen sections except one were reported as showing benign necrotizing granuloma formation. the first case was initially reported as malignant and the operation was abandoned. a benign diagnosis was confirmed on paraffin sections in all four cases with the first patient undergoing successful surgical resection at a later date. to the best of our knowledge these are the first reported cases of necrotising granuloma in the liver secondary to prior instrumentation of the liver and leading to intraoperative histological assessment. we highlight this as a potential pitfall in frozen section interpretation undertaken ahead of planned potentially curative surgery which can lead to overstaging of otherwise resectable disease or to the interpretation of a potential diagnosis of tuberculosis. these risks can be reduced with greater surgical and pathological awareness of this entity. inclusion body fibromatosis, also known as infantile digital fibroma, is a benign, predominantly myofibroblastic tumour primarily found on the digits of infants. clinically, these lesions present as asymptomatic cutaneous nodules, rarely larger than 2cm in size, classically on the dorsal or dorsolateral aspect of the second, third and forth digits. they have a high recurrence rate, reported as between 61 and 75%, although this can be reduced by undertaking complete wide local excision. we report a case of inclusion body fibromatosis in an 11 month-old boy, presenting with an enlarging, firm lesion on his left second toe. following surgical excision, the lesion showed classical histological features of inclusion body fibromatosis -spindle cells arranged in interlacing fascicles in collagenous stroma and numerous pink intracytoplasmic inclusions. the lesion appeared incompletely excised and the patient will be kept under review on account of the high risk of recurrence.decreased expression of the mitochondrial bcat protein correlates with improved patient survival in idh wild-type gliomas me conway 1 ; j hull 1 ; m el hindy 1 ; sc taylor 1 ; f el amraoui 1 ; c paton-thomas 1 ; p white 1 ; m williams 2 ; p hr haynes 3 ; sm hutson 4 ; km kurian 3 key: cord-221669-uokr4mjq authors: barnes, t. title: the shir model: realistic fits to covid-19 case numbers date: 2020-07-28 journal: nan doi: nan sha: doc_id: 221669 cord_uid: uokr4mjq we consider a global (location independent) model of pandemic growth which generalizes the sir model to accommodate important features of the covid-19 pandemic, notably the implementation of pandemic reduction measures. this"shir"model is applied to covid-19 data, and shows promise as a simple, tractable formalism with few parameters that can be used to model pandemic case numbers. as an example we show that the average time dependence of new covid-19 cases per day from 15 central and western european countries is in good agreement with the analytic, parameter-free prediction of the model in the models typically employed in epidemiology to follow the evolution of a pandemic [1] , one begins by partitioning a total population of n individuals into a complete set of disjoint subsets, according to their medical histories. the simplest models of this type have only a few categories, such as 1) the susceptible but as yet uninfected population s, 2) the infected but not recovered population i, who can infect s, and 3) the recovered population r, who can no longer infect s, and can themselves no longer be infected. first-order ordinary differential equations (odes) in time are assumed to describe the coupling between these groups. once the appropriate initial conditions are specified, these odes can be integrated, which gives predictions for the subsequent evolution of the group populations. in this example, given constant rates {r ij } for the two assumed transitions, s → i and i → r, the resulting odes for this simple "sir" model are ds/dt = −r s→i s di/dt = +r s→i s − r i→r i dr/dt = +r i→r i we will refer to these two rate coefficients as r s→i = r i and r i→r = r r . on adding these equations we find that so the total number of individuals is a constant, n 0 . since these equations are linear and homogeneous, it is convenient to divide by n 0 and solve for the fraction of individuals in each category, such as f s = s/n 0 . r or * email: tbarnes@utk.edu f r can be inferred from the other two populations, using r = n 0 − s − i, or equivalently f r = 1 − f s − f i , so we need only solve the set (3), we will assume that the entire population is initially in category s (susceptible but not infected or recovered), so that f s (t = 0) = 1 and f i (t = 0) = 0. given these initial conditions we can solve the equations (3) , which also imply f r through f r = 1 − f s − f i , with the results the behavior of this system is just what one would expect from the dynamics described in the sir-type defining equations (1) : the initial population of susceptibles (s) decays exponentially at the specified rate r i as they transition into infected (i); the infected fraction increases to a maximum [2] , and then declines as it populates the final, recovered population (r). as t → ∞ one asymptotically approaches a state in which all of the population has transitioned to recovered, f r (t = +∞) = 1. as anticipated, at any intermediate time 0 < t < +∞, f s (t) + f i (t) + f i (t) = 1. as a specific example, the time dependence predicted by this sir model is shown in fig.1 , for r i = 0.5 [days −1 ] and r r = 0.1 [days −1 ] (an infection rate of five times the recovery rate). two important modifications to this simple pedagogical sir model appear appropriate for a more realistic description of the covid-19 pandemic. one is that the rate of transitions from susceptible (s) to infected (i) should be proportional to the product of these two populations, since their interaction determines this rate. this modifies the second evolution equation for the population fraction f i (t) to this "second-order reaction kinetics" in the infection process is a standard assumption in epidemic models [1, 3] . a second, more novel modification of the sir model is to incorporate the effects of a social response to a highprofile pandemic such as covid-19. in this case there has been strong encouragement from governments and medical authorities to reduce the growth of the pandemic by implementing social distancing and related measures. in this shir model these actions reduce the susceptible population by transferring them to a new category of "hidden" individuals (h) who are not susceptible to infection, which is populated from the original group of susceptibles (s) through another rate process, the individuals in h are not only the physically sequestered; simply changing behavior, such as avoiding public assemblies, crowded schools and public transportation, implementing rigorous hygienic practices, and many other measures contribute to the continued reduction of the effective susceptible population fraction f s [4] . we consider τ = 1/r h , the "social response time," to be a measure of how quickly a society responds to a developing pandemic threat. we shall subsequently find that there are interesting differences between countries in the fitted values of this parameter. the partner de for f s , similarly modified to include a second-order infection rate process as well as the new first-order social response transition term for s → h, is the final "recovery" de for dr/dt is unchanged from the sir model formula in (1) . however since the r cases in practice include mortality as well as recovery, we will instead refer to the r population as "resolved" [6] . collecting these results gives our complete set of shir model des, note that one recovers the three original pandemic des of kermack et al. [3] (their eqs.(29)) through the substitutions f (s,i,r) → (x, y, z)/n and (r i , r r ) → (κ, ℓ), and removing the "hidden population" of the shir model by setting r h = 0 and f h = 0. we note in passing that the infected (and still infective) individuals (all those in box i) are also referred to in the literature as the "active" cases, numbering a(t). here, i(t) and a(t) are identical; any individuals in box i who are no longer infective are immediately reclassified as "resolved," and moved to box r. since (8) is a nonlinear set of odes for the four population fractions f s,h,i,r , an exact analytical solution will not be possible, except in certain limits and approximations. however some important results can still be proven for this nonlinear system. one such result, obtained by adding the 4 equations in (8) , is that the sum of the four populations fractions f is a constant of motion. thus f h + f s + f i + f r = 1, or h + s + i + r remains a constant, n 0 . another useful result is that the population fractions of the two groups h "hidden" and r "resolved" can be expressed simply in terms of the susceptible (s) and infected (i) fractions f s and f i , and our principle task will therefore be to solve the two coupled equations for f s (t) and f i (t), the second and third equations in the set (8) . since the rate of increase of the infected fraction df i /dt is proportional to the product f i f s , a trivial solution of the model follows from an initial condition of no infections, f i (t = 0) = 0; this implies f i (t) = 0, i.e. that there are no infections at any later (or earlier) time. in this limit all that happens is that the initial fully susceptible population f s (t = 0) = 1; f h (t = 0) = 0 evolves into an increasingly hidden population, as a decaying exponential; in practice the fraction of infected individuals f i (t) will usually be much smaller than the fraction of susceptibles f s (t). this suggests that we use the no-infection limit (11) for f s (t) as a first approximation in determining f i (t) in the small-f i (t) limit. with this substitution, we find that the increase in df i /dt, the fractional rate of new cases per day, will be for completeness we note that the rate of decrease of the "active" (still infective) population fraction f i (t) per day due to transitions from "infected" to "resolved" status is given by so the full de for f i (t) in this small-f i (t) limit is the formula for the fractional "new cases per day" (cpd) growth rate in the small-f i (t) approximation, eq.(12), is attractive for several reasons. first, it is easily compared to data, since there is copious information available from many countries regarding the number of new cases per day. (this quantity is simply n 0 times the df (in) i /dt above.) second, since (14) is a linear, homogeneous, first-order differential equation involving only f i (t), and the associated integral over time is tractable, we may solve for f i (t) analytically in this small-f i (t) approximation. in the early and intermediate stages of a pandemic in which the infected fraction still dominates the resolved fraction, we can ignore the assumed slower transition of infected to resolved by setting r r = 0, which gives lim s>>i>>r df i /dt = +r i e −rht f i . this relation is especially amenable to comparison with data, as it relates two observed quantities, the number of new cases per day (n 0 df i (t)/dt) and the cumulative number of cases (n 0 f i (t)), assuming that n 0 f r (t) can be neglected. the shir model in this small-infectedfraction limit predicts that this ratio should be a decaying exponential in time, in the following section we will compare the expected time dependence of several pandemic models to covid-19 data, including the shir model prediction (16) . we will follow this with more detailed shir-model calculations of the cumulative case numbers n 0 f i (t) and especially the new cases per day (cpd), n 0 df i (t)/dt, and will describe detailed fits of these functions to covid-19 data from a range of countries. here we will first discuss the time dependence predicted by two familiar dynamic models of pandemics, and will show how these models can be compared to covid-19 data. we will find that there are serious difficulties in applying these simple models to covid-19. in this discussion we will consider only the cumulative number of cases as a function of time (here called p(t)), and the rate of appearance of new cases per day, dp/dt. these numbers are widely reported for covid-19, and require little interpretation. one important point is that the number of cumulative cases to time t, p(t), is the sum of the infected i(t) and the resolved r(t) populations distinguished in the previous section; as a first, simplest model of the growth of a pandemic, one can assume that the rate of change of the number of cases dp/dt is proportional to the number of cases p, the solution of this differential equation is an exponential times the number of cases at some arbitrary initial time, this simple model clearly makes unrealistic assumptions, notably that there are infinitely many potential cases. it also neglects spatial dependence or other "local" aspects of the problem, as well as the effect of transitions from infected to resolved individuals who are no longer infective. this exponential model predicts that the ratio of new cases per day to cumulative cases should be a constant, in fig.3 we compare this expectation to the ratio of new cases reported per day (∆p/∆t) to the cumulative number of cases (p) for the uk, using data from ref. [7] over the period 18 mar -04 may 2020. evidently this ratio is far from constant; it falls by about a factor of 10 over the 47-day period shown. the simple exponential model can be improved through the introduction of a limit in the number of the population that can be infected, p max . one can assume that the infection rate is initially equivalent to the exponential model, but later in the pandemic is suppressed in proportion to the number of uninfected individuals remaining, p max − p. a simple de with these properties is the "logistic model," defined by the solution of this model, which is symmetric about p = p max /2, is the inverse of an exponential plus a constant, translated by a t 0 that is determined by the initial conditions; this logistic model can also easily be tested by comparison with data on the ratio of new cases per day to cumulative cases. in this case, the model predicts that this ratio should be a simple, linear function of p, so, when data on this ratio (y-axis) is plotted against p (x-axis), this model anticipates linear p-dependence, with an x-axis intercept (where dp/dt = 0) at p = p max . in fig.4 we show an example of this plot for covid-19 data, using the usa cumulative cases and new cases per day data from ref. [7] through 15 july 2020. although this ratio initially has some qualitative resemblance to the logistic model form (23), (see fig.4 upper left), the more recent data (lower right) is clearly inconsistent with (23), and cannot be extrapolated linearly to zero dp/dt. a study of the pandemic time dependence predicted by the logistic model shows another specific aspect of the model that makes it unrealistic for covid-19; the rate of new cases dp/dt predicted by this model is symmetric about the maximum, at the p(t) inflection point t = t 0 . we shall see that in contrast the rate of new cases dp/dt observed in the covid-19 data is rather skewed, and has a faster rise to maximum than the subsequent decline. here we will test a third prediction for time dependence, which is the form predicted by the shir model of the previous section. there we noted that the infected fraction f i of the population is predicted to satisfy the differential equation in the limits of a small fraction of the population being infected (f i << 1) and the resolved fraction being small relative to the infected fraction (f r << f i ). the existing covid-19 data suggests that these are both reasonable approximations in the early and intermediate stages of the covid-19 pandemic. the expectation (24) can immediately be compared to data, since (df i /dt)/f i is just the ratio shown on the y-axis of fig.3 . the dotted line in the figure shows a decaying exponential fit of exactly the form (24), which evidently gives a very good description of the data. the parameter values found in this fit are r i = 0.233 [days −1 ] and τ ≡ 1/r h = 20.9 [days] . motivated by the good agreement between the shir model prediction of a decaying exponential in time for (df i /dt)/f i and the covid-19 data evident in fig.3 , we will subsequently discuss shir model predictions in more detail, and will show fits of this model to covid-19 data from many european countries. this will follow a short discussion of some general aspects of mathematically similar models, which will be used in the detailed discussion of shir results. the results discussed above motivate the consideration of generalizations of the simplest "pure exponential" pandemic model, in which the constant λ of that model (20) is replaced by an explicit function of time, for a given function λ(t), the defining differential equation (25) gives the predicted growth of case numbers as formally this may be regarded as exponential growth in a new (nonlinear) time variable u, defined by du = λ(t)dt. since this is a model of the growth of a pandemic, the cumulative number of cases (26) can only increase, or become flat when the pandemic has been completely halted, which occurs if and when we reach a time t f for which λ(t f ) = 0. accordingly we constrain λ(t) to be positive (pandemic ongoing, t < t f ) or zero (t = t f , pandemic halted). the inflection point of p(t) is of considerable importance, since this is the time of peak pandemic growth. the time t inf l of the inflection point is specified by the rate of change of case numbers dp/dt reaching a maximum; this occurs when the second derivative d 2 p/dt 2 is zero, on substituting the general solution p(t) from (26) into this constraint, we find an equivalent definition of the in-flection point in terms of the proportional growth function λ(t), a. cumulative cases p(t) and cases per day (cpd) dp/dt(t) in sec.i.b. we introduced the "shir" model as a generalization of the sir model, with the effects of pandemic reduction measures incorporated through a "hidden" population (h) that migrates from the initial susceptible population (s) with a rate constant r h . in the limit of a small infected population fraction (f i << 1) relative to the remaining fraction of susceptibles (f s ), and assuming that the resolved fraction f r can be neglected, we showed that the infected fraction satisfies a differential equation of the form where the shir growth rate function is a decaying exponential in time, the rate coefficients r h and r i can be interpreted respectively as a characteristic "social response time" τ = 1/r h for the implementation of pandemic reduction measures, and an initial condition (at some t = 0) on the growth rate function λ(t); if we multiply the infected fraction f i by the total population n 0 , and similarly scale the fractional new cases per day, we can compare the results for the cumulative cases p(t) and new cases per day dp/dt directly to the data as normally reported. given the form (31), on solving (29) we find that the predicted number of cases as a function of time in the shir model is a nested exponential, this may be simplified by replacing the t = 0 initial condition λ(0) by an equivalent time shift which gives a simple three-parameter form for the general solution of the cumulative number of cases in the shir model, this function increases monotonically from p = 0 at t = −∞ to p max at t = +∞, and has a single inflection point at t = t 0 . this is an example of a gompertz [8] curve, y = ab q x [9] ; these are often encountered in problems in which a proportional rate of growthḟ /f is driven by an exponential. the rate of appearance of new cases per day (cpd) in the model, dp/dt, can be evaluated directly from p(t) above, and is this rate of growth is zero at t = −∞, increases to a maximum value at the p(t) inflection point t 0 , and then decreases with time, approaching zero exponentially as t → +∞. dp/dt integrates to a finite number of cases p max as t → +∞. the maximum number of cases per day, at the time t 0 of the p(t) inflection point, is given by the three parameters that specify a solution of the shir model, p max , τ and t 0 , all have simple interpretations; they are respectively the height, width scale, and time shift of the pandemic profile p(t) (or of the rate of new cases, dp/dt). p max is by definition the maximum value of p(t), and gives the total number of cases, which is p(t) at t = +∞. the social response time τ sets the scale for all time-interval features of p(t) and dp/dt, since changing t 0 only translates these functions. the time shift t 0 is the time of the inflection point of p(t), this inflection point t 0 may be considered the peak of the local pandemic, since the number of new cases per day estimated by the fit is then a maximum. for discussion purposes a convenient choice for t 0 in (36) and (37) is t 0 = 0, which places the inflection point of p(t) at t = 0. plotting p(t) or dp/dt against t in units of τ , normalized to their maximum values, gives a parameter-independent "universal profile" for each of these functions. the universal form for p(t), scaled by its maximum value p max , is shown in fig.5 . the inflection point of p(t) at t = 0 is indicated, where both p(0)/p max and its slope (in the dimensionless time variable t/τ ) equal e −1 . this last property implies that the total number of cases p max and the cumulative number of cases at the time of the p(t) inflection point (pandemic maximum), p(t 0 ), are related by p max = e · p(t 0 ) . this provides a simple estimate of the total expected cases p max from the number observed up to the pandemic peak, p(t 0 ), which may already be known. some additional times of interest are those at which p(t) has reached 10%, 50%, 90% and 99% of the total cumulative cases; these are respectively (t − t 0 )/τ = −0.8340, +0.3665, +2.250, and +4.600. the universal curve for dp/dt, the new cases per day, is shown in fig.6 , scaled by its maximum value of p max /eτ . the mean and variance of t for this distribution are < t >= γτ (γ is euler's constant) and σ = (π/ √ 6)τ . the skewness [10] of dp/dt is 12 √ 6 ζ(3)/π 3 ≈ +1.140, a positive value indicating a more heavily weighted rhs. several additional interesting features of dp/dt are the fwhm, 2.446τ (indicated in fig.6 ), the rise time of dp/dt from half-max to peak, which is 0.985τ ; and the much slower decline of dp/dt from peak to half-max, which requires 1.461τ . the asymptotic approach of p(t) to p max cases at large times is exponential, as is the (decreasing) rate of growth of new cases at large times, we note in passing that these results suggest an improved estimate of the asymptotic total number of cases p max which has accelerated convergence, although this improved behavior would likely be masked in practice by the background of sporadic cases. in this section we will show results from fitting the predicted early pandemic time dependence from the shir model as derived in sec.iiia to data for the confirmed covid-19 case numbers from many different countries. we will primarily consider fits of the new case numbers per day (cpd) to the shir model prediction, eq.(37). as an initial example we will show results for austria, including fits to both the cpd and the cumulative case numbers. following this we will give the results of shir model fits to the cpds reported by a representative set of western countries, and will compare and contrast these results. we will also discuss procedures for fitting more complicated datasets that show evidence for multiple infection sites, and will show examples of such fits. finally we will show how datasets from multiple countries can be combined, and will use this procedure to test the predicted shir model profile for new cases per day, displayed as a universal (parameter-free) curve. as a first application we will fit the three-parameter shir model to the data on the number of new daily covid-19 cases per day in austria, as reported on the worldometer website, ref. [7] . austria was chosen because it represents one of the earliest covid-19 outbreaks in europe, so their data allows us to follow both the earlier and later stages of the pandemic. for this fit we will use the entire austrian cpd dataset from the initial website date of 15 feb 2020 through 06 may 2020, comprising a nominal 82 data points. (for most countries we consider, including austria, there are actually many "null" or zero entries in the early dates.) as a reminder, in the small fraction of infections limit assumed here one may solve the model for the cumulative case numbers of infected people p(t) and hence the rate of new cases per day dp/dt in closed form, given by eqs. (36,37) . the three free parameters in these functions are p max (total number of cases), τ (social response time), and t 0 (the p(t) inflection point; time of the maximum cases per day). our common t = 0 reference time in all these numerical fits and discussions is taken to be 18 mar 2020. the fits are all simple, unweighted leastsquares without data errors. the result of this fit for austria is shown in fig.7 . this is a surprisingly good fit to the full range of the cpd data, including the rapid initial rise after the early cases, the peak region, and the subsequent slower decline; all are reasonably well described by the model [11] . the shir model parameters resulting from this fit to the cpd data from austria are given below. these and other fitted parameters are typically quoted to three-place accuracy in this paper, to allow numerical tests such as debugging of numerical routines and checking analytic results. there is evidently reasonable agreement between the shir model and the data with these parameters for the number of new cases per day, although there is considerable scatter in the data. the numerical value of the peak predicted cpd, p max /eτ , and the time of the peak, t inf l = t 0 are indicated by a single large point in fig.7 . these values are respectively 728 [cases/day] and 7.32 [days] (measured from t = 0 on 18 mar 2020), giving 25 mar 2020 as the peak date for new cases per day in austria implied by this fit. the fitted value of the austrian "social response time," τ ≈ 7.5 [days], is of special note. this is the smallest value (hence the fastest social response) we have found in our fits to the covid-19 pandemic numbers from a large set of 15 representative central and western european countries (see table i ). this suggests that austria deployed an especially rapid and effective response to this pandemic, which presumably had a strongly limiting effect on the total number of cases. a minor discrepancy between the model and data is evident at the early times in fig.7 that are characterized by small case numbers. (see times before about t = −4 [days] in the figure.) the number of early cases is clearly underpredicted by the fit. this may represent their growth from a small number of initial cases, closely monitored and hence developing rather slowly, which were evident before the primary pandemic contribution became dominant. we will note a similar, more prominent effect in the early data from denmark and norway at about the same date. an alternative approach would be to fit the cumulative number of reported cases as a function of time, which is typically reported together with the new cases per day. since one is the derivative of the other, these should give similar parameter values. of course the results will not be identical, because the model is not exact, and the cumulative cases data will give higher fitting weight to the later stages of the pandemic. the choice of which dataset to fit, cumulative cases or new cases per day, may be suggested by the subject of greatest interest; the peak of the pandemic is best determined by fitting the new cases per day, but the final number of cases will be best determined if constrained by the large total case numbers reported in the later stages of the pandemic. here as an example of the second fit we give the shir model results that follow from fitting the cumulative case numbers from austria. we again use a set of 82 points of austrian data [7] from the initial reporting date of 15 feb 2020 through the fitting date, 06 may 2020. the shir model fit to the austrian cumulative cases data is shown in fig.8 evidently the shir model also provides a reasonably accurate description of the cumulative cases for austria. the parameter values resulting from the two fits, to cpd data (44) and cumulative cases data (45), are quite similar [12] . the total case numbers p max differ from their mean by ±2%, the fitted social response times τ next we will follow the same procedure described in the first austrian fit above, and will fit the shir model to the cpd data for each of a representative set of central and western european countries. for each fit, as discussed for austria, we have again used 82 pts of daily new case numbers per day [7] for the period 15 feb -06 may 2020 as the only input data. this data fitted to the threeparameter shir model form for dp(t)/dt, (37). this fixes the three model parameters p max (with units of [cases]), τ [days], and the inflection point t inf l = t 0 [days] (measured from t = 0 on 18 mar 2020) separately for each country. the fitted parameter values for the entire set of 15 central and western european countries considered here are given in table. i, together with the calendar date of the inflection point t 0 , and the fitted peak number of new cases per day, which occurs at t 0 . perhaps the most remarkable aspect of the shir model fits to 15 central and western european countries in table. i is the wide range of values found for the "social response time" τ , and how it appears to correlate with national coronavirus policy. as τ sets the width of the pandemic peak, it is a measure of how long a country will have to endure the pandemic. (recall for example that the fwhm of the new cases per day, dp/dt, is about 2.4·τ .) the extreme cases are austria (τ ≈ 7.5 days) and sweden (τ ≈ 26 days), over 3 times longer than austria. the swedish cpd data and fit are shown in fig.9 . the very slow swedish response time is apparently the result of a rather controversial covid-19 policy promoted by the swedish state epidemiologist, a. tegnell [13] , which did not impose the strict school closings, border controls, and restrictions on large gatherings that characterized the policies of other scandinavian countries. tegnell instead advocated a more open society during the pandemic, to promote herd immunity and a quick economic recovery. although the swedish policy was evidently successful in broadening the curve, their per capita number of cases is over twice that of denmark and nor-way, and the swedish fatalities per capita are larger by a similar amount. this argues that an aggressive national policy of minimizing the social response time τ , for example through contact tracing, extensive testing, rapid medical response and isolation of positive cases through quarantine, may be the most effective in reducing the total case and fatality numbers. it may also prove easier to sustain a strict policy over a short period of time than a more lax one over a longer period. the uk and dutch social response times τ are also quite large, and can also be correlated with their early national policies that promoted "flattening the curve" and developing herd immunity. to quote the dutch prime minister regarding covid-19 policy in a national address [14] , (in translation) "... we can slow down the spread of the virus while at the same time building group immunity in a controlled way." in a national address [15] the uk prime minister stated that "it is vital to slow the spread of the disease. that is the way we reduce the number of people needing hospital treatment at any one time." of course the argument that slowing the spread of the pandemic is advantageous because it "flattens the curve" is specious. this tacitly assumes that the total number of cases p max is constant, so a broader rate curve must have a lower mean. in practice there is instead evidence that a longer τ may result in more infections per capita, as seen in comparing sweden with denmark and norway. if so, flattening the curve by slowing the spread of the pandemic unfortunately increases the total number of cases and fatalities. this complicated and perhaps counterintuitive issue certainly merits careful future investigation. we have also carried out fits of the shir model to covid-19 data for three north american countries, canada, mexico and the usa. these three countries were treated as a separate exercise because of concerns that european and north american policies differed considerably, which might lead to very different fit parameters and pandemic curves. this was indeed found to be the case. most of the fits of the shir model to european countries described above were carried out in may 2020, using 82-point worldometer coronavirus datasets [7] covering the period 15 feb -06 may 2020. initially we used the same procedures and datasets for the three north american countries. this appeared satisfactory for canada and the usa, however the data for mexico showed that this country was still in the early stages of the pandemic, and had a very long social response time of about 63 days, with a predicted pandemic peak in late july. for this reason we decided to carry out a fit to mexico incorporating later data, specifically a 151-point worldometer cpd dataset for the period 15 feb -14 july 2020, and have fitted the corresponding data for canada as well. recent developments in the usa have shown a dramatic departure from a single peak model, which suggests that the usa should be treated as a superposition of several more localized outbreaks. accordingly, here we quote shir model fit results for new york state alone, as a representative component of the usa with an effective social response. the cpd data in this case is available from 12 mar 2020, so we fitted a dataset covering the period 12 mar -14 july 2020, comprising 125 data points. the results of these fits are given in table. of course each of the national pandemic datasets we are studying is in some sense the sum of multiple events, displaced in time and encountering special local conditions. in the usa for example the early data is dominated by events on the west coast, but these were superseded by the major outbreak involving new york city, and then by a smaller event in new jersey, and a larger number of local events in the midwest and south. fitting the data from many local outbreaks as a single event is clearly problematic. a series of local events separated in time but still overlapping significantly, if fitted as a single outbreak, could give a misleadingly large τ . separating the individual events will require accurate local data, and a more detailed investigation. despite this concern, the shir model does nonetheless allow a reasonably accurate fit to most of the national datasets we have considered. of the 15 european countries we have discussed here, just two showed clear evidence of a more complicated pandemic history that could not be fitted as a single-peak event. these were denmark and norway. in both countries there was evidence of an early, short-lived outbreak, which led to about 800 cases of covid-19 in each country, both during the period of 10-13 mar 2020. in both countries this early outbreak was rapidly suppressed, presumably through contact tracing and quarantine. we found that the cpd data from denmark and norway could be described by using eq.(37) to represent two independent outbreaks, a short, early one, with a very short τ ≈ 1.2 [days], and a dominant second outbreak, with the more typical covid-19 parameters listed in table.i. the resulting two-peak fit for norway is shown in fig.10 [11] . . the total cases predicted for denmark and for norway are simply the sum of the early and later peaks, thus 11.01k and 7.72k cases respectively. the idea of simultaneously fitting multiple countries to the shir model seems appealing, particularly as this model gives a good description of the case numbers from many different european countries. unfortunately there are complications with this procedure; individual countries vary considerably in the fitted values of the total cases p max , the characteristic social response time τ , and the local time of the pandemic peak, t 0 . these differences suggest a scaling approach, in which we determine these parameters separately for each country in a shir model fit, and then translate (by t 0 ) and scale (by p max and τ ) the datasets for each country separately. if the model and the data are both accurate, these translated and scaled datasets should provide points that fall on the universal curves shown in figs.5 and 6. of course there are indications that the covid-19 data itself is occasionally problematic, with considerable scatter, single-bin spikes, and in some countries a modulation with a 7-day period that may reflect a weekly reporting regimen. nonetheless it is of interest to generate this translated and scaled scatterplot, to see whether a common pandemic profile is indeed evident in the european data. this scatterplot is shown in fig.11 for the 82-point cpd datasets of all 15 european countries in table. i. each national dataset was translated and scaled separately by its particular values of t 0 , p max , and τ (as given in the table) before being added to this figure. the data from denmark and norway before 20 mar 2020 was excluded from the plot, to suppress contributions from their initial early outbreaks. table. i, translated and scaled to fit on the universal plot for dp/dt, fig.6 . the (parameterfree) shir model prediction for dp/dt, eq.(46), is shown as a solid line in this figure. the scaled, centered universal curve for dp/dt is shown as a solid line in fig.11 , and is given by dp dt we can use this scatterplot to combine the data from the 15 european countries we have considered, since they have now been superimposed through translating in time and scaling both axes. on binning in the scaled time variable (t − t 0 )/τ , we can calculate the average and variance of the scaled cpd datapoints in each bin, which provides an average and error for dp/dt at that value of (t − t 0 )/τ . as an example, we have used bins of width ∆(t − t 0 )/τ = 0.5, centered on t − t 0 = 0, which gives the result shown in fig.12 . this appears approximately consistent with the parameter-free shir model result for dp/dt (46), although there may be relatively minor discrepancies at onset ((t − t 0 )/τ ≈ −2) and well after peak the europe-averaged covid-19 data for the new cases per day shown as points with errors in fig.12 can be used to calculate expectation values under this common covid-19 dp/dt distribution. some of these results are given in table. iii, where they are compared to theoretical values predicted by the shir model dp/dt, eq.(46). evidently the simpler expected values are in reasonably good agreement, notably the displaced value of the mean from the maximum. there is however a discrepancy in the cubic expected values, which are sensitive to the relatively less well determined wings of the distribution at larger |t − t 0 |/τ . fig.12 . here for simplicity we abbreviate (t − t0)/τ = x. we have shown that a shir (generalized sir) model and its associated differential equations give rather good closed-form results for the covid-19 data on new cases per day and cumulative cases, when solved under certain assumptions. these assumptions can be relaxed in future studies. one important assumption was to ignore the effects of the "resolved" population fraction f r . this quantity, which is certainly of interest in planning economic recovery, is simple to evaluate from the "infected" fraction we have discussed here; it is simply the integral in eq.10. this can be evaluated analytically, together with the full de for f i , including the r r term, to accommodate transitions out of "infected" and into "resolved." the cumulative case numbers p(t) discussed in relation to covid-19 data should then be defined by p(t) = n 0 (f i + f r ), i.e. "infected" plus "resolved," since f r is no longer assumed to be negligible. calculations of mortality rates are certainly of interest, and are implicitly included in the transition from the infected population i (or i s ) to the resolved population r. mortality can easily be treated separately, for example by specifying separate i → r rate parameters for mortality and for recovery. in the extended shir model of fig.13 , mortality would presumably only contribute to the combined rate for i s → r. the mortality rate coefficients may of course depend strongly on the procedures followed at each country's medical facilities. numerical methods can be used to extract shir model results for the full model, including the s − i quadratic infection term, and can test the importance of the "small fraction of infections" approximation used here. a straightforward generalization of the model could accommodate the effect of "asymptomatic" infected individuals on the dynamics of the covid-19 pandemic. recent antibody results from italy [16] suggest that the asymptomatic cases are a large but not dominant fraction of all covid-19 cases. once better data on the number of asymptomatic cases becomes available, it may prove useful to extend the model to include this population. this group follows a progression s → i a → r, and while in the i a population presumably contributes to the infection of other individuals still in s. although adding this branch to the model may indicate that the fraction of resolved individuals f r is much larger than the shir model alone suggests, the pathway through symptomatic cases will remain as before. the most visible predictions of the model, for the number of symptomatic cases (now i s ), may not change significantly. a block diagram of this extended shir model is shown in fig.13 . finally, although we have primarily considered covid-19 in central and western european countries, largely because of an assumed similarity in standards for reporting data, there is extensive data available from many other countries which could be addressed using this type of model, undoubtedly with interesting results. in this paper we have introduced a generalization of the sir model in order to incorporate the effects of pandemic reduction measures. this "shir" model adds a rate process that allows transitions from the susceptible population (s) to a new "hidden" population (h), which we assume is not susceptible to infection. we have solved this model analytically in the limit of a small infected population fraction, and showed that the predicted time dependence of cumulative cases and new cases per day is in surprisingly good agreement with covid-19 data. (we primarily considered central and western european countries in this study.) the solution of the model with our approximations involves three free parameters; one is the "social response time" τ , which varies widely between countries, in a manner that correlates with their coronavirus policies. we are also grateful to e.s.swanson for assistance with calculations of expected values and errors in the shir model, and with the model block diagrams. dynamics of infectious diseases in this simple sir model the time at which fi is a maximum and that maximum value can be solved for analytically, and are respectively t = ln(ri/rr)/(ri − rr) and fi = (ρ/(ρ − 1))(ρ −1/(ρ−1) − ρ −ρ a contribution to the mathematical theory of epidemics protected groups similar to the "h" box of fig.2 have previously been considered in generalizations of the sir model. for example, the effects of vaccination are discussed using an sirv model [1], and the 2003 sars model of lipsitch transmission dynamics and control of severe acute respiratory syndrome the use of "resolved" for the post-infected population r, the sum of deceased and recovered, was suggested by p.ardaury. this population is also referred to as "closed cases worldometer coronavirus updates on the nature of the function expressive of the law of human mortality, and on a new mode of determining the value of life contingencies from mathworld -a wolfram web resource skewness is a dimensionless property of a distribution we note in passing that a single "outlier" point in the austrian cpd data, 1321 new cases on 26 mar 2020, lies above the upper cutoff of 1000 cases per day in fig.7. it was of course included in the fit. similarly, the norwegian cpd data includes an outlier of 399 new cases on 27 mar 2020 and a negative entry of -44 new cases on 08 the cumulative "cas" data fit are much smaller than in the preceding new cases per day "cpd" fit. the cas parameter errors are likely underestimated, since the cas data points represent a running sum, and are therefore highly correlated. the cpd parameter errors are presumably more realistic national address british prime minister boris johnson coronavirus address suppression of a sars-cov-2 outbreak in the italian municipality of vo' key: cord-015372-76xvzvdg authors: nan title: national scientific medical meeting 1996 abstracts date: 1996 journal: ir j med sci doi: 10.1007/bf02945204 sha: doc_id: 15372 cord_uid: 76xvzvdg nan zero months months months months group a 5.53 5.09* 5.66** 6.11"* 6.26** 4 zero months group b 6.06 6.4** values are means; *p<0.05 and **p<0.0l~ most other studies show neutral effects on insulin sensitivity, with minimal incidence of glucose intolerance. this may be partly explained by the diversity of age, diagnosis (whether insufficiency or deficiency state), other pituitary deficiencies and replacement therapy, and possibly by dosage ofgh utilised in these studies. clostridium difficile-associated disease (cdad) is primarily a nosocomial condition. community-acquired disease has been described but the incidence is low "). during a recent outbreak, we prospectively reviewed all new cases of cdad to determine what proportion of cytotoxin positive cases were community or hospital acquired. during a 4 month study period, 73 cases were identified. history of diarrhoeal episodes were recorded for each case. selected isolates were typed using pyrolysis mass spectrometry (pms). community-acquired cdad was defined as diarrhoea, on or within 72 hours of admission, in association with a positive stool cytotoxin test for c.difficile and in the absence of hospitalisation within 60 days. sixty-five cases (89%) were hospital acquired. fifteen patients (20.6%) had cdad on admission; 8 (11%) were community acquired, 7 (9.6%) had been recently hospitalised (4 at st. james's hospital, 3 at other hospitals). pms typing of faecal isolates revealed that 2 predominant strains were responsible for the hospital outbreak; one of these strains was also isolated in community-acquired cases. this study suggests that the incidence of communityacquired cdad may be higher than previously reported. we suggest that all newly admitted or transferred patients with diarrhoea should be screened for this organism. several studies have confirmed that activated protein c resistance (apc-r) occurs in 20-60% of thrombosis patients and is therefore more common than congenital deficiencies in the inhibitors of coagulation such as atii1, proteins c and s. homozygosity for the factor v (fv) gene mutation is associated with a 50-100 fold increased risk of venous thrombosis while heterozygosity is associated with a 5-10 fold increased risk. the mutation, however, is highly prevalent in the general population, a prevalence of 5% has been reported in several european countries. its frequency in the population of northern ireland (ni) has not yet been reported. we screened a group of 72 generally healthy elderly individuals (av. age 81.3; range 76-97 yr on several occasions for apc-r using an assay based on the prolongation of activated partial thromboplastin time by the addition of apc. a mean ratio of 2.64 (range 1.72-3.46) was measured. seven individuals (9.8%) had ratios <2.3 (av. 2.11; range 1.72-2.28). these subjects were then analysed for the fv mutation by pcr amplification and restriction analysis. the 4 individuals with the lowest ratios (av. 2.0; range 1.72-2.15) were found to be heterozygous for the mutation. none of these 4 individuals were deficient for protein c, protein s or atiii. the frequency of apc-r (5.8%) within this ni elderly group is similar to that reported by others in the uk whose studies would have included a generally younger population. the successful ageing of these individuals perhaps underlines the low risk associated with heterozygosity. alternatively a higher prevalence of the mutation may exist in the general population of ni, where the incidence of heart disease is one of the highest worldwide. the insulin-like growth factor ii gene (igf2) is imprinted. thus, in contrast to most genes where both maternal and paternal copies (alleles) are transcribed into rna and expressed, 1gf2 is normally only expressed from the paternal copy (monoallelic expression). alterations causing biallelic expression of igf2 may lead to excess growth factor production, and thus, to tumourigenesis. this study evaluated igf2 expression in a series of childhood cancers. to date 41 tumours have been evaluated using pcr based methodology (26 wilm's tumours, 12 rhabdomyosarcomas, 3 miscellaneous). dna was extracted and a polymorphic site apal within the igf2 gene was amplified and digested. this identified 10 samples as heterozygous for igf2, meaning that separate maternal and paternal alleles were distinguishable. rna from these informative samples was extracted, pcr amplified and restriction digested, to identify monoallelic versus biallelic profiles at the expression level. samples with normal imprinting (monoallelic) displayed allele a (236bp) or allele bl/b2 (176/63 doublet). biallelic samples displayed both alleles. using this approach 3/5 informative wilm's tumours and 2/ 4 rhabdomyosarcomas demonstrated biallelic expression. in conclusion, biallelic expression of igf2 was detected in a significant number of wilm's tumours and rhabdomyosarcomas, and should be considered; with other genetic alterations, as a candidate mechanism in tumourigenesis. the proinflammatory cytokines, tnf~, il8 and il-6, may mediate host metabolic and immune responses to cancer possibly leading to paraneoplastic phenomena such as cachexia. the cellular origin of these cytokines in the cancer patient, in many cases, remains unknown. we examined proinflammatory cytokine levels intracellularly, using flow cytometry, in pbmcs from oesophageal cancer patients (n=14) and age and sex matched controls (n=10). tnf~ and il-6 levels were significantly increased (p<0.05) in pma stimulated t cells and monocytes from the cancer patients when compared to the healthy controls. these results were confirmed using standard elisa assays. following cotlagenase digestion, increased levels of tnfa and il-6,were detected in oesophageal tumour infiltrating t cells when compared to cells from normal mucosa. there was also increased production of tnf~ and il-6, but not il-ib, in malignant epithelial cells when compared to normal and halothane and maintained by 50% nitrous oxide-oxygen, 0.75-1.5% halothane, with spontaneous ventilation. tc rose marginally in group 1 and fell in group 2 (not significant, ns). tp in groups 1 and 2 at induction and 20 and 30 minutes were, respectively, (mean + sem, celsius) 31.4+0.33 vs 31.6+0.3, ns; 33.9+0.3 vs 33.4+0.3, ns and 34.5+0.4 vs 33.2+0.3, (p<0.05). overall incidence of shivering-was 14.%, but not significantly different between the groups. the data suggests that preemptive application of the space blanket increases tp in paediatric patients during general anaesthesia and tends to conserve tc. chronic actinic dermatitis (cad) an uncommon, eczematous, photosensitive eruption is diagnosed on history, clinical examination, skin biopsy and abnormal light tests. drug induced photosensitivity may look identical clinically, have a similar history and patients with cad may be treated with potentially photosensitising drugs. we therefore reviewed all patients with cad and compared their monochrumator light tests with patients who had drug induced photosensitivity. phototesting was performed on unaffected skin of the back with an irradiation monochromator; the minimal erythema dose (med) determined for a series of wavelengths between 300 and 400 nm, in 14 patients with drug induced photosensitivity and 7 patients with cad. of ten females, four males with drug induced photosensitivity, age range 40-77 (mean 61 yrs), ten (71%), were photosensitive in the uva range (320-370nm), the implicated drugs including, quinine, sparfloxacin, amiodarone, doxycycline, mefenamic acid, nalidixic acid, fenbrufen, diclofenac, enalpril and prochlorperazine maleate. three patients with rosacea were photosensitive to doxycycline. the re/nainder (29%), were tested after discontinuation of the drug and their light tests were normal. in the cad group, (four males and three females), age range 38-86 (mean 71.5 yrs), three patients (43%), were sensitive to uva, uvb and visible light, four (57%) to uva and uvb. in conclusion, uva dissociated from uvb photosensitivity seems a relative but not absolute sign of drug induced photosensitivity. this pattern of light tests should prompt a detailed drug history to elucidate the causative agent. phototesting should be performed while on the offending drug as testing days or weeks after discontinuation will give normal results. patients at risk of bone fractures by measuring bone mineral density (bmd) and markers of bone turnover and to assess the correlation of these with the severity of cld. twenty three patients with cld had bmd measured by dual-energy x-ray absorptiometry scanning of hip and lumbar spine. bone formation was assessed using serum levels of procollagen type 1 peptide, osteocalcin and bone alkaline phosphatase, and bone resorption was assessed using 2 hour urinary excretion of hydroxyproline, pyridinoline and deoxypyridinoline. 48% and 39% of patients had evidence of osteoporosis at the lumbar spine and femoral neck respectively. biochemical results showed that 74% of patients had an increase in all 3 bone resorption markers and 42% had an increase in markers for bone formation. bmd at the lumbar spine was lower in patients with cholestatic liver disease compared to patients with other types of'liver disease (p=0.004). no correlation was found between bmd and patient age, bilirubin, albumin, inr or duration of liver disease. conclusions: osteopenia occurs in up to 62% of patients with cld due to a high bone turnover state where bone resorption exceeds formation. osteoporosis is most severe in those patients with cholestasis. a detailed profile is presented of all 632 leukaemia and multiple myeloma (icd-o code 169) patients registered by the southern tumour registry during the 8-year period 1983/1990 "). annual age-adjusted rates of 13.9 and 8.7 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk of 1 in 68 for males and 1 in 116 for females. the main morphological sub-types registered were multiple myeloma (31%), cll (25%), aml (18%) cml (9%) and all (8%). one, two and five-year survival rates were examined; age at diagnosis and lesion type were extremely significant factors in relation to patient outcome. the overall incidence levels indicate that irish rates were relatively high by internatiomil standards r we assessed effects of reducing the volume of hyperbaric bupivacaine by giving half the volume as isobaric bupivacaine. when using 0.5% hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected. 40 patients undergoing elective surgery were studied in a prospective, randomised, double-blind trial: group 1 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine and 0.5% isobaric bupivacaine respectively; group 2 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine. there was no significant difference found between the two groups with regard to maximal height of block (group 1, mean (range), t7 (t4-ti 1); group 2, t8 (ts-t11)), rate of onset of blockade, or time to maximal blockade (group 1, mean (sem), 23.55 (2.41) rain; group 2:20.89 (2.95) min). there was no difference found between each group in either cardiovascular stability or vasopregsor usage. the administration of a mixture of 0.5% isobaric bupivacaine and 0.5% hyperbaric bupiv/~caine confers no advantages over administration of the same volume of 0.5% hyperbaric bupivacaine alone. propofolis used as a sedative during regional anaesthesia. providing titrateable sedation, it can compromise haemodynamic stability. a propofol ketamine combination provides stable haemodynamics during total intravenous anaesthesia, avoiding emergence phenomena m. we compared two sedative regimes in patients having spinal anaesthesia. following informed consent, 40 patients, asa i-ii, undergoing spinal anaesthesia were randomized to one of two groups (n=20). group i (propofol-ketamine) received loading doses of 0.4mg/kg propofol, 0.1mg/kg ketamine followed by an infusion of 1.2mg/kg/h and 0.3mg/kg/h respectively. group ii (propofol) received bolus 0.5mg/kg and infusion 1.5mg/kg/h. subsequent infusion rates were titrated to effect using a sedation score. heart rate, blood pressure, oxygen saturation, end tidal c02 and oxygen requirements were recorded. observation continued for the recovery period and patients visited the following day. data were analysed using t-test, chi 2 test and anova. groups were demographically comparable. sedative and respiratory indices were similar for both groups. there was no difference in total propofol requirements between the groups; group i -146_+194mg, group ii -137_+1:52mg (mean _+ sd). there was a large difference in mean arterial pressure, being much lower in the propofol only group. both groups had an uneventful recovery without emergence phenomena. our results do not confirm the described additive effect of andketammc . ketamine.with propofol for sedation propofol ' =~2) confers haemodynamic stability during spinal anaesthesia. we designed a controlled study to investigate whether there is a direct relationship between the degree of postoperative pain and the development of negative middle ear pressure in adults following tonsillectomy. middle ear pressure was measured by tympanometry. pressures were classified as type a (o to -99 mmh20), type b (flat) or type c (-100 to -350 mmh20) tympanograms. 30 patients with type a tympanograms, undergoing tonsillectomy were enrolled in the study. patients had daily tympanometry whilst in hospital and then weekly until amrmalisatign. a questionnaire incorporating visual analogue pain scores was filled in at the same time. a control group of 26 patients with type a tympanograms, undergoing appendicectomy and endotracheal intubation was used. follo~v up was available on 26 patients. 13 patients (50%) developed type c tympanograms, 5 patients 09%) type b and 8 (31%) patients remained unchanged. no member of the control group developed any change in middle ear pressure (chi squared = 27.5, p < vol. 165, 1996 irish journal of supplement no. 2 medical science 0.001). there was no relationship between pain scores for throat pain or otalgia and the development of negative middle ear pressure. 8 patients recorded higher pain scores for throat pain at day 7 then day 1, only 3 of this group had negative middle ear pressure. middle ear pressure reverted to normal at day 7 in 15/18 patients and in the remaining 3/18 it was normal at day 14. this study demonstrates the development of transient negative middle ear pressure following tonsillectomy in 69% of patients. this change is unrelated to the degree of postoperative pain nor is it associated with otalgia. postoperative ward analgesia remains suboptimal. this may be partially related to inadequate early use of opioids to attain minimum effective analgesic concentration (meac). we examined the incidence and predictors of severe postoperative pain on admission and discharge from our postoperative recovery room (rr). verbal pain scores were obtained in a pilot study of 202 patients on rr admission and discharge. procedures were classed as open cavitary, laparoscopic, orthopaedic, ent or body surface surgery. intraoperative use and dosage of narcotics and nonsteroidal (nsaid) analgesics, anaesthetists' experience (prefellowship or post-fellowship nchd, consultant) were noted, and rr opioid usage recorded. pain scores and analgesic use were examined using mann-whitney, ~2 analysis and logistic regression. moderate or severe pain was experienced by 25% of patients on either arrival or discharge. median intraoperative morphine dosage was 5 mg. opioid use was slightly (median morphine dosage 8 mg, p < 0.05) higher in patients undergoing cavitary surgery; these patients had the highest pain scores on rr arrival and departure. 21 patients (10%) received > 10 mg morphine intraoperatively. discharge scores of 6/10 or higher occurred in 24 patients (12%). opioid usage and pain scores were unrelated to level of training. nsaid use/nonuse was unassociated with differences in opioid use or rr pain scores. no morbidity attributable to analgesic use (desaturation, slow respiratory rate) occurred. nonattainment of meac is frequent after open cavitary surgery. conservative opioid dosages continue to be employed despite inadequate early postoperative pain relief; this does not change with increasing experience. reporting such findings in departmental audit may help to alter perioperative management; such data may serve as a baseline for future interventional studies. diclofenac is frequently used for analgesia after tonsillectomy. recently concern has been expressed about the effect of diclofenac on prolonging bleeding time. one recent retrospective study found its use in tonsillectomy was associated with an increase in reactionary haemorrhage. we designed a randomised controlled study to compare the effects of rectal diclofenac and im pethidine given at induction with pethidine alone, in children undergoing tonsillectomy. fifty nine patients were entered into the study. there were 26 males and 33 females, mean age 6 years, range (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . patients were randomised according to chart number. thirty five patients received rectal diclofenac after induction. twenty four patients acted as controls. there were no significant differences in operating time or operative blood loss between the two groups. in the recovery room the diclofenac group was significantly less restless than tl~e control group (p < 0.05, chi squared test), with less crying, movement and agitation. there was no difference in postoperative recovery and no primary or reactionary haemorrhage. one patient in the diclofenac group developed a secondary haemorrhage. this study demonstrates a significant reduction in restlessness in the recovery room in children receiving rectal diclofenac. no increase in reactionary haemorrhage was demonstrated. diclofenac remains a safe and effective analgesic agent in children undergoing tonsillectomy. elderly patients have decreased dose requirements for many drugs compared to the young. few studies have examined dose requirements of opioids in the elderly when administered via patient controlled analgesia (pca) 1. we compared the pca morphine requirements between young and elderly patients. records were retrospectively analysed from 1,000 consecutive patients receiving pca for post-operative pain. inclusion criteria (i) age less than 10 years or greater than 60 years, (ii) upper abdominal surgery, and (iii) morphine pca usage. 238 patients fulfilled the inclusion criteria. 89 patients were young and 149 were elderly. the mean age in the young was 29 years and in the elderly was 69 years. 51% were female in the younger group, 40% were female in the older group. pain scores at rest and on movement were similar in both groups 3.8 and 6.7 respectively in the young, 3.2 and 6.2 in the elderly. (p > 0.05, students t-test). morphine usage over 48 hours was 58 + 32.1mg in the young, and 37 + 20.3 in the elderly. (mean + s.d.) (p.< 0.05, students t-test). elderly patients required significantly less morphine via pca to achieve the same pain scores as the young. these findings are consistent with studies showing decreased requirements of other drugs in elderly. the erythrocyte sodium-lithium countertransport (slc) is abnormal in essential hypertension and some other forms of cardiovascular disease (cvd) but the considerable overlap in its activity in patients with these conditions and in normotensive healthy subjects remains a strong point against its possible utility as a marker for cvd. we sought to address this issue in greater detail. twenty-nine hypertensive patients (19 with family history of cvd) aged 56.4+1.9 years (mean + se) and 32 normotensive subjects (12 with family history of cvd) aged 45.9 + 1.9 years, participated after informed consent. slc were determined in 35, 50, 70, 100 and 140mm sodium chloride; and the vmax and km of the transporter determined. hypertensive and normotensive individuals with family history of cvd (n = 31) had higher slc activity (0.331 + 0.011 vs 0.238 + 0.011, p < 0.0005), greater vmax (0.473 + 0.019 vs 0.397 + 0.018 mmol/lcell.h, p < 0.006) and lower km 56.0mm (median) vs 95.5 (p < 0.001) than hypertensive and normotensive subjects without such a history (n = 30). however, none of these parameters was sufficiently discriminatory as evidenced by the considerable overlap in the scattergrams for the two groups. on the other hand not only was the median quotient vmax/km significantly different 7.89 vs 4.20 (p < 0.001), but also the scattergram separated the two groups. this may reflect an effect of hereditary factors on the identified rate-limiting step in the transport system. capsaicinadministration results in depletion of substance-p sensitive nerves. this study was carried out to observe the impact on the morphology of mitral valve endothelium. the experimental group received capsaicin i.p. on day four of life; control animals received drug vehicle only. animals were anaesthetised by chloral hydrate and hearts were removed following perfusion of 4% glutaraldehyde, and were routinely processed for scanning electron microscopy. normal endothelial morphology showed an ordered and structured pattern, with large raised nuclei covered in discrete microappendages: no zoning was observed over the valve surface. following capsaicin administration, valves were seen to be torn and possessed a denuded endothelium. nuclear bulges changed in both apparent height and area, with the surface partially denuded of microappendages. one month following systemic administration of capsaicin to neonatal rats, a serious alteration of mitral valve endothelium morphology and integrity had occurred. depletion of substance-p may have resulted in mechanical insufficiency of the mitral valve. this study was funded by the health research board. with expanding applications and increasingly aggressive stress protocols, concerns about the safety of dobutamine stress echocardiography (dse) have arisen. the purpose of this study was to analyse prospectively the safety, adverse event profile, and complication rate of dse. prospective data was recorded in a consecutive series of 309 patients undergoing dse for diagnostic evaluation of chest pain, for risk assessment following myocardial infarction or for detection of hibernating myocardium. the maximum dose of dobutamine used was 50 mcg/kg/min in 24.6% of patients and 40 mcg/kg/min in 73.1%. atropine was used in 6. 8% long-term outcome following coronary artery bypass grafting may be related to the prevalence of major risk factors and their treatment following surgery. we aimed to establish the prevalence and current management of coronary risk factors in a group of consecutive patients attending our hospital. this is a report of the first 100 patients. data was collected by a structured patient interview, chart review, physical measurements and blood sampling. there were 74 male and 26 female patients, average age 61.5 years. the mean length of time since surgery was 2.2 years. thirty-nine patients had a recurrence of angina and this occurred on average ll months after surgery. as regards risk factors, 22 were active smokers, 56 were ex-smokers and only 22 had never smoked. two thirds of the patients were taking regular exercise; only 6 took no exercise at all. seventy-two percent of patients had a cholesterol greater than 5.5mmol/l, yet only 10 of the patients were on lipid lowering drug therapy and a further 16 were on a lipid towering diet. twenty-nine patients had a systolic bp > 160 or a diastolic bp > 90 and 15 of these were on antihypertensive therapy. seventy-seven patients were overweight but most of these had received specific advice regarding weight reduction in the preceding year. our results show a high prevalence of treatable risk factors in this high-risk group with inadequate treatment in many cases. new combined primary and hospital care strategies for cardiac rehabilitation and long-term secondary prevention of coronary heart disease are required. if the goal of modern therapy of acute myocardial infarction (ami) is preservation of myocardium, the occurrence of cardiac failure could be regarded as a treatment failure. in recent studies of iv thrombolysis and primary ptca the frequency of left ventricular failure (lvf) following ami has been as low as 3.5%. this very low rate might be explained by selection bias in patients recruited to randomized trials. the purpose of this study was to examine the frequency of lvf in a consecutive alterations in nitric oxide (no) synthesis have been implicated in cardiomyopathy, ischaemic heart disease and septic shock. recent work has suggested a possible role for nitric oxide in cardiac arrhythmogenesis. the effects of inhibiting no synthesis with l-name (n c -nitro -l -arginine methyl ester) on cardiac electrophysiology have not been fully determined. the dominant frequency of electrically induced ventricular fibrillation was determined in l0 anaesthetised pigs (30-42 kg) using a fourier transform. the dominant frequency (7.8 _+ 0.5 hz in lead ii) was not altered by treatment (8.5 _+ 0.5 hz) with l-name in a group of 16 pigs (45-58 kg) the effect of l-name (10 mg/kg) was assessed in relation to energy required to defibrillate. there was no significant difference in the energies required to achieve successful defibrillation on 80% of attempts between the l-name group (104.9 _+ 8.0 j) and the control group (111.1 _ 11.9 j), and on 100% of occasions, l-name (122.5 + 11.5 j) and control (137.5 _+ 13.1j). the results show that inhibition of no synthesis has no significant effect on the dominant frequency of ventricular fibrillation or on the efficacy of defibrillation in the pig heart. received either 100mg (7pts) or 200mg (19 pts) oral flecainide followed by a maintenance dose of 100mg bd. all pts were euthyroid. none had significant hypertensive, valvular or ischaemic heart disease. 23 pts had normal echocardiograms, 3 had mildly dilated atria. of the 26 pts 17 (65%) converted to sinus rhythm, 15 within 72 hrs and 2 at 12 and 21 days respectively. 2 subsequently, he was found to have paralysis of all the muscle groups of his right upper limb apart from some flexor movements in his fingers with areflexia and sensory loss over the c5/c6 dermatomes. the findings were thought to be in keeping with a brachial plexus.lesion. an mri scan showed a "haematoma" or "fibrosis" around the brachial plexus~ emg studies revealed a complete lesion from c5/c7 with evidence of partial function at c8 and a good function at c4. despite physiotherapy, at follow up 2 months later there was no improvement in the emg findings. though brachia~ plexus injury has always been considered a complication of central venous line insertion <l), there have been no cases described, in the literature in the last 10 years. central lines are mandatory irt major surgical cases but one must always be aware of the possible complications caused by them. body weight (bw) in icu patients fluctuates due to alterations in body mass (1) and water (2) and is usually estimated rather than measured. we hypothesised that (i) estimated weights are inaccurate and (ii) changes in true body mass (tbm) are masked by changes in water content. on admission, icu patients whose expected length of stay (los) was > 5 days were placed on a bed incorporating 4 load cells (accuracy of _ 0.1 kg). bw was estimated by icu staff. mbw was then recorded 12 hourly under standard conditions. changes in mbw due to pack insertion/dressing changes were excluded. we calculated tbm as mbw minus cumulative fluid excess, corrected for insensible losses c2~. patients received standardized nutritional support from day 3 and urinary nitrogen on day 6 was calculated we studied 20 patients whose mean (sd) age, mbw, los and apache ii score were 44.3 yr (18.54), 70.5kg (14.39), 10.0 days"(6.78) and 18 (3.8) respectively. mean error in estimated weight on admission was 10.3% (nurses) and 12.2% (doctors). mean protein and calorie intake was 64g and 1670 kilocals/day. mean decrease in mbw during icu stay 0.22 kg/day (range 0.21 kg (gain) to 0.49 kg). mean reduction in tbm was 0.56 kg/day (range 0.28-0.86 kg/ day) and this correlated with urinary nitrogen loss (r=0.7). in icu, (i) estimated weight is significantly inaccurate and should not be used in physiological calculations and (ii) rapid and significant decreases in body mass occur which may be underestimated due to fluid accumulation. dopamine appears to influence pituitary function and is associated with decreased circulating human growth hormone and insulin-like growth factor i m which could exacerbate catabolism, and therefore wasting, during critical illness. at 12 hrly intervals from admission, patients whose expected length of stay exceeded 5 days, were weighed (measured body weight, mbw) under standardized conditions,-using a bed incorporating 4 electronic load cells (accuracy + o.1 kg). standard demographics, apache ii score and use of dot~amine by infusion was recorded. changes in weight due to removal./ insertion of prostheses, packs/dressings were excluded mbw was converted to true body mass (tbm) using measurements of cumulative fluid balance (1000 ml = 1 kg) and insensible lossr patients received nutritional support, starting on day 3, based on their admission mbw. there were no significant differences in mean age, weight, length of icu stay, admission apache ii scores and mean daily protein/calorie intake between 14 patients who had received dopamine by infusion (group d) and 18 who had not (group nd). mean (sd) decreases in mbw during icu stay were 0.42 (0.08) kg/day (group d) and 0.20 (0.05) kg/day (group nd) (p<0.0i). mean decrease in tbm was 0.55 (0.10) kg/day and 0.35 (0.07) kg/day respectively (p<0.05). thus group d were losing an additional 1.4 kg body mass per week relative to group nd. the use of dopamine by infusion is associated with an accelerated loss of lean body mass during critical illness. adhesion of polymorphonuclear leucocytes (pmns) to pulmonary endothelial cells is an initial step in the inflammatory process characterising the adult respiratory distress syndrome. previous studies using human umbilical vein endothelial cells (huvecs) have suggested that lipopolysaccharide (lps) is a potent stimulus for pmn adhesion to endothelial cells. the aim of this study was to investigate the effect of lps on pmn adhesion to human pulmonary artery endothelial cells (hpaecs). human pmns were coincubated with hpaecs _+ lps (0.001-10mg/ml) with 2% serum for 1 hour. the effect of phorbol myristate acetate (pma) (125 ng/ml) was also examined. percentage adhesion stimulated by pma was 66+10sd. lps did not significantly increase adhesion at any of the concentrations used. to confirm the activity of lps pmns were incubated at 37~ with 0.01-10mg/ml lps + 2% serum for 1 hour and labelled with flourescent antibodies to the mac1 adhesion molecule complex (cd 18/cd 1 lb). facs analysis indicated upregulation of both cd18 and cdllb. thus in the system used, lps did stimulate pmn adhesion molecule expression, indicating that the lack of adhesion reflects a difference in hpaec response to lps compared to that reported for huvecs. this work was supported by the health research board ireland. although inhaled corticosteroid therapy is of undoubted benefit in the management of asthma, dysphonia is a recognised sequela. this study was designed to examine longitudinally the effect of inhaled steroids on the voice and vocal cords of newly diagnosed and previously untreated asthmatics. twenty subjects were recruited and underwent voice and vocal cord assessment prior to and 3 months after starting inhaled steroid treatment. the assessment consisted of 1) rating dysphonia using a visual analogue scale, 2) acoustical analysis of the voice and 3) videostroboscopic examination of vocal cord activity. prior to commencing inhaled steroid therapy for their asthma 14 subjects had normal voices, 5 subjects were mildly hoarse and one was moderately hoarse. vocal cord pathology was noted in 12 subjects, 2 patients had vocal cord nodules and the remainder were noted to have mildly oedematous cords together with a glottic chink. at 3 month follow up, improvement in voice was noted in 2 subjects, one patient felt more dysphonic but there was no change in vocal cord appearance. one subject was noted to have developed a mid glottic chink with no associated change in voice. one subject had clearing of mild vocal cord oedema and improvement in voice. this study demonstrates that 60% of subjects commencing inhaled steroid therapy for asthma have mild vocal cord pathology. voice is more likely to be improved following use of inhaled steroids for 3 months then made worse. although the relationship between elastin degradation and emphysema is well known, recent evidence suggests that a more complex process of pulmonary remodelling occurs within the emphysematous lung. the aim of this study was to assess the extent of extracellular matrix remodelling by ultrastructural examination of its two major components, elastin and collagen. emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase (1.2u/g body weigh0 and human lungs were obtained at surgical resection for lung carcinoma. emphysema was confirmed histologically in both animal and human samples by measurement of the mean. linear intercept. matching sections were immersed in 2.5m naoh and 88% formic acid to digest elastin and collagen respectively. scanning electron microscopy with stereo-pair imaging allowed 3-d visualisation of elastin and collagen frameworks. the distribution of emphysema was primarily panacinar in rat lungs a'nd centriacinar in human lungs. as expected in both types of emphysema, elastic lamellae were disrupted and perforated with multiple fenestrations. accompanying this disintegration was a marked increase in thickness of collagen fibrils which in some cases coalesced irish journal of medical science imparting a sheet-like appearance to the airspace walls. unique to human centriacinar emphysema, collagen formed helices which spiralled around alveolar septae to form bulky walls between adjacent airspaces. in conclusion, these findings lend support to the novel concept of aberrant collagen remodelling in the pathogenesis of emphysema. small cell lung carcinoma (sclc) is the most aggressive of the four common cell types of lung carcinoma. less than 10% of patients with sclc are alive two years after diagnosis. staging procedures, treatment regimens and survival results were reviewed in a small regional centre to make a comparison with larger treatment centres. thirty-one cases of sclc seen by one physician from 1985 to 1993 were reviewed. staging was clinical. treatment was undertaken in conjunction with the local oncology and radiotherapy services. 42% of patients had limited disease where as 58% had extensive disease at diagnosis. in patients with limited disease, 20% were alive at 18 months and there was a 10% long term survival rate i.e. greater than three years. average length of survival in limited disease was 456 days. survival results were comparable with those treated with chemotherapy alone and combination chemotherapy and radiotherapy. in patients with extensive disease the best results were from those treated with a combination of chemotherapy and radiotherapy with art average survival of 353 days. these figures compare favourably with those from larger multidisciplinary centres. the factors contributing to our relative success may relate to continuity of care achieved in a smaller centre. nasal cpap is a very effective therapy for osa, but is cumbersome, and compliance varies. we prospectively evaluated 91 consecutive osa patients treated with ncpap, who were asked to complete questionnaires before, and 2 to 12 months after starting therapy. this stttdy intended to examine both the patient's subjective response to ncpap and their bed-partner's impressions also. replies were received in 84 (92%) patients. patients were divided i~to 4 groups depending on whether they had a bed-partner, and according to their response to an initiat question assessing overall improvement in sleep quality and daytime al'ertness with ncpap, ranging from 0 (minimal/none) to +4 (excellent). patients were called responders if they scored > 2. group a (45 pts, 54%) were responders with bed-partners; group b (10 pts, 12%) non-responders with bed-partners; group c (15 pts, 18%), were responders (12 pts, 14%) and nonresponders (3 pts, 4%) without bed-partners; and group d (14 pts, 17%) had stopped ncpap. nine questions were directed at the bed-partner, and assessed their perception of changes in both the patient's and their own sleep quality, daytime alertness, mood and quality of life, and also to changes in the relationship between patient and bed-partner following institution of ncpap. these questions scored from -1 (worse) to +3 (marked improvement). significant improvement in all parameters for the patient (mean + sd = 2.4 + 0.7) were noted in group a. in addition, group a bed-partners reported ~ubjective improvement in the same parameters (1.7 + 1.1). group b improvements were less, (1.8 + 1.0 in patients, and 0.6 + 1.1 in partners). overall, the data indicate a subjective success of therapy in 68% of patients, but the bed-partner's replies indicate this figure underestimates the true response rate. furthermore, the results show significant improvements in the bed-partner's sleep quality, daytime alertness, mood and quality of life, indicating that successful treatment of osa patients with ncpap also gives significant benefits to their bed-partners. vincent's hospital, dublin. neutrophil collagenase (mmp 8) is a member of the matrixmetalioproteinases (mmps), a family of highly homologous zinc endopeptidases which play a crucial role in many physiological processes. the aim of this project was to develop a purification system for mmp 8 from purulent sputum and raise polyclonal antibodies. after initial extraction, contaminating proteins were removed with a zinc chelate affinity column. mmp 8 was then separated, from another closely related mmp, gelatinase b, on a q sepharose ion exchange column using a nacl gradient. the final purification step was carried out with an orange sepharose affinity column. sds-page analysis indicated the presence of purified protein with bands corresponding to latent neutrophil collagenase (85kd) and products of coll~igenase autodegradation at lower molecular weights (55kd & 57kd). a 10fold increase in specific activity was observed, with a 20% final yield, which provided mg quantities of pure enzyme. this work is funded by forbairt and the. irish american partnership. cd30 is a protein first described as a surface marker on hodgkin's lymphoma cells. recently cd30 has been demonstrated on th2-type t lymphocytes (produce il-4, 5, 6, l0 and 13), which have a pro-inflammatory cytokine profile. but is not found on thl-type t lymphocytes (produce il-2 and ifn-gamma). its ligand, cd30l, has also been described, cd30-cd30l interaction has been shown to aid the development oft lymphocyte clones into a th2 rather than a th 1 phenotype. th2-type cytokines are inextricably linked to the aetiology of inflammatory airway disease. firstly we investigated serum cd30 levels in various patient groups. we have demonstrated significant differences in serum cd30 levels in the following groups, atopic asthmatics (mean = 149 iu/l, n = 62), non-atopic asthmatics (mean = 107 iu/l n = 13) and atopic rhinitis/dermatitis (mean = 90 iu/l n = 36) and normal controls (mean = 14 iu/l, n = 9). secondly we cultured peripheral blood mononuclear cells from allergic individuals and normals. when these cultures were stimulated with house dust mite antigen (der p 1) and il-2 or der p 1 with both il-2 and i1-4, surface expression of cd30 on t lymphocytes could be demonstrated using fluorescent staining and flow cytometric analysis, after 9 days culture in the allergic individuals but not in normals. the presence of i1-4 in the culture increased the degree of surface cd30 expression. these results are important as they show that allergic individuals have an expandable population of memory t lymphocytes which respond to allergen by expressing cd30 and developing th-2 phenotype. most work on cd30 and th-2 cytokines has hitherto been carried out an t cell clones. we have developed a relatively simple in vitro system of looking at t lymphocyte response to allergen which will allow the testing of novel therapeutic interventions with a view to modulating the immune response in allergic disease. our work also suggests that even non-allergic patients with inflammatory airway disease may have increased th2 activity, which has not been shown previously. scimitar syndrome is a rare congenital disorder consisting of a spectrum of abnormalities including hypoplasia of the right pulmonary artery, dextroposition of the heart, anomalous pulmonary venous drainage of the right lung into the inferior vena cava and anomalities of the right diaphragm. bronchiectasis and respiratory tract infections on the right side are the usual clinical presenting features. a 70 year old male patient was referred to the outpatient clinic with a history of recurrent chest infections which were slow to resolve following antibiotic therapy. physical examination revealed decreased air entry, coarse crepitations and a prolonged expiratory wheeze in the right lower lobe. the only abnormalities on routine biochemical and haematological screening were an elevated esr of 69 and a slightly raised white cell count of 15. a chest x-ray revealed hypoplasia of the right lung when compared to the left. in addition to this there was a vascular shadow present in the right lower robe representing an anomalous pulmonary vein which appeared to drain to below the diaphragm on the night side. bronchoscopy showed a normal left bronchial tree. on the right, no apical segment was detected in the right lower lobe. otherwise no endobronchial lesion was seen. a dynamic computerised axial tomographic scan of the thorax was performed. this showed a dilated anomalous right lower pulmonary vein which was clearly seen to enter the inferior vena cava below the diaphragm. in addition the right lung was again noted to be hypoplastic when compared to the left. these findings were pathognomonic of the scimitar syndrome. "the patient was treated symptomatically and is presently stable. conclusion: scimitar syndrome, with its wide spectrum of abnormalities should be considered when reviewing plain chest x-ray in patients with recurrent right lower lobe respiratory tract infection. recent studies indicate that the ability of circulating neutrophils to regulate surface levels of adhesion molecules may be altered in disease situations. the aim of this study was to determine if changes in neutrophil responsiveness accompanies chronic inflammation in cf. neutrophils in blood samples from 17 cf patients and 13 age-matched control subjects were analysed by flow cytometry for expression of l-selectin and mac-1 (cd1 lb) following stimulation by interleukin-8 (il-8) and fmlp. as expected, both il-8 and fmlp provoked a decrease in surface levels of l-selectin and an increase in cdi lb levels. however, the magnitude of these changes was significantly lower in cf patients than in control subjects (table) . these results suggest that chronic exposure to inflammatory stimulii in vivo may alter neutrophil responsiveness in cf. given the emphasis on rational prescribing, we reviewed drug use in a 208 bedded long-stay unit. prescribing patterns were analysed on an appointed day thereby obtaining a "snapshot" of prescribing practices. one hundred and ninety four long-stay residents, with a mean age of 78, were on 1188 drugs, the maximum number of drugs per patient was 12, the minimum 1 and the average 6.1. sixty percent of prescriptions fell within one of the following therapeutic categories:-central nervous system (cns) preparations (321 prescriptions), analgesics (145), gastrointestinal preparations (139) and cardiovascular preparations (i 25). there were 19 ,prescriptions for respiratory drugs and only 36 prescriptions were for antibiotics. the most commonly prescribed cns preparations were anti-psychotics (127), benzodiazepincs (1 t 3), anti-depressants (30). 43% of all analgesics prescribed (63) were nsaids. the most commonly prescribed h2 blocker was cimetidine (33). nuseals aspirin (43), digoxin (35) and captopril (24) were the most commonly prescribed cardiovascular drugs. 25% of drugs were issued on an as required basis, i.e. "prn". the most commonly prescribed prn therapeutic classes were analgesics (100 prescriptions) followed by gastrointestinal (74) and cns preparations (68). these results contrast with prescribing patterns in hospitals and general practice and may provide an insight into the challenges and realities of management in long-stay units. supported by the health research board. evaluation of physician requests to hospital based clinical pharmacist for (1) drug information, (2) possible adverse drug reaction (adr) was undertaken over a two year period from jan '94 to dec '95 (admissions -1,667, opd attendances -5,908). overall 55 requests were made. (1) drug information: advice/information on new drugs, formulation, dosage, safety consideration prior to drug prescribing was given in 23 cases. (2) suspected adr: a total of 32 suspected adverse drug reactions were investigated. in 4 cases, no adr link was established, after extensive literature/data base search. adr's were confirmed in 28 cases of which 11 were reported to n.d.a.b. regular on-going interaction between physicians and clinical pharmacy allowed critical analysis of new drugs and heightened awareness of, potential adverse drug reactions in current clinical practice. we previously demonstrated that commonly prescribed medications are not easily identified by patients, doctors or nurses in the hospital setting o,2~. we then investigated the ability of hospital pharmacists, 33 in all, to identify the same 12 commonly prescribed branded and generic drugs. correct identification as follows:-bendrofluazide k (32/33), cimetidine (33/33), diazepam 5mg (31/33), diazepam 5mg generic (30/33), digoxin (24/33), ferrous sulphate (14/33), frusemide (16/33), mefenamic acid (33/33), paracetamol (27/33), prednisolone (1 l/ 33), temezepam (33/33), theoph3/lline (25/33). pharmacists had 78% correct answers compared with 62% for nurses and 42% correct for doctors. the pharmacists had no difficulty recognising drugs with brand names written on them e.g. cimetidine, but like nurses and doctors had difficulty identifying the plain white tablets e.g. prednisolone. generic drugs were tess well recognised. a number stated that they were unwilling to definitively identify medication with no clear marking. pharmacists were also asked to list the top 5 prescribed drugs, l0 got 1/5 correct and 23 got 0/5 correct. in contrast 36 out of 80 doctors got 1/5 correct, 25 got 2 right and only 4 got 3 right. we conclude that hospital pharmacists are generally better than doctors or nurses at identifying commonly prescribed drugs but their knowledge of the top 5 prescribed drugs is not as good at that of doctors. all professionals need assistant in this important task. suggesting reduced activity or more iranians with inherited variants of cholinesterase. one iranian subject with very low activity (dibucaine number below 20, atypical) had a history of apnea. these data indicate that the frequency of atypical and heterozygote genes for cholinesterase activity leading to prolonged apnea with succinylcholine (suxamethonium) is much higher in iranian than irish populations. this study emphasises the importance of ethnic pharmacology. it is has been advocated that funding for the prescibing of methadone in general practice should be provided separate from the indicative drugs budgeting scheme on the assumption that this may act as a disincentive to g.p.s to take on care of drug addicts. the objective was to analyse the current level and cost of methadone prescribing in 550 general practices in the eastern health board over a six month period. there was a review of methadone prescriptions for gms patients from jan. to jun. 1995. 1,087 persons received prescriptions. 3,945 scripts were issued. the age-specific prescribing rate for the total population was 6/10,000 (males 10/i 0,000, females 3/10,000). males aged 25-34 years had the highest age specific rates (44/100,000). the cost of methadone prescriptions amounted to s for the six months there was a trend towards an increase in the number of g.p.s who prescribed methadone over the period. only four of the 70 g.p.s (5.7%) who prescribed methadone issued in excess of 50 scripts for the period studied. for a small number of g.p.s methadone prescribing is a significant cost item on their budget. in the light of this, government policy should be reviewed with a view to excluding methadone from the indicative drug budgeting scheme. sciences, mashhed, lran. there is increasing evidence that some of the wide variation in the response to medicines has a genetic origin which may be expressed on a racial basis. to further study inter-ethnic differences in pharmacology we compared the activity of an enzyme responsible for the breakdown of endogenous substances and drugs -serum cholinesterase (pseudocholinesterase), dibucaine and fluoride numbers -in 200 irish and 200 iranian healthy subjects. irish subjects had significantly higher serum cholinesterase activity (7.28 + 0.14 vs 5.22 + 0.09 u/ml, mean + sem, p < 0.01 drug prescribing data may reflect changes in therapy and disease pattern. we reviewed current drug use among patients (n = 578) in a dublin teaching hospital in "snapshot" fashion on a designated day, and compared it with that obtained in 1985. in 1985, patients received an average of 7.8 different drugs each, with 41% on 5 or more and 3% on none. by 1995, the average was 6.8 (range i -19), with 62% on 5 or more. the percentage of patients receiving heparin fell from 42% to 13%, due mainly to a reduction in use on the medical side. the proportion of patients prescribed hypnotics fell from 55% to 37%, while ssri's are now the most used anti-depressants. antibiotic choice changed from amp/amoxicillin to coamoxiclav and the cephalosporins. diuretics remained the most frequently used cardiovascular agents, accounting for 4% of all drugs used and prescribed for around one quarter of patients. digoxin use remained constant, and by 1995, 20% of patients were on anti-platelet doses of aspirin. at least four different agents were in use in each of calcium antagonist, beta blocker and ace inhibitor classes. some of these changes in therapy reflect therapeutic advances, changes in disease management, greater choice of therapy and amendment of less than desirable therapeutic practices. on the other hand, some may reflect fashion or pharmaceutical promotion, rather than change as a consequence of evidence-based practice. acknowledgements: pharmacy staff, st. james's hospital and the health research board. studies of in vivo endothelial function in humans have usually involved intraarterial cannulation and the subsequent administration of substances that stimulate the endothelium to produce nitric oxide (no). such techniques are invasive and potentially hazardous. an alternative non-invasive method would be of benefit. animal studies have indicated that reactive dilation of vascular beds may be at least partially endothelium dependent. this study aimed to determine whether reactive hyperaemia in the human forearm was an endothelial dependent process with the potential to be used as a non-invasive method of stimulating the endothelium. ten volunteers underwent brachial artery cannulation and randomly received either placebo or n-monomethyl-l-arginine (l-nmma) (8~mol/min), an no synthase inhibitor, for 10 minutes. following this reactive hyperaemia was induced by the inflation of an arm cuff to 200 mmhg for 5 minutes and the response to this was measured by strain-gauge plethysmography. when flows had returned to baseline the process was repeated with the remaining substance. results were analysed by repeated me~isures anova. l-nmma resulted in significant reduction of basal forearm blood flow (p<0.01). there was no significant difference in reactive hyperaemia with either l-nmma or placebo. in conclusion, no does not contribute to reactive hyperaemia in the human forearm. dublin 8. home-based infusion therapy has been widely recognised as the optimum for treatment of disease states that require daily intravenous therapy from a patient-care aspect. conditions necessitating intravenous therapies in hiv disease include: cmv retinitis, intractable cryptosporidial diarrhoea, azole-resistent candidosis, nutritional support with total parenteral nutrition, chemotherapy for aids-related malignancies and palliative care in the terminal phase of the disease. the need for such therapies is increasing as patient survival improves. in 1993, the home-infusion service was set up in recognition of the need to treat patients, requiring intravenous therapy, in the home environment. this has been brought about by the development of small, light-weight pumps suitable for ambulatory use, the development of a service for aseptic compounding and the availability of permanent in-dwelling venous catheters. we describe the impact of this service on our patient cohort. to date, sixty-five hiv positive patients have received parenteral therapy at home. patients' age, sex, risk group, cdc stage, cd4 count, indication for therapy, complication rate and response to treatment are described. the provision of this service has reduced the number and length of patient admissions with associated improvement of quality of life. in addition, it recognises that patients prefer to be treated in the home environment aided by a co-ordinated multidisciplinary approach. since blood alcohol levels over 80 mg/100 ml are now illegal for vehicle drivers we have investigated if the commonly held "safe" limit of two drinks will bring the young adult over the legal limit and if this amount of alcohol will affect their psychomotor skills. following informed consent 33 healthy volunteers, nonhabitual drinkers on no medication (16 male, 17 female), with a median age of 24 (range 20-27) years participated and refrained from alcohol for at least 2 days. each drank within 15 minutes two standard drinks (35.5 ml each ) of 37.5% vodka (2.7 units of alcohol) plus 60 ml of orange juice at about 90 minutes after a standard mid-day meal. their psychomotor performance was estimated by the number connecting technique at 45 minutes after alcohol consumption and they were also asked to rate their feelings (which included alertness, clear-headedness, competence and attentiveness) using a visual analogue scale of 1 to 10. blood samples at one and two hours later were collected from the antecubital vein and analysed on the same day for alcohol content using enzymatic methods. mean (+ sem) blood concentrations of alcohol at 1 and 2 hours respectively were 25.39 + 6.19 and 21.62 + 4.58 mg/100 ml. in males and 36.43 + 11.89 and 38.66 + 3.43 mg/l-00 ml in females. values were significantly higher in females. blood concentrations in females were also higher (p < 0.01) than in males when expressed per kg body weight. while the blood alcohol in both the genders was considerably lower than the current legal limit in ireland their psychomotor skills as estimated from their task completion time and their answers to questionnaires were indicative of an impaired cns function. thus while 2 drinks may keep many subjects below the legal limit, there is considerable inter-individual variation with females showing higher concentrations and both genders have evidence of impaired performance at these lower levels. a non-linear approach was used to develop an hrv parameter, robust to both data non'-st~itionary and missing data points. unlike the standard chaos approach, using higher dimensional embeddings and time-delays, we employed a onedimensional correlation integral plot. the parameter thus obtained, allows an estimate of the spatial spreading of the attractor (ssa) or spatial variation of rr intervals along a straight line. heart rate data from 18 volunteers (22:00 to 06:00 hr), ~ifter oral placebo or propranolol 80 mg, investigated the ability to detect drug effect. vitamin e (~-tocopherol) is the most important dietary antioxidant in lipid and cell membranes and its intake reversely relates to the incidence of coronary heart disease and certain cancers. estrogen regenerates oxidized tocopherol radical in vitro m but such interaction has not been investigated in postmenopausal women receiving estrogen containing hormone replacement therapy (hrt) although estrogen containing oral contraceptive may reduce plasma vitamin e levep. we studied 21 healthy post-menopausal women (aged 46 -56) ammenorrheic for at least one year. fifteen subjects took a combination of harmogen provera therapy and 6 acted as a control group. blood samples were taken from all subject at baseline and after 4 weeks. in the hrt group, serum fsh levels were greatly reduced (86.46 + 25.27 vs 68.67 + 11.59 iu/1, p < 0.04, mean + sd, after hrt) with an increased serum oestradiol level (< 20 -28.68 vs 603.67 + 343.56 umol/1, p < 0.0001). no change occurred in the control group. vitamin e status, measured either as plasma or red cell ~-tocopherol respectively showed no change in both groups (hrt group 26.48 + 5.42 vs 27.17 + 3.39, 23.79 + 5.19 vs 24.93 + 4.37 gmol/1, p > 0.05). we conclude that in post-menopausal women, 4 weeks estrogen containing hrt did not alter vftamin e concentrations in vivo. we assessed the clinical benefit of the newer markers of bone formation: osteocalcin (oc), procollagen 1 carboxyterminal peptide (picp), bone alkaline phosphatase (balp), and bone resorption: carboxyterminal telopeptide of type 1 collagen (ictp) and urinary deoxypyridinoline crosslinks (dpd) over traditional assays such as total alkaline phosphatase (talp) and urinary hydroxyproline (oh/pr) in 23 patients with primary hyperparathyroidism (phpt). patients were sampled basally, then at 2, 6, 24, 48 and 72 hours post surgery and again at 1.5, 3, 6 and 12 months post op. the mean basal p1cp level was 89+26 ug/l (normal: 38-202) this increased to a peak at 48 h (168+_74 ug/l), then declined to normal at 6 weeks (116+56 ug/l). mean basal urinary dpd levels were raised at 8.6+1.2 nm/mm cr. (normal 2.0-6,0), they had normalised by 6 months to 5.9-+1.4 nm/mm cr. mean balp levels were always normal, although normal the yearly mean oc level was significantly lower than the basal value. mean 1ctp, oh/pr and talp levels were always normal. therefore bone turnover in phpt is best assessed by the newer markers picp and dpd. we have previously described seasonal variation in fibrinogen with higher levels in winter. as fibrinogen is an acute phase reactant, the winter rise may be a response to seasonal infections. the present study investigates this hypothesis by examining seasonality infibrinogen and markers associated with infection: white cell count (wcc), interleukin-6 (1l-6), human herpes virus 6 (hhv6) and herpes simplex virus (hsv) antibodies. monthly blood samples from healthy volunteers age 75 and over were measured for fibrinogen, wcc, il-6, hsv and hhv6 reactivation over a 1 year time period. a rhythmometric method was used to examine the data for seasonality. statistical significance was measured using the fstatistic. a highly significant seasonal variation (sv), peaking in mid-february, was found for fibrinogen (n=24; sv=0.629 g/ 1; f=76.148; p<0.01). no significant seasonal variation was present for measures of wcc (n=24; sv=0.209 e9/l; f= 1.940; p>0.05), hhv6 (n=24; sv=0.065 au; f=0.653; p>0.05), hsv (n=7; sv=6.055 au; f=2.048; p>0.05) or il-6 (n=7; sv=1.235 pg/ml; f=0.558; p>0.05). the present investigation does not support the hypothesis that seasonal variation in fibrinogen is a direct effect of the acute phase response, initiated by a seasonal variation in level of infection. the explanation for the seasonal changes in fibrinogen remains unknown. increased plasma homocysteine and reduced plasma antioxidants are risk factors in the development of vascular disease. design: 131 subjects drawn from elderly people living in the community (median age 85 yr, range 60-102 yr; 81 female). total plasma homocysteine, vitamin c, gamma tocopherol, retinol and beta carotene were measured by high pressure liquid chromatography. homocysteine levels in elderly males [median (range) = 12.35 um (4-18.1), n=241 were significantly higher than in vol. 165, 1996 supplement no. 2 irish journal of medical science elderly females [8.45 um (4.8-24.8), n=30]. these values were also higher than in a younger (30-49 years) male cohort [mean = 7.85 um, n= 610]. no correlations to vitamin concentrations were found, nor was there a correlation to age within the elderly cohort. within the elderly females, a significant negative correlation with age was found in vitamin c, gamma tocopherol and beta carotene (p<0.05). however a significant increase in retinol was noted. a very strong correlation between vitamin c and gamma tocopherol levels was noted in the elderly population sample (p<0.001 after multiple regression). conclusion. homocysteine levels in the elderly are higher than in samples of a younger population. a gender difference is maintained in the elderly. the provision of extended care forms one part of a spectrum of health care for older people. in the eastern health board area all patients over the age of 65 must be assessed by the multidisciplinary geriatric team prior to placement. we report on the experience of the total number of referrals for assessment for extended care to one department of geriatric medicine in a 268 bed teaching hospital. ninety-eight patients listed for extended care in 1994. the mean number of days between listing for long term care and placement was 49 _+ 45 days (range 3 to 206). almost one quarter of patients died while in hospital awaiting long term care: this underlines the frailty of patients who are admitted to hospital and request long term care. two patients were transferred to other institutions and 10 patients were able to get home. of the remaining patients 35 (63%) were placed in statutory or voluntary long term care accommodation and only 37% were eligible (usually financially but in some cases due to significant disability) for nursing home care using the terms of the 1993 nursing home act. patients who are listed for long term care through a general hospital are in general very frail, they tend to have a very extended length of stay and the provisions of the nursing home act only apply to a minority. these findings underline the need for provision of adequate statutory and voluntary extended-care places within the eastern health board area. there are over 40 screening assessments for cognitive function and choosing the most appropriate may be difficult. increasingly the importance of behavioural dysfunction is recognised. can any of the cognitive assessments help to predict behavioural dysfunction.'? we compared and contrasted the folstein mini mental state examination (mmse) and the cognitive assessment schedule (cas) of the clifton assessment procedures for the elderly (cape) and compared them with the behavioural rating scale (brs) of the cape. the study was carried out on a total of 21 referrals to the occupational therapy departments by geriatricians in the meath hospital and st. james's hospital. all subjects were over 65 and medically stable. the time scale involved was may-july 1995. the mmse and the cas were administered within the one sitting and each was timed. brs was rated the same day by either a staff or family, member. the average time to complete the mmse (416 + 124 s) was longer than the cas (342 + 171 s) but this was not statistically significant. the mmse and cas were significantly correlated (r = 8208, p < 0.0001). the cas was significantly correlated with the behaviour scale (r = 0,551, p < 0.01) whereas the mmse was not. these results suggest that equivalent assessments of cognitive function may be made with the mmse or cas, but a low cas score will be a better prediction of behavioural dysfunction. a spectrum of neurological and myopathological changes are associated with patients in intensive care units. we observed several patients post discharge from icu who presented with unexplained dysphagia which we suspected may be associated with the neurological complications of sepsis. the particular complication of dysphagia as a neurological manifestation of sepsis has not been documented. our descriptive study presents a series of three patients with persistent dysphagia which may represent a similar phenomenon. we selected patients for the study ranging from 75-87 years of age and on the basis of medical history including icu stay, sepsis, and intubation. all patients presented with dysphagia as observed on videofluoroscopy. we studied the video findings in-depth in order to ascertain if similar swailow patterns were present in these patients and if this could be correlated with their medical history. each of the three patients presented with similar dysphagia signs. the oral phase of the swallow was moderately atypical but the pharyngeal phase was significantly atypical. it was felt that intubation alone was not the sole causative factor of this dysphagia. the polyneuropathy associated with sepsis in icu may explain the atypical swallow patterns observed in these patients. the severity of the persistent dysphagia can cause serious respiratory and medical consequences. there is a need for further investigation of this phenomenon to identify patients who are at risk. little attention has been paid to the prevaience and phenomenology of behavioural disturbances among medical patients despite awareness of the high prevalence of cognitive vol. 165, 1996 supplement no. 2 impairment in this patient population. we screened 79 consecutive admissions to a department of acute geriatric medicine. patients were evaluated over a 2 week period using a modified version of the brief agitation rating scale. medication use, cognitive function and impact on nursing time were also measured. the prevalence of behavioural disturbance in this population was 19/79 (24%). the most frequent behavioural abnormalities were restlessness (12), complaining (12) and screaming (6). the most common underlying disorders were dementia, stroke disease, personality disorder and paranoid psychosis. the behavioural disturbance was only documented in the medical notes in 7 patients (37%) and in only 5 cases was a psychiatric consultation sought. these findings demonstrate that behavioural disturbances are not only common but also under-documented in elderly medical patients and there is a need for training in the detection and management of behavioural symptoms in this patient group. in lower limb trauma where there is severe compound fracture, the successful treatment of this depends on adequate bone and soft tissue debridement. as a result, subsequent bone defects can lead to instability and often require large amounts of bone grafts, and major soft tissue reconstruction is reaquired to obtain skin cover. large soft defects can by reduced bv primary bone resection and shortening of the limb. this will improve the chance of bone healing if performed in the presence of an external fixator, then lengthening at a site away from the traumatised area can gradually restore limb length. two cases are presented to demonstrate .the effect of compression / distraction techniques on soft tissue and bone injuries in these difficult situations. wegener's granulomatosis -wg (15), churg strauss syndrome -css (4), polyarteritis nodosa -pan (2) and unclassified (5). using the chc definitions, the diagnoses were wg (5), microscopic polyangiitis -mpa (10), pan (1) and undefined (10). there was concordance in only 5 patients (all wg). there is significant discordance between these two criteria sets. since the acr criteria does not recognise mpa, they tend to overdiagnose wg. in addition, the chc criteria cannot be applied without a biopsy and therefore surrogate features which predict the underlying histology are required to allow more practical application of the chc definitions. the objective was to determine the value of examination of dried freshly produced saliva, under light microscopy, in patients with xerostomia related to secondary sjogrens syndrome. ten patients with known connective tissue disease or rheumatoid arthritis attending rheumatology clinic were enrolled into the study, all with symptomatic xerostomia and dry eyes. all had an abnormal schirmer's test. five normal patients were enrolled, all of whom were without clinical evidence of rheumatological disease. control patients were enrolled who had no clinical evidence of rheumatological disease, a salivary sample was collected and examined by light microscopy. serum was also examined for the presence of anti-ro/la, rheumatoid factor, and anti-nuclear factor. all ten patients demonstrated 'reindeer horn' type ferning of saliva, a pattern of shorter thicker clubbed branches of crystallised mucus, in contrast to the normal ferning pattern of the healthy subjects. conclusion: we have shown in this preliminary report that light salivary microscopy is a simple test easily performed in an outpatient setting which could be a useful diagnostic procedure in sjogrens syndrome. recently, two sets of criteria have been proposed for the nomeclature of primary vasculitides, the 1990 american college of rheumatology (acr) classification criteria and the 1992 chapel hill consensus conference (chc) definitions. the aim of this study was to determine the concordance of these two systems in a cohort of patients with primary systemic vasculitis. patients with systemic vasculitis were recruited who had a biopsy proven diagnosis or, who had typical clinical features associated with a postive antineutrophil cytoplasmic antibody (anca). the case notes were reviewed and patients were classified according to both sets of criteria. twenty-six patients were recruited, 21 of whom had a positive biopsy. applying the 1990 acr criteria, the diagnoses were, primary pulmonary hypertension (pph) typically affects young individuals, and has a high morbidity and mortality. secondary pulmonary hypertension complicating connective tissue diseases likewise carries a poor prognosis. we evaluated the acute and chronic effects of ketanserin, a selective serotonin type-2 receptor antagonist in 2 patients with pulmonary hypertension in the acute study ketanserin was administered as a peripheral venous infusion during right heart catheterisation. following encouraging results during catheterisation oral administration of ketanserin 160mg daily in divided doses was instituted. in patient 1, a 30 year old female with probable pph, serial cardiac catheterisations over a 1 year period showed a significant, sustained reduction in both mean pulmonary artery pressure from 49 mmhg at baseline to 24 mmhg at 1 year (normal 10-20 mmhg) and pulmonary vascular resistance 1118 units at baseline to 288 units at 1 year (normal <200 units). in patient 2, a 61 year old female with limited scleroderma (crest) echocardiography after 1 month's oral ketanserin showed a reduction in estimated peak right ventricular systolic pressure from 60 mmhg at baseline to 13 mmhg (normal range 15-30 mmhg). the acute and long term response to ketanserin with improyement in pulmonary haemodynamics in these patients suggests that if a beneficial effect is detected during catheterisation long term oral therapy may be worthwhile. levels were low (< 7.9 iu/1); normal though above average (>-8 -<10 iu/l) and moderate high (>10 -<15 iu/l) respectively. gonadotrophins for ovarian stimulation were commencing initially at 225 iu for group a & b and at 450 iu for group c. ivf performance was poor in most aspects (total follicles, oocytes & embryos transferred) in group b comparing with group a or c; the cumulative ongoing pregnancy rate (pr) over 2 ivf cycles in group b; was 15% comparing with 27.6% in group a (p < 0.05) however there was no significant difference in pr in group c (20%) comparing with other two groups. cycle day 3 fsh screening is predictive of follicular development in ivf. high initial dose of gonadotropins help to improve the pregnancy rate in the presence of moderate high level of fsh. the purpose of this study was to evaluate patient satisfaction with antenatal care provided in the perinatal day centre (pndc). a self administered questionnaire was administered to 61 consecutive patients. the main indications for referral were suspected small-fordates (34%), non-proteinuric hypertension (23%), glucose tolerance testing (15%), reduced fetal movements (10%) and post-term evaluation (7.5%); 51% were nulliparae. thirty-two percent of patients were reviewed in the pndc on the day of referral; the rest within 7 days. twenty eight percent of patients lived more than 10 miles from the hospital and 48% spent more than 30 minutes in travelling there. eighty five percent of patients scored their level of satisfaction with the service provided in the pndc as > 7 out of 10; only 6.5% would have preferred admission; 42% said that they would prefer to visit the pndc 5 times per week to avoid admission. the main area of dissatisfaction related to the waiting time for review prior to discharge, with 69.5% of patients waiting over 3 hours. patients attending the pndc report a high level of satisfaction; changes to reduce the visit duration have been introduced. to examine the change of taking-up the essential preconceptual measurements; rubella immune status, cervical cytology and prophylactic folic acid intake; following specific advice and publicity through 3 general public meetings with new patients prior to in vitro fertilization (ivf) programme. in 1994 we studied 281 new couples for ivf for the presence of some specific pre-conceptual data (group a). in this study we follow-up the same intake in another 229 new women interviewed to commence ivf programme from january 1995 till september 1995 (group b). in group (b) the taking-up measurements were dramatically improved. however, 30% and 18% stilldid not have rubella immunity test and cervical cytology performed; compared to 54% and 36% in group (ai respectively (p<0.001). while folic acid intake was sustained at >90% in both groups. following specific advice the rate of taking-up of preconceptual measurements prior commencing ivf programme was improved. there is a future need for continuous enhancement of the publicity and advice regarding the importance of preconceptual measurements. the aim of this study was to introduce icsi to ireland for treatment of specific cases of male factor infertility. following an introductory proving period using the bovine model, thirtyeight couples with infertility attributed to the male were selected for an icsi attempt. ovulation induction, oocyte retrieval and luteal management were as described for conventional ivf tm. the average age of patients selected for icsi were 34.5 + 3.7 years and 36.4 + 4.6 years for the female and male respectively, with an average duration of infertility of 4.8 + 2.6 years. a 64 year old woman presented with a three day history of parasthesia in her lower limbs and difficulty walking. neurological examination revealed sensory loss in her limbs and truncal ataxia. rombergs sign was positive. pelvic examination revealed a large pelvic mass that was distinct from the uterus. routine blood investigations were normal. csf culture, ct brain and serum electrophoresis were negative. anti-purkinjie cell antibodies were not present. ca-125 levels were elevated at 159 micrograms/litre. laparotomy revealed a 20 cm left ovarian tumour. a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy was performed. histology revealed a poorly differentiated clear cell adeno-carcinoma of the left ovary. the capsule was intact and peritoneal washings were negative. she made a good postoperative recovery. she received six courses of carboplatin without ill effect. her neurological symptoms resolved. subacute cerebellar degeneration can occur as a paraneoplastic disorder in ovarian carcinoma. the mechanism by which cancer can cause neurological disorders is not fully understood. paraneoplastic cerebellar degeneration occurs with or without the presence of purkinjie cell antibodies. the aim was to review all red cell transfusions in gynaecological surgery in 1994. a retrospective review of blood bank records and individual charts was carried out. 4611 patients underwent gynaecological surgery; 97 (21%) were cross matched and 60 (1.3%) were transfused. 184 units were transfused. there were no single unit transfusions. the mean number of units transfused per patient was 3. this accounted for 33% of all units transfused this year. 92% of patients were undergoing elective surgery. the overall cross match/transfusion ratio was 2. intraoperative difficulty was recorded in 56% of cases. 64% of patients were transfused perioperatively and 36% postoperatively. the percentage of patients requiring blood transfusions in the the main individual operation categories was as follows: radical surgery: 57%; total abdominal hysterectomy and salpingo-oopherectomy, 17%; vaginal hysterectomy and repair, 16%; subtotal abdominal hysterectomy, 30%; vaginal hysterectomy alone 12.5%; and total abdominal hysterectomy alone 5%. adverse reactions to transfusions were seen in 5% of patients. conclusion: the majority of patients transfused were undergoing elective surgery. vaginal hysterectomy was associated with greater blood loss than abdominal hysterectomy. only half of all units cross matched were transfused. dilatation and curettage (d+c) is the most common operation performed in the u.k. the liberal use of d+c has been criticised. the objective of this study was to evaluate the use of outpatient endometrial pipelle biopsy and determine its safety in terms of detecting abnormalities. complications and financial costs were also evaluated. data was reviewed from an active gynaecological unit from february 1993 to january 1995, using theatre and outpatient records. a total of 303 d+cs and 104 endometrial pipelle biopsies were performed in this period. 9 malignancies were detected by d+c and 1 by pipelle biopsy. a total of 24 and 3 benign abnormalities were detected by each method respectively. there was a higher complication rate in the d+c group but the failure rate was higher in the endometrial pipelle biopsy group. the monetary savings over this period is estimated at s there were no missed malignancies to our knowledge over the 8 year period since endometrial pipelle bioposy was introduced to this hospital. our study indicates that outpatient endometrial pipelle biopsy appears to be safe, efficacious and economical. while ultrasound findings may sometimes be in conflict with clinical examination, it is the case that there are instances when ultrasound findings have, following subsequent laparotomy, been found to be wholly incorrect. it is therefore not surprising that there remain some gynaecologists who view ultrasound with scepticism, preferring to rely solely of their clinical findings. there have been few studies that directly compare clinical, ultrasound and surgical findings in the detection of pelvic masses. the objective of the study was firstly to directly compare the reliability of clinical and ultrasound examination findings in the detection of pelvic masses proven by subsequent laparotomy and secondly to determine the accuracy of ultrasound in detecting malignancy. this was as a retrospective review of 86 women who underwent a laparotomy because of a pelvic mass between january 1993 to february 1995. information was obtained from theatre and patient records. real time abdominal ultrasound was used. findings at laparotomy were correlated with clinical and ultrasound findings. the sensitivity and specificity of ultrasound in detecting a uterine mass was 94% and 99% respectively. this contrasts sharply with clinical examination (sensitivity = 74% and specificity = 94%). similar findings were obtained when ultrasound was compared to clinical examination in detecting ovarian masses. ultrasouud is capable of predicting benign disease with reasonable confidence but the prediction of malignancy is less reliable. in conclusion, ultrasound is more sensitive and specific in detecting pelvic masses compared to clinical examination. vincent's hospital, dublin. osteoporosis occurring during pregnancy or lactation is a rare event despite the homeostatic demands of the foetus for calcium. we investigated the case of a 24 year old woman who, immediately following vaginal delivery of her first child, developed severe back pain due to a vertebral compression deformity of the second lumbar vertebrae. bone mineral density (bmd) was measured by dual-energy x-ray absorptiometry. calcium metabolism and bone turnover were studied. there was a severe reduction in bmd in the spine (z-score = -4.8) and f e m o r a l neck ( z -s c o r e = -3.6); but, serial measurements showed no further reduction in bmd. indices of calcium metabolism and bone turnover were normal. pregnancy-induced osteoporosis is a severe but self-limited disorder in calcium homeostasis of unknown aetiology. women with low bmd prior to pregnancy may be at increased risk. in view of increased demand, supplemental calcium and vitamin d should be considered during pregnancy and lactation. crumlin, dublin 12. the aim of this study was to assess the clinical status on admission and the critical care m a n a g e m e n t of children p r e s e n t i n g with m e n i n g o c o c c a l i n f e c t i o n . t h i s was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+3.46). the average duration of symptoms prior to admission was 20.4 hours (+11.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a wood gas analysis on admission had a metabolic acidosis (bases excess < -5.0). the mortality rate was 10.9%. 80% of deaths were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of cardiovascular compromise, 78% were admitted to the high dependency unit, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. in this study children presenting with m e n i n g o c o c c a l infection have a high incidence of cardiovascular instability. successful management is dependent on early presentation and initiation of therapy and on aggressive intensive care monitoring and support of the cardiovascular i11 and vital organ systems. the normal crying curve and incidence of colic for term infants are well known. we studied prospectively the crying pattern and the incidence of colic in preterm'infants to determine if prematurity influenced these behaviours. the subjects were consecutive preterm infants admitted to the cork neonatal units for two and a half months from july 1995. a continuous 24 hour diary was completed on each infant by the neonatal nurses, when the babies were on full oral feeding and no longer required intensive care. the parents completed the diaries 'after discharge. colic was defined according to wessel's rule of threes. two unwell babies were excluded. the duration of follow up was from 2 to 16 weeks. fifty infants were recruited and 45 completed the study (4 lost to follow up and one withdi'awn due to sepsis). their mean (range) gestational age was 31 (26-36) weeks and birthweight was 1.52 (0.64-2.4) kg. the mean (range) age of crying onset was x(y-z) weeks; crying, peak 'was x(y-z) weeks and crying offset was x(y-z) weeks. one baby developed colic in the period of follow up. conclusions: the incidence of wessel's colic was less than expected in these preterm babies. the crying pattern according to chronological age was different from that clescribed in term babies. in general preterm babies had a delayed onset of crying, but the pattern became similar to term babies when allowance was made for gestational age. the findings'suggest that the crying patterns of early infancy have a developmental basis. we re-evaluated 18 children who had had rs (1979-86), with their closest-age siblings using the wechsler scales, coopersmith self esteem inventory and achenback child behaviour checklist (acbc) (duffy j. et al 1991). the rs patients' means were consistently lower than that of their sibs. however, comparison of mean raw data, using "t-tests", yielded significant differences only in the acbc scores, in that rs children exhibited significantly more problem behaviours than their sibs (p=0.033). after categorisation of iq data, further comparisons between the groups (using x2), found rs patients were significantly more likely to score "below average" in tests of verbal iq compared to their sibs (p=0.04). age of onset and clinical stage were also found to be more important predictors of outcome. children less than 1 year of age at onset of rs had significantly lower iq scores on all measures of cognitive ability (p=0.003) and more problem behaviours (p=0.01) than children over 1 year of age. no significant differences were found in comparison with sibs. clinical stage to which rs progressed affected only verbal iq scores. children in whom consciousness had been impaired had significantly lower iq scores than both their sibs and rs children in whom consciousness was less impaired (p=0.02). in conclusion outcome remains cautiously positive, with 13/18 rs children attending mainstream schools or in employment without apparent difficulties. a national breastfeeding policy was introduced by the department of health in 1994. factors identified for promotion of breastfeeding were based on the who/unicef "ten steps for successful breastfeeding". we present clinical cases which suggest that one step may need to be modified. the charts of breastfed babies admitted to the special care baby unit were reviewed for one year following the introduction of the national breastfeeding policy to this hospital. thirteen term breastfed babies were admitted because of fever and dehydration. none of the babies had water or bottle feed supplements. ten of the thirteen mothers were primigravida. eleven babies were admitted in the six months following the introduction of an exclusive breastfeeding policy. the nursing staff were then alerted to the risk of dehydration, but two further babies of mums committed to exclusive breastfeeding were admitted in the subsequent six months. routine biochemistry, haematology and a limited septic screen was performed in all babies. three of the thirteen babies had lumbar punctures. the mean (range) weight loss on admission was 9.5 (4.1-16)%. the mean (range) plasma sodium level was 147.6 (138-156)meq/1 and the mean (range) urea was 6.5(2.1-13.4)meq/t. there was no growth from the cultures of the blood, urine, csf and swabs. all the babies were given intravenous fluids and parenteral antibiotics for 48 hours. the outcome was satisfactory in all babies and breastfeeding was reestablished in eleven of the thirteen babies. conclusions: the common factor to these babies was inadequate fluid intake prior to admission associated with stricl~ adherence to the policy, and avoidance of all supplements including water. we conclude that the who/unicef step 6 "not to give food or drink other than breastmilk unless medically indicated" is too restrictive in the immediate postpartum period. 42.5% of children reported headache in the previous 12 months 44.8% of girls and 40.5% of boys reported headache (p < 0.0001). 0.6% of children reported daily headache 5.9% of children reported weekly headache 9.4% of children reported monthly headache 22.5% of children reported headache less often than monthly the percentage of children with headache at each frequency, other than daily, increased with increasing age. in girls headache showed a marked rise at ages 14, 15 and 16 years. reported prevalence of headache in the past year in 5 to 15 year old aberdeen children was 66% and 48% was recorded for swedish children aged 8, 11, 13 and 17 years old. this study is the first community based prevalence study of headache in irish schoolchildren. our aim was to test the hypothesis that there is no correlation between the type of feeding & swallowing disorder the child has with the neurological diagnosis or the radiological findings. a further purpose was to develop a classification of the feeding & swallowing disorders which would guide us towards a management plan. a retrospective analysis of the data collected between the years 1988-94 from the feeding & swallowing clinic at booth hall children's hospital was done. 73 children were included in our study 9 ages ranged from 4 months to 17 yrs. all the children were assessed by the members of the feeding & swallowing team and had videofluroscopic assessment by the same radiologist. neurological signs, speech therapy assessments & videofluroscopy findings were compared between children with spastic quadriparesis & those without. significant differences were noted. a clinical classification was devised using cluster analysis. we conclude that there is no causal relationship between the neurological diagnosis & the type of dysphagia. there are three distinct groups of children who require different strategies of clinical management. surveillance commenced in january 1995 to continuously monitor the incidence of surgical site infections (ssi). employing modern optical scanning technology (formic for windows version 2.0, formic limited, london) a questionnaire was designed, which required minimal completion time. the questionnaire includes relevant data based on the american national nosocomial infections surveillance system for ssi including the ssi risk index. surveillance commences at the time of surgery and continues until the patient's discharge. optical scanning technology allows rapid reading of surveillance questionnaires thereby bypassing the bottleneck of manual data entry. by october 1995, details of 2,156 procedures had been recorded. the crude ssi rate for these patients was 2.7%. the patient risk index used demonstrated that there were increased chances of developing ssi in certain patient groups. seventy-nine per cent of ssi had presented by the 10th post-operative day. the length of stay increased by an average of 4 days in patients developing ssi. regular feedback to individual surgeons, theatre and ward staff maintains awareness and highlights possible problems. we recommend optical scanning technology to all those engaged in surveillance work. this system would be especially useful were data collected is transported from outlying hospitals to a central receiving centre for collation and analysis. in 1872 francis crumpe published a paper ~ in which he described the therapeutic effect of poisonous mussels (psp) on a case of tetanus. he obtained the mussels from tralee ship canal on the occasion of its infrequent emptying, and entertained the idea of using them in tetanus after treating a young girl with psp who recovered after 24 hours. prior to the use of psp he described it's paralytic effect in two cockrels who both recovered. in concluding his successful use of psp he speculated as to the clinical nature and role of the toxin tralee ship canal was opened in 1846. the water was relatively stagnant and would have contained plenty of nutrients. as such it would have been an ideal habitat for toxic algae which may have been brought across the atlantic as spores in bilge water 2. the emptying of the canal may well have been done at times of algal blooming. this is the first irish report of psp and a most remarkable use of saxitoxin (?) in the treatment of tetanus, antedating the current management by seventy years. this study was carried out to quantify the published research on smoking in irish medical journals; to ascertain the type of research carried out; and to identify the authors of that research. during the 35 years under study, 50 papers explicitly dealing with smoking were published. only 2 papers appeared in forum. there was a decline in published papers in the eighties with a resurgence in the early nineties. of the 50 papers, a majority were observational and ten were editorials. only one paper dealt with smoking cessation, and one with preventive work. general practitioners were poorly represented as authors. one doctor (prof. r. mulcahy) published at least one paper on smoking in each quinquennium since 1960. this study underlines the relative insignificance of smoking as a topic for research in ireland. a major sea change in attitude will be required if the government's targets for smoking cessation are to be realised, particularly if they are to be achieved by relying on the medical profession. radiology represents a major cost centre within a hospital. lack of awareness of cost amongst doctors may result in the inappropriate requesting of radiology services. this study assesses doctors knowledge of the cost to a tertiary referral hospital of commonly performed radioj'ogical procedures and investigations. doctors were asked to estimate the cost of 9 items namely -chest,x-ray, arch aortogram, ultrasound abdo., lumbosacral spine, barium enema, ct brain, ct abdo., ultrasound abdomen, i.v.p. and percutaneous gastrostomy tube insertion under radiological screening. 60 doctors in st. vincent's hospital were surveyed. doctors as a group overestimated the cost of all 9 individual tests, by margins ranging from 10% (i.v.p. & gastrostomy insertion) to 100% (ct brain/ct abdo.) the total cost to the hospital of all 9 items was s consultants'overestimated this total cost by 107%, followed by registrars, interns and s.h.o's who overestimated the total cost by margins of 51%, 43% and 12% respectively . conclusion: doctors tend to overestimate what radiological procedures and investigations cost a public hospital, often by quite wide margins. thus, any excessive requesting of radiology services by docto.rs is not due to a lack of awareness of their true cost to the hospital. (ded) in dublin. secondly, to identify the major cancers contributing to years of potential life lost (ypll) for the ehb, each cca and ded in males and females. in ireland little work has been done to date on disease specific premature mortality. crude death rates weight all deaths equally; in comparison, ypll emphasise deaths among younger persons and provide a measure of the burden of premature mortality. premature mortality for the 322 deds in dublin for the years 19988 and 1989 was estimated using ypll, which was calculated by subtracting the date of death form 65. ypll due to each of the major disease groups were ranked for each ded. seventy-one thousand four hundred and sixty-eight &471468) years of potential life were lost in dublin in the 1988 and 1989. 21.9% of ypll was due to injury and poisoning, 20.64% to cancer, 15.6% to circulatory disease. however, when the major cause of ypll was established for each ded, injury and poisoning was the number one cause of death in 25.5% of the deds; cancers 24%, congenital and perinatal conditions 21.5% and circulatory disease 14%. by emphasising deaths in younger individuals ypll is a valuable tool for planning and monitoring local health promotion initiatives. serum total homocysteine (thcy) levels are inversely associated with dietary intake of folic acid and b vitamins and raised levels have been linked with chd. we have examined the association between thcy concentration and the risk of chd in middle-aged men in 18 british towns. we used a nested case control study design, within an ongoing prospective study, thcy concentration was measured in serum samples, stored at entry to the study, from 110 incident cases of myocardial infarction and 118 controls. cases and controls were frequency matched by town and age group. levels of homocysteine [geometric mean (95%c1) were significantly higher in cases than'controls: homocysteine 13.5 (12.6 -14.3)gmol/l vs 11.9 (11.3 -12.6)lamol/l; p = 0,005. there was a graded increase in the relative risk (odds ratio; or) of chd in the 2nd, 3rd and 4th quartile of thcy (or 1.4, 1.9, 2.2; trend p = 0.006) relative to the first quartile. adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol and serum creatinine did not attenuate this association, (or 2.1,2.3, 2.7; trend p = 0.04). the findings suggest that thcy is an independent risk factor for chd with no threshold level. in summer 1995, a diagnosis of cryptosporidiosis was made 25 in a child who had visited a pet farm. this child had participated in a summer project involving 161 children and nine adults. reports of a similar illness among other project members, prompted an outbreak investigation. a cohort study consisting of two phases was initiated. 95% (162/170) of project participants responded to a self-administered questionnaire in the first phase. thirteen children met the case definition, of whom seven had cryptosporidium detected in their stools. illness was significantly associated with having visited a pet farm. (p<0.000005). 75% of those ill sought medical attention, of whom two were hospitalised the second phase of the cohort study was conducted among those who had visited the pet farm. 95% (52155) were interviewed. illness was significantly associated with play in sand, to which animals had access, at a stream's edge beside a picnic area (p<0.005). contact with various animals was not statistically significantly associated with illness. however the small numbers involved may have obscured such an association. this outbreak highlights a potential hazard for children visiting pet farms and also that cryptosporidiosis is a significant but often overlooked cause of morbidity in healthy children. managers of pet farms need to be aware of the potential for transmission of disease to visiting children. strict implementation of hygiene measures is essential to minimise risk. the mrc vitamin trial highlighted the importance of folic acid in the prevention of neural tube defects(l). since 1993, the department of health has recommended periconceptional folic acid supplements. the objective of this study was to document the knowledge and behaviour of women in child bearing years to periconceptional folic acid. a cross sectional community survey was conducted using an interviewer administered questionnaire in dublin. three hundred and thirty five women took part in the study. approximately two thirds 213/335 (63.6%) had heard of folic acid. knowledge was significantly associated with higher social class and higher education (p<0.o5). few 18/335 (5.4%) had been advised to take folic acid before pregnancy. only 9/335 (2.7%) of the women in the study were currently taking folic acid supplements. three quarters of the group (75.9%) would be willing to take periconceptional folic acid supplements if they knew it would reduce the risk of malformations. the majority (77.4%) would prefer to take folic acid in tablet form. this study clearly shows that few women in childbearing years have been advised on folic acid. however, if advised appropriately the majority would be willing to take periconceptional folic acid in tablet form. future publicity campaigns involving all health professionals should address these issues. unstable intra-articular fractures with or without dislocation of the phalangeal joints often lead to joint stiffness ana loss of function. nine patients with comminuted intra-articular phalangeal fractures were treated in our unit by dynamic external fixator using "pins and rubber bands traction system". the mean age was 32.3 years, and the follow-up average was 4.6 months. five patients had full and good range of motion in the involved joints. three patients had poor results, and one patient underwent open reduction one week following the original procedure. the technique and our results are discussed. this dynamic frame is compact, comfortable for the patient, easy to apply and allows early mobilisation. careful selection of patients and close follow-up in the first few weeks are needed. this study examines how gp's store and handle vaccines. all 144 gp's in a health board region were invited to take part. gp's were interviewed in their practice premises about how they dealt with vaccines fridges were examined and temperature recorded post interview. oral polio was taken from 20 randomly selected fridges for potency testing. cold chain monitors and freeze watch indicators were used to monitor batches of vaccine stored. of the 144 gp's, 142(98.6%) agreed to participate, 140 used 111 fridges to store vaccine, 2 store vaccine at room temperature. of the 111 fridges, 6 (5.4%) had the power supply safeguarded, 9 (8.1%) had thermometers, 36 (32.4%) had vaccine only stored therein. during defrosting, vaccine was inadequately protected in 22 (19.8%). of the 138 gp's who use multi-dose vaccine vials, 133 (97.2%) keep them for further use at the end of a day/session, 3 store them at room temperature. 42 (37.8%) fridges had temperatures outside the recommended range. 13 (28.2%) coldchain monitors indicated vaccine exposed to more than 10~ 18 (90%) of the oral polio samples showed a reduction in total titre of live virus, however, none were below the minimum acceptable. this study indicates that vaccine potency could be seriously compromised due to breaks in the cold-chain and suggests the need for guidelines to be drawn up, implemented and monitored to ensure the integrity of immunisation schemes. comparison was made with a study carried out in 1983. in addition the range of antimicrobial agents tested included new oral cephalsporins and quinolones that were not then available. three hundred microorganisms isolated from mid stream urine (msu) samples were examined by standard microbiological techniques. antimicrobial susceptibility testing to 12 antimicrobial agents was performed on significant pathogens (>105 organisms per ml) by disc diffusion test, and minimum inhibitory concentrations of antibiotics was carried out by e test on organisms found resistant by disc testing. by comparison with 1983 resistance amongst e.coli, the most commonly isolated pathogen, had increased for the following: ampicillin by 6% to 61%, co amoxyclav by 4.4% from 0%, trimethoprim by 18% to 28% and nitrofuradantin by 5% from 0%. no increase in resistance occurred to cephradine (4%), or nalidixic acid (5.4%). resistance to cefixime, ofloxacin and ciprofloxacin, was 3%, no resistance was encountered to cefotaxime. for proteus species resistance to ofloxacin, ciprofloxacin and cefotaxime was 4%, and for enterobacter sp 20%. enterococci were sensitive to ampicillin and augmentin but the numbers were small. pathogens isolated from patients domiciled in the inner city were significantly more resistant to nalidixic acid (20%), cefotaxime (6%), cefixime (14%), ofloxacin and ciprofloxacin (12%) than those isolated from patients in rural areas. the purpose of this study is to examine the relative importance of obstetric complications (0c%) in the aetiology of schizophrenia and mania. using the dublin psychiatric case register, birth records of 635 patients with an icd-9 diagnosis of schizophreniaor mania were obtained. these records were evaluated, for obstetric complications using two scales, the lewis, owen and murray scale (lom) it~ and the parnas scale 121. the mothers of those going on to develop schizophrenia did not differ from those going on to mania as regards maternal age, parity, social class, or period of pregnancy. however, males who developed schizophrenia when compared to males developing mania, experienced significantly more oc's when rated by the lom scale (p=0.007) and.more frequent oc's on the parnas scale (p<0.001) of greater severity (p=0.018). no significant differences were found between females with schizophrenia and those with mania. dublin. the aim was to evaluate the diagnosis, symptomatology and level of functioning of patients presenting with a first episode of psychosis to a catchment area service and a private psychiatric hospital. all patients presenting with a first episode of psychosis were assessed using the scid-p,,the positive and negative syndrome scale (panss) and the global assessment of functioning scale (gaf). fifty-eight patients (34 male, 24 female) ranging in age from 15 to 65 years (mean + sd = 28.4 + 10.8) were included in the study. the mean total panss score was 84.8 (sd + 21.6) and was strongly correlated with the gaf score (p < 0.001) but independent of age (p = 0.16). males had a significantly lower gaf score compared to females (p = 0.04) but there was no gender difference in the total panss score (p = 0.20), twenty-five patients (43%) had a lifetime prevalence of drug abuse or dependence but only 12 patients (21%) had signs of drug abuse or dependence in the month prior to presentation. level of functioning was strongly influenced by the severity of psychopathology. substance abuse is common in individuals presenting with a first episode of psychosis. the aim was to evaluate the presence of involuntary movements in patients presenting with first episode psychosis to a catchment area service and a private psychiatric hospital. patients presenting with first episode schizophrenia and schizophreniform psychosis were assessed for involuntary movements using the involuntary movements scale (a.i.m.s.). 28 patients (17m., llf.), age range 15-67 years (mean=30.5 years.) were included in the study. one patient (3.5%) satisfied the strict criteria of schooter and kane for spontaneous dyskinesia. 7 patients (6m., if.), had minimal involuntary movements in at least 2 body areas, predominantly orofacial. the total a.i.m.s. score was positively correlated with the number of days spent in hospital per year of follow up (p=0.01). the group with involuntary movements were found to have spent more days in hospital per year of follow up, 94 v. 55 days (p=0.047). for patients with first episode schizophrenia or schizophreniform disorder spontaneous dyskinesia is not common. however involuntary movements at presentation may be a predictor of poorer outcome. psychiatry has moved from custodialcare towards care in the community. adequate reprovision will have to be made in order to discharge the remaining continuously hospitalized patients. the objectives of this study were to describe a.n entire long-stay hospital population, to examine the differences between the old and new long stay groups within this populatian and to evaluate the needs for community residential and day care facilities in order for hospital closure to take place. the study group consisted of the total long-stay population of st. davnet's hospital, monaghan. the patients were assessed using the community placement questionnaire (cpq) one hundred and twenty four patients were included in the study. fifty-six were female and 89% were single. the mean age of the total group was 68.7 years. the majority suffered from schizophrenia. the assessment revealed a globally disabled group with multiple handicaps. the new long-stay group were disabled as the old long-stay group. the patients were characterised into four groups with regard to placement recommendations. these were a specialist unit for chronically disturbed geriatrics, a geriatric unit, a high support hostel and a medium support hostel. the remaining population of this hospital were highly dependent with multiple handicaps but would live in community with adequate support. there is little difference between the needs of the old long-stay and those of the new long-stay. failure of the immune system to identify self peptides is likely to lead to the development of an autoimmune reaction. susceptibility to autoimmunity is strongly influenced by genes clustered in the hla region (chromosome 6p) particularly class i (a, b and c) and class ii (d, q and p). it has been suggested that there is an autoimmune component in the aetiology of schizophrenia. of many conflicting reports from case/control studies using hla antigens the most consistent finding has been an increased frequency of hla-a9 (now split into a23/a24). additionally, a susceptibility locus for schizophrenia has been reported near the hla locus. to attempt to confirm the hla a9 hypothesis, we have genotyped a preliminary sample of 63 familial schizophrenic probands and 77 unrelated controls at the hla-a region, using a pcr-ssop technique. the frequency of hla-a24 (the major component of a9) in patients and controls respectively was 12.5% vs 15.5%. these findings do not support the hypothesis. some of the discrepancy may be due to unspecific cross-reactions produced by commercial antisera used in the microlymphocytotoxicity method of previous studies. however it is also possible that the hla associatton with schizophrenia may reflect linkage disequilibrium with unidentified gene(s) within the hla region which is less strong in the irish population. schizophrenia is a common mental disorder affecting about 1% of the general population with a devastating disturbance of mind and personality. family, twin and adoption studies have demonstrated that the disease is largely genetic with a polygenic mode of: transmission. dopamine receptors have been implicated in the aetiology of the disease. as yet 5 dopamine genes have been identified (d1-d5). in particular the d3 receptor is expressed in the limbic regions of the brain, implicated in the control of emotions. association studies of a d3 polymorphism (glycine to serine substitution at position 9,) with schizophrenia have produced conflicting findings, many of which, however, have demonstrated a significant excess of homozygosity, or excess of the 1 -l genotype at this polymorphism. in this study, 200 familial schizophrenics and 239 irish unrelated controls were genotyped. the result show a small increase in the frequency of the 1-1 genotype which did not attain statistical significance (patients, 41.5% vs. controls, 38.5%). homozygosity (alleles 1-1 and 2-2) was also slightly increased in the patients (patients, 55.5% vs. controls, 48.9%). the small increase in the frequency of the 1-1 genotype and of homozygosity in the patients is in keeping with earlier findings but suggests that the effect, if any, of d3 sequence variation in the development of the disease is small. lack of information about general practitioners' (gp's) ability to prescribe psychotropic medication may affect patients' compliance. in this study, 73 out of 75 patients attending a psychiatry out-patient clinic completed a questionnaire which documented how many had run out of medication, the steps taken if they had and the role each patient thought their gp played in their treatment. 82% indicated that their gp knew what their current medication was but only 46% thought that their gp could provide them with a prescription if they did not have one from the clinic. this figure was similar in those who had (21%) and had not (79%) run out of medication in the past. on running out of medication, 42% of patients waited until their next appointment, 29% attended their gp and the remainder either contacted the department or went to a chemist. in conclusion, many patients do not appreciate the entitlement of their gp's to prescribe psychotropics for them. literature regarding whether or not the social class distribution of patients with psychiatric illness may differ from the general population remains controversial. we sought to clarify this by examining social class at the time of birth, to see whether patients with serious psychiatric illnesses (schizophrenia and mania) differ from the general population. paternal occupation of 450 schizophrenic patients, and 77 manic patients, from the dublin psychiatric case register, were obtained from birth registration details and categorised according to central statistics office criteria, the same-sex previous live birth was used as a matched control. there was no difference between the social class of patients with schizophrenia or mania (p=0.32). neither patients with schizophrenia (p=b.31) nor mania (p=0.153) differed from controls in social class distribution. paternal social class was found to be related to amount of time spenr in hospital (p<0.001', mean=439.62), and educational age (p<0.001, mean=15.01) and "age at onset of the illness" (p=0.03, mean=31.59). these results suggest that social class of origin may not be related to the development of either schizophrenia or mania. however, social class of origin may be relevant in terms of presentation of schizophrenia for treatment. cognitive function is widely recognised to be impaired in schizophrenia but there is an ongoing debate as to whether this impairment is generalised or localised, progressive or static, similar in both sexes, or related to symptoms. using the positive and negative syndrome scale (panss)'we measured psychopathology in 48 chronic in-patients (27m, 21 f; mean age 68.0+12.7) who satisfied feighner criteria for schizophrenia. subsequent to this, we assessed their global cognitive function using the mini-mental state examination (mmse) and their frontal cognitive function using a new instrument, the executive interview (exit). poor performance on the exit was associated strongly with increasing severity of negative (r=-0.74, p<0.001) but not positive (r=--0.09, ns) symptoms,'in both males (r=--0.73, p<0.001) and females (r=--0.76, p<0.001). overall exit performance declined modestly with increasing age (r=--0.32, p<0.05) but this phenomenon was co'nfined to females (r=--0.64, p<0.01; males: r=--0.13, ns). mmse performance was also associated with negative symptoms (r=--0.67, p<0.001) but decreased mm:e prominently with age (r=--0.59, p<0.001) and showed no gender difference. frontal dysfunction in schizophrenia appears to be intimately related to negative symptoms over the course of severe chronic illness, and may reflect among males a more static' trait deficit than is accessed by the mmse. this study was supported by the stanley foundation. while determinants of the course of schizophrenia are unclear, emerging evidence suggests that the longer psychosis proceeds unchecked before initiation of anti-psychotic therapy, the poorer may be long-term outcome. we have reported that, among older in-patients, increasing duration of initially untreated psychosis in the pre-neuroleptic era was associated with a deterioration to a state of muteness (after controlling for intervening variables). the current survivors of this population have now been examined more extensively using the positive and negative syndrome scale (panss), the mini-mental state examination (mmse) and the executive interview (exit). among these 48 patients (mean age 68.0+12.7), after controlling for age and for the duration and continuity of subsequent antipsychotic treatment, increasing duration of initially untreated psychosis was associated with greater severity of negative symptoms (p<0.005) and with lower scores on the mmse (p<0.05) but not with executive dysfunction on the exit (p=0.3). increasing duration of initially untreated psychosis appears to be associated with the evolution of more prominent negative symptoms and cognitive impairment in a manner consistent with an active, morbid process in schizophrenia that can be ameliorated by anti-psychotic drugs. this study was supported by the stanley foundation and the health research board. patients who are selected and who agree to participate in the royal college examinations play an important role. as psychiatrists and exam organisers, we should be aware of the potentially stressful experience which this might present. the purpose of our study was to elicit attitudes to the exam, and also knowledge of the examination procedure. a questionnaire comprising 10 questions was circulated to 30 patients who had participated in the royal college examinations. responses were received from 28 (93%) of the 30 patients. there were 14 males and 10 females in the responding group. none of the patients had previously participated in the examinations. all of the respondents (n=28) felt that the candidate had been polite towards them during the interview. 38% (n=9) of the patients were nervous prior to the examination, and this group was predominantly female (n=6). 21% of the patients (n=5) did not know that they would receive payment for their participation. 67% (n=l 6) did not know that they might be physically examined as part of the examination procedure. 17% (n=4) of the patients described experiences which had been upsetting for them the results of our study suggest that on the whole patients tolerate the exam procedure quite well. one of the central issues concerning physiotherapists in stroke rehabilitation is the emergence of abnormal tone. rehabilitation involves re-establishing a normal postural control mechanism (ncpm)"~. abnormal tone may develop in the presence of severe sensory and proprioceptive loss. the patients' attempts to move and find a stable base can lead to compensatory movement patterns and asymmetrical postures. positioning is used by physiotherapists to influence the distribution of muscle tone and facilitate symmetrical postures. it is essential that the patient is made aware of his 'position in space' as failure to do so presents no feedback regarding movement resulting in inertia ~2~. standardised positioning charts have been used in hospitals. the physiotherapist liaises with nursing staff regarding correct use on a 24 hour basis. this study looked at the role of a more individualised approach to positioning in the form of a photograph. patients were randomised to two positioning groups group a standard vs group b photograph, and their positioning was scored by a 'blinded' research physiotherapist over an eight week period. the results of a pilot study on a small number of patients revealed that nursing staff preferred a positioning chart individually tailored to the patient's problems. from a physiotherapy perspective, improved postural awareness correlated with better positioning scores in group b. the prevention of compensatory movements, posture is critical in stroke rehabilitation, the use of an individualised positioning chart requires further evaluation. we discuss the case of a fourteen year old boy who presented with bilateral ptosis present since birth, microcephaly and pigmentory retinopathy"!. he was found to have mild facial and proximal limb weakness. creatinine kinase and ldh were raised. muscle biopsy showed ragged red fibres consistent with a mitochondrial myopathy ~2~. electron microscopy showed abnormal mitochondria. the term mitochondrial myopathy describes a diverse range of clinical disease ~3~ and this is discussed. she developed a vasculitic skin rash with pruritis and oedema associated adenopathy, low grade fever and mouth ulcers. lab tests showed leucocytosis, eosinophilia and abnormal liver function. skin biopsy indicated an inflammatory picture without vasculitis. ct thorax confirmed axillary and para-aortic adenopathy. lymph node biopsy confirmed a reactive lymphadenopathy. the aim of. this study was to assess the characteristics of patients referred for pudendal nerve studies over a one year period. 31 consecutive patients were asked a standard questionnaire and nerve studies were performed as described by kiff and swash m and swash and snook~2k of the 31 patients, only 3 were male. the age range was from 21 to 79 (mean 50). 10 presented with constipation and 19 with faecal incontinence. two had both symptoms. bladder incontinence was presented in 10 of 31 patients. of these, faecal incontinence was the cardinal symptom in 8 patients, constipation in 2. 12 patients were nulliparous. of the remaining 17, 14 had a history of complicated births involving forceps (7), caesarean section (4), post partum haemorrhage (1), breech without forceps (2) . 14 patients had pelvic surgery and one had major trauma. 22 of 31 patients had bilaterally delayed pudental nerve terminal motor latency (pntml). 7 of 31 people had unilaterally delayed pntml, 2 were right sided, 5 were left sided. 2 had normal studi~s. the range of measurements was 1.8 -9.2 ms with a mean of 3.65 ms. in conclusionl delayed pntml was seen in 11 of 12 patients with constipation and 19 of 20 with faecal incontinence. pelvic surgery and a complicated obstetric history were significant. urological symptoms were also a common association. the p50 component of the middle latency-auditory evoked potential is attenuated in response to the second of paired clicks in a normal population. in schizophrenia, this attenuation is minimal. in alzheimer's disease (ad), the results have varied between centres depending on the frequency of the stimuli and the interclick interval. we studied 10 ad patients, 10 elderly controls (ec) and 10 young controls (yc) using a paradigm of 32 sets of paired clicks. in contrast to previous studies, our study demonstrates significantly larger absolute p50 generation and recovery amplitudes in ad patients compared to elderly controls and young controls. the purpose of study was to establish a simple screening vol. 165, 1996 irish journal of supplement no. 2 medical science test to identify asymptomatic intracranial aneurysms (icas). an association between atherosclerosis and icas is recognised. elevated serum lipoprotein (a) [lp(a)] is an independent risk factor for atherogenesis. we aimed to assess the degree of correlation between serum lp(a) and the occurrence of sporadic ruptured aneurysms and familial asymptomatic aneurysms. lp(a) levels were measured in (a) 50 patients with icas and 42 normal controls, (b) 25 first degree relatives of patients with familial subarachnoid haemorrhage (sah). icas were detected by cerebral angiography. patients with sporadic icas had significantly elevated lp(a) levels when compared with matched controls. mean level was 20.1 mg/dl in patients and 10.8mg/dl in controls. in the familial studies, 10 out of i1 subjects with asymptomatic icas had elevated lp(a) levels. one young female with elevated lp(a) had a pre-aneurysmal dilatation at operation. six out of 14 subjects without icas had elevated lp(a) levels; four of these were in the second or third decade of life and may yet develop aneurysms. conclusions: lp(a) has potential as a biological marker for icas. follow-up studies are required on angiographically negative subjects. we have begun a genetic case-control study to establish if particular apoprotein (a) gene polymorphisms can be correlated with the occurrence of icas. post mastectomy breast reconstruction has undergone several changes in the recent years. attitudes have changed towards the problem from both the patient and the reconstructive surgeon, as aesthetic outcome receives a greater emphasis than previously. there is a shift towards using autologous tissue as a means of reconstruction; these new technically difficult procedures entail a longer learning. centralization of this type of reconstruction in highly specialized centres only will serve the patient better. we share our experience of post mastectomy breast reconstruction spread out over the past five years. seventy-eight consecutive cases of breast reconstruction are included in the study. different techniques of breast reconstruction were used with a recent switch to transverse rectus abdominis myocutaneous (tram) flap; we feet that tram flap is the gold standard of breast reconstruction as far as the ultimate cosmetic result is concerned. ours is only a moderate sized study compared to some published, yet it is representative of the experience of most of the plastic surgery units in the british isles. clinically significant paraneoplastic neurologicaldisorders are rare, most are associated with small cell lung, female genital tract and breast carcinoma. the malignancy is often silent and the neurological manifestations vary from encephalomyelitis, cerebellar degeneration, sensory neuropathy to neuromuscular block. prognosis is usually poor. pathogenesis is thought to be related to cross reaction with neurons of antibody produced to tumour antigens. detection of these antineuronal antibodies in serum has assisted diagnosis of paraneoplastic encephalomyelitis in which anti-hu antibodies are present and cerebellar degeneration, in which anti-yo are found. in our lab, we used avidin-biotin-complex immunocytochemistry to detect anti-hu (cortical neuron antibodies) and anti-yo (purkinje cell antibodies) in patients' sera. tests were performed on human frontal cortex and cerebellum, at 1 in 500 and 1 in 4,000 dilutions, with positive and negative controls. of 68 sera, 6 were positive. two patients had repeat positives; in one, antibody titre rose in the second sample. subsequent patient review showed 3 positives (2 patients) had identifiable carcinoma with paraneoplastic cns signs, 2 had no identifiable malignancy but had no other cause of their cns disorder and are being followed up; details of one patient were unavailable. these results are similar to other centres. the proliferation of tumour cells despite the presence of tumouricidal mediators could be due induction of a heat shock response, a universal cellular defence mechanism in host cells and possibly tumour cells. protection may be mediated either by increasing intracellular levels or surface expression of heat shock proteins (hsp). the aim was to assess the effect of heat shock induction on tumour cell protection against host effector cells. the heat shock response was induced in sw707 colorectal cells by either sodium arsenite (0-320~tm for 6hr) or by hyperthermia (42~ for 20 rain). monocyte (m0)-mediated cytotoxicity or flow cytometry to evaluate surface expression of hsp60 and hsp70 were assessed. cytotoxicity showed a significant decrease in all treated groups (p<0.002) when compared to the control value. there was also a significant decrease in all groups (p<0.001) when compared to the 42~ value. no significant alteration in surface expression of either hsp60 or hsp70 was seen. conclusion: heat shocking tumour cells significantly protects them from m0-mediated tumour cell lysis. since the flow cytometric data indicate that there is no concomitant increase in surface expression of hsp60 and hsp70 on the tumour cell following heat shock, it can be inferred that induction of intracellular hsp levels are responsible for the protective effect on the tumour cells. a 4 week qol study in 157 consecutive advanced cancer patients was undertaken to compare the subjective 28 question fact-g with simple subjective global tools (visual analog, categorical scales: vas, cas), objective tools (spitzer qli and ecog performance status) and verbatim patient description. we anticipated the high drop out rate enrolling 157 to achieve 87 complete study patients for statistical purposes. the study sample appeared representative of the advanced cancer population in the usa. generally qol was satisfactory despite the severity of illness. there were significant differences in all measures between those who described qol in verbatim responses as positive and negative, particularly cas, vas, and qli (p<0.0001). there were significant intercorrelations between qli and ps (observer rated), vas and cas (subject rated) respectively (p<0.0001). taking patient description as the gold standard, simple, global qol measures e.g. vas or cas are as effective as multidimensional ones (fact-g and qli). irish journal of medical science males had more dysphagia. survival from diagnosis was greater for females </=65 years (female median 18 months, male median 13 months, p<0.0016). anorexia was associated with reduced survival since diagnosis in the total population and in males. anorexia anddysphagia were associated with reduced survival from referral. advanced cancer patients were polysymptomatic. symptom prevalence differed with age, gender and cancer site. the pattern of g1 symptoms was related to gender and severe weight loss. specific symptoms were associated with reduced survival. there was a gender difference in survival favoring females. the administration of recombinant interleukin-2 (rll-2) is limited by the induction of increased microvascular permeability. the in vivo antineoplastic effects of taurine in combination with rll-2 were investigated and its impact on the associated vascular leak was examined. lung metastases were established in mice via tail vein injection. ten days after injection mice were randomized into treatment groups. on day 18 the mice were sacrificed; lungs were removed, weighed and metastases counted. treatment of tumour-bearing mice with rll-2 alone resulted in a significant reduction in tumour nodule incidence compared to a control group, while the~group recei'~ing rll-2 + taurine showed an even fuither reduction in the incidence of lung nodules. animals receiving rll-2 showed a significant increase in mean wet lung weight compared to control lung weight, while mean wet lung weight of the rll-2 + taurine group was significantly less than that of the rll-2 group.and comparable to control values. animals receiving ril-2 + taurine in vivo demonstrated significantly enhanced splenocyte-mediated antimelanoma activity ex vivo compared to animals receiving rll-2 alone. thus taurine may have an important role in modulating both metastatic growth and the associated toxicity of rll-2 immunotherapy. a prospective analysis of symptoms in 1,000 patients on initial referral to the palliative care service of the cleveland clinic was made using the paradox relational database. the median number of symptoms per patient was 11 (range 1-27). the 10 most frequent symptoms were pain 82%, easy fatigue 67%, weakness 64%. anorexia 64%, >10% weight loss 60%, lack of energy 59%, dry mouth 55%,'eonstipation 51%, dyspnea 51% and early satiety 50%. patients 65 years and under had more pain, sleep problems, depression, anxiety, vomiting and headache (all p<0.01 ).'the prevalence of early satiety, nausea, vomiting and anxiety were greater in females; dysphagia 3nd hoarseness in males. patients with >/= 10% weight loss had more gi symptoms; of these females had more nausea, early satiety; the progn.o'stic significance of abnormalities in the p53 tumour suppressor gene and in the expression of its protein in colorectal carcinoma may be influenced by the method of analysis used. we studied p53 abnormalities in 66 patients with colorectal cancer followed for more than 10 years. single-strand conformation polymorphism analysis (sscp) was used to detect alterations in exons 5-8 of the p53 gene. paraffin sections were examined immunohistochemically for p53 overe,xpression with the monoclonal antibody do-7 (dako) both with and without microwave antigen retrieval. abnormalities of the p53 gene were found in 41% of cases by sscp analysis but were unrelated to age, sex, tumour size or differentiation. outcome was unrelated to sscp abnormalities (p=0.19). overexpression of p53 protein was seen in 47% of cases by immunohistochemistry without microwave antigen retrieval and in 52% of cases with microwaving. poor long-term survival was related to immunohistochemical expression of p53 protein either with (p=0.03) or without (p=0.02) microwave antigen retrieval. these results suggest that immunohistochemical detection of the p53 protein product may be more useful than sscp analysis of the encoding p53 gene in identifying those at high risk of colorectal cancer recurrence and death. dublin 9. the anti-tumour activity of tumour infiltrating lymphocytes (tils) is known to be poor and therapeutic manipulation of these cells has met with little success. suppressor macrophages (smo) influence t cell cytotoxicity and proliferation. we hypothesized that smo are a component of the lymphoreticular infiltrate and that these cells may be related to lymphocyte numbers within the tumour. tgf-b may influence macrophage phenotype. colorectal and breast tumours were obtained within an hour of resection. tumours were dissaggregated with collagenase and dnase for three hours. antibodies was used to identify smo (rfd 1 and rfd7) and t cell subsets (cd4 and cd8) by flow cytometry on the resulting cell suspension. pre-op blood was collected from patients and tgf-b levelsdetermined by elisa. conclusions: we have shown for the first time that smo, defined by the antibodies rfd1 and rfd7, are present within breast tumours. we have also shown that the balance of t cell subsets is different in these tumours and may be related to smo content. circulating tgf-b levels are increased in breast cancer and associated with greater smo numbers. this was not found to be the case in colorectal cancers. these results imply the existence of a fundamental difference in the make-up of the lymphoreticular infiltrate between these cancers. swelling of the upper limb is an uncommon but well irish journal of medical science recognised complication of breast cancer treatment. in severe cases, patients have limited arm function and feel disfigured. in a pilot study, the incidence of arm swelling following complete axillary clearance in the immediate post-operative period and at long term follow-up was investigated. arm volume measurements were performed using an opto-electronic volometer (bosl medizintechnick, hamburg). both ipsilateral and contralateral arm volumes were assessed. the expected volume of the ipsilateral arm volume was calculated using the formula vr = v1 = 31 mls for right handed people and v1 = vr = 25 mls for left handed people (vr and v1 = volume of the right and left arms respectively). the difference between the expected and actual volume of the ipsilateral arm was expressed as a percentage of the expected volume. twelve patients undergoing axillary clearance for breast cancer were prospectively evaluated pre-operatively, 48 hours and 5 days post-operatively. a second group of 48 patients who had had axillary clearance at least 3 months previously (range 3 to 116 months) were also evaluated. there was no significant change in arm volume in the immediate post operative period. clinically detectable arm welling was found on 3 patients who had undergone axillary clearance at least 3 months previously but none had any impairment of arm function. we conclude that axillary clearance can be performed safely and that arm swelling is an uncommon complication. a larger study is planned to investigate factors such as the influence of pectoralis minor division, duration of the operation and the number of axillary nodes retrieved on upper limb volume. epidermal growth factor (egf) is a potent mitogen and has been shown to accelerate healing of epithelial damage both in the skin an.d the gut. in the skin egf is not produced locally as the requisite mrna is not present but egf receptors are present on the surface of basal keratinocytes. egf is produced in various sites in the gi tract including the submandibular salivary glands. we have hypothesised that as there is upregulation of salivary egf production in some enteropathies a similar situation may occur in disorders of the skin with an associated enteropathy. using a sensitive radio-immunoassay, egf activity was estimated in stimulated saliva from 87 patients with various skin disorders, 49 patients with gastrointestinal disease, 8 patients with mixed dermatological and gut disease and 41 normal healthy volunteer controls. elevated egf activity was found in the following groups of patients : skin cancers, psoriasis, acne, oesophagitis and ulcerative colitis. the hypothesis of up-regulation of salivary egf production in skin associated enteropathy was rejected but the discovery of elevated egf activity in skin cancers and psoriasis may have aetiological and therapeutic implications. the malignant fibrous histiocytoma (mfh) is considered an uncommon malignancy. its potential for invasion, metastasis and death of patient has been reported in literature. it can be confused with other tumours including fibrosarcoma. salient histologic features include cells of both the fibrocytic and histiocytic series. mfh with its high recurrence rate and lethal potential merits an aggressive evaluation and treatment. we present unusual case of recurrent mfh treated in our unit with an open question as to what qualifies to be adequate primary surgical excision. the recommended management of localised merkel cell carcinoma has been wide surgical excision, combined with adjuvant radiotherapy in selected cases. the risk of recurrent regional disease is reported to be between 30% and 45%. a 70 year old woman with merkel cell tumour on the cheek is presented; this patient was treated exclusively with radiotherapy to a total dose of 50 gy over 18 days. the tumour regressed rapidly during the treatment, and there were no signs of local or regional recurrence. the patient is still alive and free of disease for 45 months. immune in origin with a heightened cutaneous immune response to ultraviolet light. the coexistence of cad and pbc is a new association which has not previously been documented and may not be fortuitous given the similar pathogenesis of both diseases. chronic actinic dermatitis (cad) is a rare photosensitive disorder which primarily affects elderly men resulting in an eczematous reaction to ultraviolet-radiation and sometimes visible light. the pathogenesis has been attributed to an autoimmune process, possibly in response to a photoallergen which has yet not been identified. we report a 71 year old female patient who developed cad four years after being diagnosed with primary biliary cirrhosis (pbc). abnormal monochromator irradiation tests were detected with narrow band ubv, uva and in addition visible light wavelengths. phot0provocation tests induced florid vesicular eczema and multiple patch and photo-patch tests were positive, findings typical of cad. immunoglobulin g was elevated at 2290 mg/dl and liver histology was typical of pbc with an elevated anti-mitochondrial antibody. routine biochemical and immunological tests were ~ormal and porphyrin screen was negative. azathioprine 50 mg/day induced remission of cad. pbc is an auto-immune disorder where cell-mediated immunity is impaired, suggesting that sensitized t lymphocytes may cause damage to bile ducts. the pathogenesis of cad may be autowe present two cases of cutaneous polyarteritis nodosa (pan) associated with seronegative arthritis: the first patient, a 50 year old male presented in 1993 complaining of a7 year history of pain, stiffness and swelling affecting his right ankle. he also noted intermittent tender nodules on the dorsum of his foot and over his ankle over the preceding three years. the second patient, a 51 year old male, presented .in 1994 complaining of a 5 month history of tender nodules on his shins, and pain and swelling of his right ankle. skin biopsies of the nodules in both cases showed medium vessel vasculitis consistent with polyarteritis nodosa. neither patient had any symptoms or signs to suggest systemic involvement. the only abnormality.0n laboratory investigations for vasculitis was elevated esr. x-rays showed periosteal elevation and new bone formation in case 1, and were normal in case.2. bone scan demonstrated increased uptake at the talo-navicular joint in case 1 and at the fight ankle in case 2. synovial biopsy and mri confirmed the presence of an inflammatory arthropathy in patient 1. joint involvement has been a prominent feature throughout the course in both cases requiring aggressive treatment with vol. 165, 1996 irish journal of supplement no. 2 medical science cyclophosphamide and systemic corticosteroids in case 1. cutaneous pan is a localised cutaneous vascular disorder with a benign chronic relapsing course. in one reviewl, 12 of 23 patients had arthralgias but an association with arthritis has not been emphasized in the literature to date. we conclude that this condition may present as a seronegative arthropathy in which the joint symptoms may be the most prominent feature and aggressive immunosuppresive therapy may be required for control. cardiac transplantation patients have an increased risk of skin disease. in our centre, 119 heart transplants were performed with a 5 year survival of 75%. eighty three patients are now alive and 39 have required dermatological assessment. the mean age of patient was 48.3 years, (range 11-64 years); 99 males, 20 females. skin infections were diagnosed in 13 of 39 patients. drug side effects, including sebaceous hyperplasia and steroid acne, were common. in the patients who developed skin cancer, mean time from transplant to development of lesions was 3.1 years. eleven of 36 patients had 32 non melanoma skin cancers (nmsc), 28 squamous cell carcinomas (scc), 4 basal cell carcinomas (bcc), giving scc/bcc ratio of 7:1. three of 11 patients had multiple skin cancers, one had 12 tumours. nine of 39 patients had actinic keratoses, two thirds of whom had sccs. nineteen of 39 patients had viral warts, two of whom had sccs. viral warts, premalignant and malignant lesions were located on sun exposed sites. skin complications of cardiac transplantation though mild were very common. the observed incidence of nmsc in age matched cardiac transplant recipients, appears much higher than the expected incidence of 171.38 per 100,000 population (national tumour registry 1994). regular dermatological assessment of cardiac transplant patients is necessary to detect skin disease and early skin cancer. the increased incidence of warts and skin cancer in renal transplant recipients {rtr} is well known. the oncogenic potential of unusual human papilloma virus {hpv} types has been postulated from warts and in both premalignant and nonmelanoma skin cancer (nmsc). the possible etiological role of sun-exposure in facilitating the development of hpv associated skin disorders is also suggested. a clinical study to assess the risk factors for development of these lesions in 50 rtr attending the dermatology servic& age and sex matched haemodialysis patients were similarly examined as controls. 40 male and 10 female patients with a mean duration of transplant of 10.6 years, range 1 to 25 years. a total of 191 nmsc (range 1 to 57),175 scc and 16 bcc, ratio 10.9:1, were excised from 26 rtr of which over 95% had viral warts, mosle commonly occurring on sun exposed sites and always predated the development of neoplastic lesions. both were associated with mean duration from transplantation, 4 years for warts and 10.8 for skin cancer and not the type of immunosuppressive treatment. none of the control patients had similar findings. conclusions: the close clinical association of viral wart lesions and development of skin cancer in these patients suggests a close relationship to immunosuppression, in addition to exposure to ultraviolet radiation. this study highlights the high rate of nmsc in rtr. these patients justify early and regular skin assessments soon after transplantation with advice on sun protection. sensitivity to ultraviolet (uv) light may be established by exposure to broad band uva and uvb radiation. the minimal erythema dose (med) can be determined at individual wavelengths using a monochromator. uv action spectra of photosensitive disorders may thus be constructed. we examine the value of this process in distinguishing two clinically similar photosensitive disorders. the radiation from a xenon arc is separated into component wavelengths using the monochromator. each wavelength is focused on unaffected skin, on the patient's back. the patient is exposed to a range of doses of w radiation. the med is determined for a series of wavelengths from 300 to 400 nm. chronic actinic dermatitis (cad) and drug induced photosensitivity are photosensitive disorders which may have similar clinical history and presentation. ten cad and 14 drug induced photosensitivity patients were tested. uva photosensitivity was seen in 71% of the latter group. the remaining 29% had normal mlts as the implicated drug had been discontinued prior to testing. cad patients were sensitive to both wa and wb radiation. forty-three percent of these patients were also sensitive to visible light (400 to 800 nm). monochromator light test (mlt) results show that uva photosensitivity dissociated from wb photosensitivity is indicative of a drug induced light sensitive disorder. sensitivity to both wa and wb however indicates a diagnosis of cad. mlts can therefore distinguish between clinically similar photosensitive disorders. bartholomew's hospitals, london. patients with mpd have an increased incidence of both thrombosis and haemorrhage suggesting a pivotal role for; platelets in these conditions. this study aimed to examine platelet activation antigen expression in stable patients with mpd and to examine the predictive value of these antigens prospectively. 52 patients with mpd had p selectin and gp53 measured using a refined minimally manipulative flow cytometric technique. expression of p selectin -median 5.1% (inter quartile range 2.1-10.9), control 2.0% (1.3.-4.0) and gp53 -median 4.0% (1.8-5.7), control-2.1% (1.0-2.9), were significantly elevated p<0.01. patients were followed for a median of 26 months. 15% experienced thrombosis and 6% bleeding during follow up. at entry to the study 48% of patients had previously experienced thrombosis, median disease duration 8 deaths, 3 of which were caused by thrombotic events in which the mpd was a major risk factor. increased expression of p selectin or gp53 expression failed to predict thrombosis or bleeding in this study. nor was any significant retrospective relationship demonstrated. however, previous thrombotic events were strongly associated with future events (p<0.05). this association was independent of disease, duration, age and medication. not surprisingly disease duration was also correlated with thrombotic/bleeding events. taurine levels fall in gut mucosal cells during critical illness. however, taurine transport into human intestinal cells is poorly understood. the aim was to establish the efficiency of taurine uptake by enterocytes, and to examine uptake under stressful conditions. to investigate efficiency of taurine uptake, confluent caco-2 cells were incubated for time points up to 10h. in a second study, cells were incubated for 9h with medium containing dexamethasone and / or cytokines. media for both studies was supplemented with [3h]-taurine. radioactivity was related to mg/ml protein to calculate rate of taurine uptake for each time point. study 1: uptake exhibited a steady linear response which approached saturation at 7h. maximal uptake occurred at 9h after which the rate levelled off. study 2: dexamethasone alone reduced taurine uptake by 75.4% (p<0.001) and in combination with tnf-c~ and ifn ~/ it decreased transport by 66.3%. (p<0.001). lps alone impaired uptake by 56% (p<0.001). conclusion: we have established the time course over which taurine transport reaches its maximum rate in caco-2 cells, and that corticosteroids and cytokines significantly impair uptake of taurine in these cells. elderly individuals have an increased risk of infection suggesting that immune responsiveness is altered with age. changes in the level of proinflammatory cytokine production may be an important indication of any such age related change. using flow cytometry we examined intracellular tnfet, il-lf~ and il-6 in pbmcs from normal healthy volunteers of different ages ranging from 22 up to 85 yr (n=28). tnf and il6 levels from pma stimulated cd3 positive cells (t cells) were shown, using this technique, to increase in an age dependent manner (p<0.05). no il-113 was detected in any t cell sample. no significant differences were observed between the different age groups for tnfa, il-1 g or il-6 in cd 14+ cells (monocytes). the age related changes detected by flow cytometry have been confirmed using conventional elisas. this novel method of proinflammatory cytokine detection has detected increased tnf and il6 levels in t cells from elderly healthy volunteers which may help explain some of the exaggerated inflammatory responses seen in elderly patients. detection of proinflammatory cytokines by conventional elisa or bioassay is problematic due to the presence of naturally occurring biological inhibitors. flow cytometry allows the simultaneous detection of both intra and extracellular antigens thus intracellular cytokine levels can be quantified while cell surface markers allow cell type identification. a range of monoclonal antibodies were examined for tnfcx, il-lg and il-6 using saponin permeabilisation oft cells (cd3), monocytes (cd14) and epithelial cells (ber-ep4). t cells and monocytes were grown in ~culture, t~p :to 72 hr with or without pma activation, and intracellular cytokine levels were shown to increase with time, with the stimulated samples producing more cytol<ine than the spontaneous.samples (p<0.05). elisas performed on these same samples (n--:24) correlated well with the flow cytometric results (r=0.52). tnf~x, il-113 and il-6 are detectable !n monocytes while only tnfa and il-6 have been detected in tcells and epithelial cells using this methodology. this novel technique will allow us to identify the cytokine producing cell while also avoiding the influence of the extracellular millieu, thus being useful for a number of research and diagnostic applications. all the viral hepatitides are said to be common in the middle east. hepatitis a viru s (hav) seroprevalence is said to be 100% in the adult population of gulf countries. hepatitis b, c and d seroprevalence are taken from blood banks data which are biased by selection criteria and the background of blood donors. very little is known about hev in the uae. vol. 165, 1996 supplement no. 2 irish journal of medical science the aim was to investigate the seroprevalence of hav, hbv, hcv, hdv & hev in the uae. we prospectively recruited volunteers drawn from all the uae, socioeconomic classes and from different age groups in both sexes. each donated 10ml. of venous blood and using an indirect enzyme immunoassay were tested for total ig to hav; hbsag using monoclonal antibody t.o hbsag; for hcv antibodies using recombinant antigens hc-34, hc-43, c-100 and ns-5, (3rd generations); for total ig to hdv and for total ig to hev. positive samples were retested in duplicate for confirmation. this large sero-epidemiological study clearly shows that hav is decreasing in the uae. hbv is low but significant, hcv is similar to southern european countries, hev is lower than expected and hdv does not exist in hbsag carriers in the uae. reactive oxygen and nitrogen species produced by inflammatory cells plays a role in tissue injury and inflammation. taurine, a bamino acid present in high concentrations in leukocytes functions as an anti-oxidant by scavenging the mpo derived oxidant, hypochlorous acid, to form taurinechloramine. experimental colitis was induced in male sprague-dawley rats and randomised into two groups. one group received tap water whilst the second group was supplemented with taurine (4%). these groups were sub-divided into those receiving tnbs/ ethanol (n=8) or saline (n=8). an increase in wcc count, particularly neutrophils was found in colitis. tat~rine was found to reduce the severity of the disease (ns) usin.g a colon macroscopic score. in the first group, respiratory burst activity was reduced in animals that had developed colitis (p<0.05) although mpo activity was augmented (p<0.05). nitric oxide production was 2.2 and 1.8 fold respectively higher in inflamed and non-inflamed areas of the colon than that by normal colonic mucosa. animals supplemented with taurine demonstrated attenuated m po activity (p=0.001). administration of taurine did not lead to a reduction in nitric oxide production. thus, taurine appears to have a beneficial role in reducing the severity of disease. a reduction in mpo and respiratory burst may be beneficial in reducing tissue damage. to women subsequently identified by a national screening programme which revealed 438 to be positive for rna of hepatitis c, type lb. 94 were referred to this centre for evaluation. 10 had moderate activity by modified knodell index, mean 8 (_+1.4), bridging fibrosis and a mean alt of 77 (42-176). six months dual therapy was administered. two patients withdrew due to depression (2) and hyperthyroidism (1). one patient di d not respond. seven responded with normalisation of alt and negative pcr. eight patients showed improved histology (kai 4 +_ 1.5), fibrosis was unchanged. adverse reactions in the treated group included marrow suppression (1) and thyrotoxicosis (1) . at six months follow up four remain in remission with normal alt negative pcr, three have relapsed, (pcr positive, mean alt 87(50-150). we conclude that despite adverse prognostic indicators in this homogenous group with chronic hepatitis c, lb and long disease duration, dual therapy with interferon and ribavirin achieved results comparable to prolonged high dose interferon in patients with more favourable initial prognoses. the aim of the study is to evaluate a 5 day course of azithromycin in combination with omeprazole in eradication of h.pylori. twenty-four patients with proved h.pylori infection and peptic ulcer disease were treated with omeprazole 40 mg daily for 2 months plus azithromycin 500 mg daily for 5 days. h.pylori was diagnosed on the basis of clo test and histology. this was done at the beginning and 3 months after the treatment. eradication was defined as negative clo test as well as negative histology. out of 24 patients, 2 patients were lost to follow-up. only 21 patients were analysed. sixteen patients responded to treatment and had successful eradication as well as healing of their ulcers. these included 8 patients with gastric ulcers (79.95%), 4 patients with duodenal ulcers, 2 patients with gastric and duodenal ulcers and 3 patients with non-ulcer dyspepsia. five patients did not respond including 2 with non-ulcerative dyspepsia. 2 duodenal ulcers and 1 gastric ulcer. however their ulcers have healed and symptoms relieved and they were treated with other h.pylori eradication regimes. conclusion. in this small study we have shown that azithromycin 5 mg oncedaily for 5 days is an alternative safe effective and well tolerated monotherapy for eradication of h.pylori. it has been suggested that hepatitis c virus (hcv) infection is associated with a high prevalence of autoimmune disease, but that this risk may be diminished in patients who received a low viral load at initial infection. the aim was to determine the prevalence of symptoms, signs and markers of autoimmune disease in a group of women vol. 165, 1996 irish journal of supplement no, 2 medical science infected with hcv contaminated anti-d immunoglobulin. 50 patients, mean age 44.3 years (range 32 -65 yrs) were examined. 7 patients (14%) were rheumatoid factor positive. ana was positive in 6 patients (12%) but titres were 1/10 in 4/6. thyroid globulin antibodies were found in 3 patients (6%) with 1 also being tm positive. 2 further patients were positive for thyroid microsomal ab. prevalence of apa, ara, sm were 4%, 2% and 2% respectively. one patient had cryoglobulins detected while antibodies against mitochondria and lkm-i were not found only one patient of 50 was symptomatic. these levels of antibodies are no greater than levels in the general population and similar to other groups infected with hcv contaminated anti-d. these results further support the hypothesis that either low viral innoculum or long duration of infection may in some way result in a low level of autoimmunity. a previous study (ilan et al, arch int. med., 1993; 3: 1588-1592) suggested that lga deficiency disposes towards hepatitis c virus (hcv) infection in some patients. it has also been suggested.that iga deficiency may occur as a result of the viral infection as a secondary event. the aim was to determine the prevalence of selective and partial lga deficiency in patients with hcv infection using a radio-immunodiffusion technique. serum lgg and igm levels were also examined. immunoglobulin levels were measured in 67 patients, 59 women and 8 men, mean age 45.3 years (range 29 -72 years) infected with blood between 1977 and 1990. none of the patients were found to have selective or partial iga deficiency. mean iga = 2.59 g/l (range 0.86 g.1 -5.19 g/l). furthermore no patient exhibited deficiency of igg or igm. conclusion : no evidence of iga or other immunoglobulin deficiency exists among this group of patients with hcv infection. while it may be possible that lga levels decrease following infection, no evidence exists that this phenomenon persists in the long term. hereditary non-polyposis colorectal cancer (hnpcc) is the most common form of hereditary colon cancer and accounts for up to 10% of the overall cancer incidence. the disease is inherited in an autosomal dominant manner andis characterised by predominantly right-sided tumours. hnpcc is caused by mutations in one of four mismatch repair genes. these genes are homologues of the bacterial mismatch repair systerfi and their function is to detect and correct defects in dna. the hmsh2 gene is reported to account for up to 60% of cases, hmlhi, hpms1 and hpms2 account for 30%, 5% and 5% of cases respectively. we have collected eighteen irish hnpcc families which satisfy the amsterdam criteria for the disease. we have screened the entire hmsh2 gene in eight of these families. a combination of single strand conformational analysis (sscp) followed by dideoxy sequencing was used to detect defects in the dna. sequence analysis revealed the presence of a novel polymorphismin exon 5 in one family and a possible splice site mutation in the remaining ten families, using a novel mutation detection technique. departments of medicine and immunology, university college hospital, galway. serial iga gliadin antibody (igaga) studies are used as a surrogate for repeated small intestinal biopsy to monitor dietary compliance in treated coeliac patients. we have correlated igaga levels with indices of hyposplenism (platelets and erythrocyte "pits") to determine if either index can be used to monitor adherence to a gluten free diet. the study population of 129 biopsy proven coeliacs was stratified according to dietary status into 114 gluten free diet (groups a) and 15 normal diet (group b). the iga gliadin antibody titres were significantly lower in group a than group b (44.5 : 126 units p = 0.007). in group a gliadin antibody titres showed a statistically significant correlation with both platelet (p = 0.039) and pits count (p --0.04) ; in group b gliadin antibody titres correlated with platelet counts only (p -0.003). those ona gluten free diet were further subdivided according t ~ reported degree of adherence to the diet (strict diet -sgfd, lax'diet -lgfd). significantly higher gliadin antibody titres were found in lgfd than sgfd (103.6; 27.9 p = 0.003), but the ptatelet and.pit counts were similar in the two groups. igaga is superior to platelet or pit coums in monitoring dietary'compliance in coeliac disease. hospital, galway. iga endomysial antibodies (igaea) are said to be more specific and sensitive indicators of coeliac disease than iga gliadin antibodies (igaga). initially the igaea substrate was monkey oesophagus and more recently umbilical cord. a new commercial product has becomeavailable using primate smooth muscle (accu track -diagnostic slides). we have undertaken a preliminary study to compare the performance of this lgaea assay and igaga elisa in treated and untreated coeliacs, coeliac relatives and controls. the study population consisted of 127 biopsy-proven coeliacs who were stratified according to "reported" dietary status into 89 good gluten free diet (group a), 24 poor gluten free diet (group b) 14 normal diet (group c) and 10 unaffected coeliac relatives (group d) and 5 controls (group e). elevated igaga (>25 units) were found in the following groups : a 37%, b46%, c 50%, d 20%. igaea were positive in a 27%, b 33%, c 50%. with respect to igaga positivity, there were 11 false positive igaeaa 18%, b 12.5%, c 14% and 23 false negative -a 8%, b 8%, c 14%, d 20%. in this preliminary study in untreated coeliac patients the performance of the igaea test was on a par with the igaga assay. alt and fibrosis may be associated with a non-alcohol steatohepatitis and these processes may be synergistic. finally, number and type of riba bands is not a predictor of inflammatory activity. ~stepping hill hospital, stockport, uk sk2 7je. 2royal oldham hospital, rochdale rd., oldham ol1 2jh. we investigated upper gut bleeding in patients aged 75 years and over. a proforma addressing demography, drug therapy, clinical status, timing of endoscopy / surgery, and outcome was used. 109 consecutive patients (median age 80 years, range 75-92) were studied over 6 months. 106 (97%) underwent gastroscopy with a 97% diagnostic yield. 32 patients had severe oesophagitis, 2 had oesophageal malignancy, 29 had gastric ulcers -5 of which are malignant and 23 had duodenal ulcers. ulcerogenic drugs were implicated in 52 patients. 38 patients were referred for surgery, 8 operated upon with one postoperative death. 51 had haemoglobins of 10g/dl or less. all malignant lesions were inoperable. the overall mortality was 6% reducing to 2% if neoplasla were excluded. co-morbidity influenced mortality. 92 patients were discharged with a median hospital stay of 15 days. information on cause of bleeding greatly influenced management. the prognosis of gut haemorrhage in the very old need not be so poor. a few require surgery but the majority respond to active medical resuscitation which is a key factor in determining outcome. we advocate low threshold for endoscopy, judicious use of ulcerogens and adherence to guidelines on management of upper gut haemorrhage. haemochromatosis (hh), a common recessively inherited disorder of iron metabolism is closely linked to the hla-a locus on chromosome 6. linkage studies have demonstrated a close association between (hh) and the hla alleles, a3 and b7 ("ancestral haplotype"). heterogeneity at the molecular level may account for the variance in clinical phenotypic expression. the aim was to evaluate phenotypic expression of hh in the presence/absence of the a3-b7 ancestral haplotype. 32 probands (26m:6f) from unrelated irish families were investigated. phenotypic variability was assessed with regard to l) age; 2) % trans.sat.; 3) serum ferritin; 4) liver bx iron grade; 5) body iron stores and 6)symptomatology. three males were homozygous for a3b7, 15 were heterozygous for a3b7 and 2 were non-a3b7. symptomatology, trans, sat., serum ferritin and liver bx grade were not influenced by homozygousity or heterozygousity for a3b7. conclusion: there were no significant differences in phenotypic expression on comparison of the three haplotype groups. no predominant genotype appears to be responsible for phenotypic severity in irish families indicating the possibility of multiple mutagenicity of the hh gene. of 1013 riba positive anti-d associated chronic hepatitis c patients, 438 were pcr positive but had surprisingly mild disease. the disease status of the pcr negative patients was hitherto uncertain and is the subject of this study. 20/72 riba positive patients referred to this centre were biopsied because of elevated alt (4) or florid symptoms which dated from inoculation (16). 1 2 3 4 histological activity index * 5,1,0,1 3,2,5(f), 2(f), 0,4(f), 2,1,2 0,2,1,2 2,2,3 no bile duct damage, lymphoid follicles or aggregates was observed. 3/20 had mild periportal fibrosis (f), 2 of these had steatohepatitis with obesity (1) and impaired glucose tolerance (1) suggesting dual pathology. we conclude that riba positive, pcr negative patients have minimal disease activity. elevated the association between the hla locus and haemochromatosis (hh) has allowed early identification of affected siblings. it is unclear what proportion of subjects who are predicted to be homozygous or heterozygous for the disease by hla typing develop the disease. studies correlating clinical features with hla type in families from ireland -a putative source of this celtic trait have not been described. the aim was to correlate clinical, biochemical and pathologic features of hh with hla typing in 67 first degree relatives of 12 probands. initial analyses identified 12 homozygous (hh), 40 heterozygous (hn) and 15 normal (nn) individuals. however, 11/40 hn individuals had stainable iron on liver biopsy, confirming hh. further hla analysis revealed 7 homozygous x heterozygous matings and identification of all disease haplotypes within each pedigree allowed final classification of 30 hh, 25 hn and 12 nn individuals. vol. 165. 1996 supplement no. 2 conclusion: this study demonstrates the importance of hla typing in the clinical management of families with hh, furthermore, in multiply affected families the incidertce of homozygous x heterozygous matings is high indicating the high degree of "pseudodominance" in the irish population. the degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (hc) damage and cell death resulting from either necrosis or apoptosis. we have previously demonstrated that tnf-ct and lps can directly lead to hc necrosis, but not apoptosis. recent, studies have shown that reactive oxygen intermediates (roi) and nitric oxide (no) are capable of inducing apoptosis in eukariotic cells. however, it is unclear whether roi or no are involved in hc cell death. the aims of this study were to evaluate the role of no and roi in hc cell death (apoptosis vs necrosis). hcs were isolated from sprague-dawley rats, and cultured with the no donor, sodium nitroprusside (snp) or the roi generation system, hypoxanthine-xanthine oxidase (hx-xod) and h202. the effect of lps, tnf-t~, and ifn-y alone or in combitmtion with different antioxidants and the no synthase inhibitor, n-methyl arginine (nma) on hcs was also assessed. snp caused a dose-dependent increase in hc apoptosis. roi generated by hx-xod and h202 did not induce hc apoptosis, but were responsible for hc necrosis. tnf-ct alone failed to induce hc apoptosis, but when ~combined with antioxidants resulted in increased hc no production and apoptosis. this effect was attenuated by nma. snp also induced hc damage and hc necrosis. moreover, tnf-ct-mediated hc damage and necrosis could be further reduced by the combination of antioxidants and nma. these results indicate that roi preferentially induce hc necrosis, but not apoptosis. induction of no resulted in both hc apoptosis as well as hc necrosis, which suggest that overproduction of no may be detrimental during the sirs. irish journal of medical science intervention was not uniform. mean albumin was 34.3g/1. mean weight was 57.8kg. poor cognitive status greatly increased the requirement for dietetic consultation time. lack of dietetic resources results in inadequate monitoring of these patients following discharge. this study highlights the need for a dedicated clinical nutrition service, for medical services for older people. periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. the preventative strategy of universal food fortification with folic acid presents the possible risk of masking the diagnosis of cobalamin deficiency in pernicious anaemia. in addition, the ultimate longterm effect of universal exposure of adult or foetal cells to a synthetic substance, ie. folic acid, is unknown. in this study, the threshold oral dose of folic acid in a number of foods above which metabolically-unaltered vitamin appeared in serum postprandially was determined in a young and elderly population by microbiological assay of serum pre-fractionated by hplc. subjects on a five-day regime of fortified cereal and bread along with their normal unfortified diet. were shown to have a threshold level of 400~g/d, abovewhich unaltered folic acid appeared in the serum. individuals given folic acid in either isotonic saline, milkor white bread exhibited a threshold level of 200~tg per serving. from patterns of food consumption in ireland, even moderate levels of fortification are likely to lead to some population groups being exposedto excessive amounts of un-altered folic acid in serum. many older people are nutritionally compromised. there is clear evidence that: nutrition intervention reduces morbidity m and mortality in older patients. to identify the spectrum of nutritional abnormalities referred for dietetic intervention and the problems associated with nutritional assessment, 50 elderly patients were alphanumerically selected from files of the department. of nutrition and dietetics. the most common dietetj.c interventions were: use of supplements 60%; high protein high calorie diet 46%; nasogastric feeding 26%; reduction fat 8%; iron/thiamine assessment 6%; high fibre diet 6%; diabetic diet 4%; nutrition swall6w programme 2%; lipid lowering diet 2%. some 60% referred required nutritional supplements, but the profile of an increase in oxidative stress in cystic fibrosis patients has been suggested. activated neutrophjls in the presence of chronic lung inflammation in addition to increased activity of the electron transport chain in cfmay. increase free radical generation. antioxidant protection against free radical attack is likely to be compromised as a i'esult of deficiencies in fat soluble antioxidants vitamins. in the present study stimulated thiobarbituric aoid reacting substances (tbars) were measured to determine the ability of plasma to withstand lipid peroxidation. copper was used to in!tiate the breakdown of lipids to lipid hydroperoxides and eventually to aldehydes, mainly malonyldialdehyde (mda). pooled cf plasm a and pooled control plasma were incubated for 0, 30, 60, 90, 140 and 200 min. mda complexes with thiobarbituric acid which absorbs at approximately 535 rim. there is a lag phase where antioxidartts vol. 165, 1996 irish journal of supplement no. 2 medical science in the plasma or tissue protect against lipid peroxidation, then a log phase where the protective effect is overcome and finally the reaction reaches a plateau when lipid peroxidation is complete. absorbance at 532 nm was measured in all samples and zero order and first derivative spectra were obtained. the lag phase appears to be longer in the pooled cf plasma compared with controls. plasma t~-tocopherol levels were within the normal range in both groups, indicatin~ an alternative protective effect in cf. mild hyperhomocysteinaemia is an established risk factor for heart disease. a source of homocysteine in humans is the essential amino acid methionine found in protein of animal origin. in an 8-week study weekly fasting plasma homocysteine levels were examined in a group of healthy male subjects (n=6) under normal dietary conditions (weeks 1 to 4) and in response to graded increased methionine intakes (weeks 5, 6, 7). nutrient intakes, including methionine, were calculated from 4x3-day food records. under normal dietary conditions weekly mean plasma homocysteine levels were not significantly different (anova) from each other ranging from 6.82+1.77 to 9.42+2.73~tm/1. doubling daily methionine intakes (supplementing with 25mg/ kg/d) did not result in a significant increase in plasma homocysteine (8.56+3.68~m/1), however, significant increases were achieved when diets were supplemented with methionine at levels of 50 and 75mg/kg/d resulting in mean plasma homocysteine levels of 13.37+5 9 and 18.051am/1+11.08, respectively. mean plasma homocysteine levels returned to baseline (8.76 + 3.42 ~tm/l) 10 days post supplementation. we conclude that supplementary methionine results in a significant increase in plasma homocysteine only when levels of five times the normal dietary intake are reached. this study is evaluating the use of a synthetic construct which encompasses primer binding sites for lpl and a variety of cytokine and other transcripts, to quantify lpl expression in cultured human monocyte-derived macrophages. following isolation of total rna at various times during cell culture, its reverse transcription (rt) using random hexamer primers generating first strand cdna and specific amplification of lpl cdna targets by polymerase chain reaction (pcr), generates products identifiable on gel electrophoresis. quantitation of message is obtained by incorporating the pawl08 construct in the rt assay in varying quantities as an internal standard with known amounts of monocyte-macrophage rna (l~tg). pcr amplification of this construct yields size distinguishable products from that produced by the monocyte-macrophage lpl cdna transcript. pcr conditions for the assay have been optimised at 29 cycles of denaturation (tmin @94~ annealing (1.5min@55~ and extension (lmin@72~ lpl mrna has been detected in cultured monocytes and macrophages throughout their differentiation. also increased expression of monocyte lpl mrna has been observed following 15 hr incubations with chylomicrons (30t~g/6x 106 mononuclear cells/ ml)-when compared with controls. interestingly, little or no lipase mrna was detectable in circulating monocytes using identical pcr conditions to preparations of mrna from the day 4 and day 8 cultured cells. this methodology will now permit investigation of the factors controlling lpl expression in cultured human monocytic cells. replacement growth hormone (gh) therapy in adult hypopituitarism is attracting increasing interest. in 1992 markussis (l) detected premature atherosclerosis by ultrasonography in the untreated patient 9 we have shown plaque regression with patients on replacement gh (norditropin) in a 4-month trial. 11 females and 9 males were recruited, mean age 49.6 years. at each timepoint plaque characteristics were measured by duplex ultrasound. 6 patients showed a large reduction in plaque size (mean 21%) after four months treatment (p value < 0~00l). similarly, highly significant values in cholesterol, hdl and ldl and apo a1 are achieved. chol hdl ldl apo a 1 pretreatment 6.54 1.23 5.04 119 posttreatment 5.64 w 1.24 w 3.79 w 109 ~ w achieve a high degree of statistical significance (p< 0.001). the significant reductions achieved in plaque characteristics in six patients studied who showed plaque formation correlates with other parameters traditionally accepted as reducing cardiovascular risk. hypertension is found in approximately 70% of patients with cushing's syndrome, but the mechanism is poorly understood. previous studies in our unit have examined levels of exchangeable sodium, plasma renin and angiotensin ii and cardiac sensitivity to phenylephrine. one previous study has demonstrated enhanced pressor responsiveness to noradrenaline in a group of patients with cushing's syndrome due to adrenal adenoma. we have investigated the blood pressure response to noradrenaline in 7 patients with pituitary dependent cushing's syndrome and in 7 controls matched for age, sex and bmi. noradrenaline was infused for 10 minute intervals at five different concentrations between 0.01 and 0.18 mcg.kg.min -~ multiple systolic and diastolic readings were recorded and the infusion was stopped if the systolic pressure became > 200 mmhg, diastolic _> 1 l0 mmhg or the systolic pressure rose > 35 mmhg. basehne blood pressure in the patients with cushing's disease (cd) was 140/88 + 6/3 compared with 122/83 + 6/6 mmhg in the normal controls (nc). in 6 of the 7 patients with cushing's disease, the test had to be stopped before completion of the protocol, whereas this was necessary in only one control subject the change in blood pressure from basehne to the blood pressure value recorded either at the time the test was stopped or at the peak blood pressure reading during equivalent noradrenaline infusions was compared between the matched pairs. the mean change in diastolic pressure was 22 + 5 mmhg in cd compared with 7 + 2 in nc (p<0.05). there was no statistically slgmficant difference in either systolic pressure (28 + 5 vs 26 + 5 mmhg) or mean arterial pressure (23 + 5 vs 14 + 3 mmhg). these results demonstrate an increased diastolic pressor response to noradrenahne tn cushing's disease. increased pressor sensitiwty to uoradrenaline may contribute to the elevated blood pressure seen in cushing's disease. increased plasma homocysteine (thcy) is an independent risk factor for premature vascular disease. patients with insulindependent diabetes have an increased prevalence of cardiovascular disease. accordingly, we measured plasma thcy concentration in 119 such patients (15-59y), randomly selected, and in 51 control subjects. in controls, thcy was higher in males than in females (supine: geometric mean (95% ci): ,8.3 (7.2,9 6) v 5.9 (5.1,7.0) i.tmol/l, p<0.001), as previously described, but there was no gender difference in patients. male patients, without microvascular complications, had lower thcy than controls (supine: 6.5 (5.4, 7.8) v 8.3 (7.2,9 6) ~mol/l, p<0.05), but values in female patients without complications were similar to those of female controls, thcy significantly correlated with age in diabetics but not in controls, thcy increased in patients with increased severity of microvascular complications, partly due to the effect of age. thcy was higher when standing.than when supine in both controls (8 0 (7.0,9.1) v 7 1 (6.4,8.0)lalmol/l, p<0.05) and patients (6.9 (6.5,7.4) v 6.4 (6.0,6.9)l.tmol/l, p<0.02). the absence of gender difference, the association between thcy and age, and higher levels with increasing microvascular complications suggest thcy could be of pathogemc significance in iddm patients, despite unexpectedly low levels in male patients without complications. differences.between supine and erect samples may be due to haemodilution of albumin-hound thcy in the latter a review of the treatment outcome of thyrotoxicosis with standard dose/doses of radio-active iodine (sdrai) in 67 consecutive patients presented to the endocrinology department, uchg, from december 1992-december 1993 was analysed. the mean pre-treatment levels of free thyoxine (ft4) was correlated with the treatment outcome. there was statistically significant difference in the pre-treatment ft4 between responders and nonresponders to the first dose rai (p = 0.001). response with hypothyroidism and/or euthyroidlsm was considered successful treatment 43/67 (64 2%) responded to a single dose rai; 26/ 67 (38.8%) and 17/67 (25 4%) responded with euthyroldism and hypothyroidism respectively 19/67 (28.4%) and 3/67 (4.5%) responded to the second and third doses of rai respectively giving a total response rate of 92.5% and 97% respectively. interestingly, 2/67 (2.99%) patients failed to respond even to the fourth dose rai 4/67 (5.97%) patients with t3 toxicosis (3 females and 1 male). two responded to the first dose (one with hypothyroidism and the other with euthyroidism), the remaining 2 required a second dose, which produced the same results. no statistically significant difference in the response rate between t3 and t4 toxicosis (p = 0.9) was observed. inherent in the st. vincent declaration targets is the need for continuous data collection and audit. we present preliminary information from the mater database, the first prospective audit of patients from a homogenous irish population. 1,051 iddms (527m:524f) were identified with the following characteristics (mean + sd), age 37.3 + 14.4 years, duration of dm 185 + 11.2 years, bmi 24.4 + 3.0 (males) and 24.2 + 4.0 kg/m2 (females), hbalc 8.6 + 1.97% (n<6.2%). no male:females differences existed in the above nor in macrovascular complication rate 10.1% (predominantly peripheral vascular &sease and lschaemic heart disease). however, males were more likely to be current smokers (34% vs 27%, p = 0 01). hypertension rates (17.2m vs 15.4%f), cholesterol > 6.5 mmol/1 (6.2m vs 9.8%f) were similar but more males had cholesterol < 5.2 mmol/l (64.8m vs 53.8%f, p < 0.01). clinical nephropathy was present in 11.4% of males vs 8.7% in females (p<0 05) 11.9% had clinical peripheral neuropathy. retinopathy will be described elsewhere. 10.4% of females and 2.3% of males had a history of hyperthyroidism and 2.5% of females vs 1.4% of males of hypothyroidism. 7.8% had history of psychiatric disease. conclusion although not a population based study, care of iddm in ireland is almost totally hospital clinic based cigarette smoking is identified as the major problem to be addressed patients with diabetes meltitus (dm) are at a higher risk of developing vascular complications, including coronary artery disease (cad). we performed a detailed analysis of predictors of cad and its seventy in patients with dm and chest pain 19 patients in total single vessel cad (svd) in 4, double vessel (dvd) in 5, and triple vessel (tyd) in 5 on cine contrast angiography clinical, biochemical and dobutamme stress echocardiographic findings are tabulated below for patients with angiographically proven coronary artery disease. patients with tvd had a longer duration of dm (19 4 years) and were more likely to have retinopathy (100%) the sensitivity of dse was excellent for severe disease. conclusion' duration of dm, retinoapthy, and a positive dse were the best predictors of severe cad in a diabetic population with chest pain the haemodynamic hypothesis for the pathogenesis of diabetic microangiopathy argues that an initial increase in microvascular flow leads to sclerosis and disturbed microvascular autoregulation. we have recently demonstrated impairment of vasoconstrictor responses to endothelin-1, a potent endothelium-derived constrictor substance, in niddm and have suggested that this could contribute to the initiation of microangiopathy the purpose of this study was to determine whether responsiveness to endothelin-i is also impaired in iddm. non-specific vascular smooth muscle contraction was assessed using high dose serotonin eleven patients with iddm and 11 control subjects underwent forearm blood flow (fbf) measurement by venous occlusion plethysmography in response to local infusions of endothelin-1 (5 pmol/min for 60 minutes) and serotonln (30 la g/min for 2 minutes) control subjects showed slow onset vasoconstriction in response to endothehn-1 reaching maximum at 35 minutes (p<0.01) the diabetic group did not respond to endothelin-i group differences were significant (p=0.02). the two groups showed similar vasoconstriction in response to serotonln. in conclusion, vasoconstriction in response to endothelin-t is impaired m iddm non-specific vascular smooth muscle contraction is preserved. impaired vascular responsiveness to endothehn-i is a possible common mechanism for the pathogenesls of microanglopathy in 1ddm and niddm. we measured total corrected (tca) and standardised ionised calcium (lca) in a population of 76 intensive care (1cu) patients (with a mean age of 57+22 years, 59% male) to determine the prevalence of abnormalities in circulating calcium and its possible determinants severity of illness was measured by the apache ii score (acute physiological and chronic health evaluation). for comparison of ica we examined 20 subjects undergoing arterial gases which proved to be normal and 40 non-critically 111 hypoxlc subjects ica was measured on arterial gas samples and corrected for ph 84% of icu patients had a total ca (unadjusted) of < 2 2mmol/l. after adjustment for serum albumin, 55% of icu patients had an tca <2 2 mmol/1 30% of icu patients had a serum phosphate of <0.8 mmol/1 ica in controls was 1.44 + 0.017 mmol/1 and 1.38 + 0.015 mmol/1 in hypoxlc non icu patments (ns) ica in icu was lower: 1.34 +0.016 (<0.003). tca and lca were not slgmficantly related. tca and ica did not sigmficantly differ between patients who died and who survived in the icu, and they were not related to apache ii score. belfield, dublin 4. from july 1987 to june 1995 there was a 16-fold increase m the annual number of specimens submitted to the virus reference laboratory because of a perceived risk of contracting hiv through a needlestick injury, blood splash, human bite, or through occupational exposure. needlestick-associated specimens also comprise an increasing proportion of 'at-risk' specimens, rising from 0.9% m the year july 1987-june 1988 to 9.4% in the year july 1994-june 1995. between july 1992 and june 1995 a total of 1787 patients had specimens submitted for hiv antibody testing after a perceived'exposure to hiv of these only 209 patients had more than 1 specimen taken. although the time of putative exposure is rarely avadable, the median interval between 1st and 2nd postexposure specimens for these 209 patients is 3 months with 136/ 209 (65%) lying between 1 to 6 months. if the risk of hiv refection from a needlestlck injury is assessed as sufficient to warrant serological investigation, the timing and number of blood samples are important. a negative report from a single early specimen may not indicate an absence of infection a basehne specimen and follow-up specimens at 6 weeks, 3 months and at a minimum of 6 months post-exposure are recommended appropriate serology for other viral mfections (hepatitis b and hepatitis c) should also be considered. since the beginning of 1995 a significant sustained increase in the numbers of hepatitis a cases has occurred. the number of cases m 1995 was 229 against 121 in 1994. this reverses the continuous fall observed over previous years. the reason for this increase remains unidentified at present this increase has occurred following a dramatic increase in the total number of hepatitis a igm tests carried out by the vrl since february/ march 1994 the increase in the number of tests carried out since this time is primarily attributable to increased hepatitis testing following receipt of hepatitis c-contaminated rhesus anti-d immunoglobuhn. analysis of the age profiles of the positive patients and of all the referrals indicates that 95.5% of positive results were found m patients aged 50 yr or less whdst 31% of all hav igm tests were performed on individuals aged 51 yr or greater. this raises the question whether greater selectivity should be employed when requesting hepatitis a tests studies report compliance rates ranging from 30 to 50% in hiv negative patients there has been no comprehensive study of compliance in hiv positive patients. accurate measurements of compliance are not easy; easy measurements of comphance are not accurate "). to determine the compliance rate in hiv posmve patients attending st. james hospital, dublin, one hundred consecutive patients attending the service were interviewed (homosexual 46, ivdu 45, heterosexual 9). the questionnaire was divided into three sections. firstly, a medical review was completed by the clinicmn which included demographic data, cd4 count, cdc staging, karnofsky index and prescribed medication. secondly, the pharmacy detailed the medication dispensed to each patient. the third section comprised a patient interwew to determine adherence to, and understanding of prescribed drug therapy. we report an overall comphance rate of 61%. this was unevenly distrtbuted between the two main patient groups (89% in homosexuals, 24% in ivdu) the following factors were found to mfluence compliance: number of medications, cdc stage, karnofsky index, dysphagia, educational and socio economic factors. we also found that poor patient understanding of the prescribed therapy significantly affected compliance. the aim was to determine the incidence of stds in patients presenting for hiv testing at the department of genito-urinary medicine, saint james's hospital. a retrospective analysis of all patient notes who presented for hiv testing between july '94 and december '94 was undertaken. according to clinic policy all patients had been screened for the following stds; neisseria gonorrhoea, chlamydia trachomatls trlchomonas vagmalis, candida, human papilloma virus, herpes simplex virus syphilis and hepatitis b in addition intravenous drug users (ivdus) were also screened for hepatttls c all patients underwent pre test counselhng. sex, age, risk groups and diagnoses were noted. 504 patients presented for hiv testing, of whom 66% were male, 34% female, wtth an average age of 28.5 years. of the total, 8% were, or had previously been ivdus. of the total 46 ivdus, 37 were heterosexual males 2 were bisexual and 4 were females 10.2% of the male patients were homosexual and 4 4% btsexual there were 15 posmve hiv tests (3% of total); 14 males and 1 female. in this group there were 5 patients with hepatitis c, all of whom were ivdus. no other stds were detected in the hiv negative group hepatitis c was diagnosed in 20, hepatttis b in 4, anogenital warts in 58, herpes gemtalis in 10, syphilis in 3, n. gonorrhoea in 9, t. vagmalis in i, c. trachomatls m 12, g vaginalis in 5 and candldlasis in 47. this study confirms the importance of std screening in all patients requesting a hiv test. of the total testing for hiv, 30% had a concurrent std diagnosis although no stds were identified in the hiv positive group this may be more a reflection on the makeup of the irish hiv positive population, the majority being ivdus, rather than a difference m the mode of sexual toxoplasmosis is the most common opportumstlc infection of the central nervous system in aids patients. in clinical practice the diagnosis depends on clinical, radiographic and serological findings coupled to chnical response to therapy brain biopsy is not routinely performed. in this retrospective review, we describe our experience with diagnosing toxoplasmosis we examined (a) the clinical demographics and presentation, radiographic findings, response to therapy and patient outcome (b) role of polymerase chain reaction (pcr) in detecting toxoplasma gondii from blood and csf samples (c) the usefulness of serology in diagnosing acute infection. all cases diagnosed as toxoplasmosls based on the above criteria were reviewed. pcr to detect toxoplasma gondn dna used primers to the b 1 gene giving a 223bp amphficatton product serological tests used were sabln-feldmen dye test and latex agglutination. there were 25 cases diagnosed (17 m, 8f, cd4 0-300; cdc iv 19). 3 panents unknown to be hiv posmve presented with cerebral toxoplasmosis 22 patients were not receiving continuous systemic prophylaxis against pce diagnostic value oft gondii pcr in blood and csf showed a sensmvlty of 20%, specificity of 100%, ppv was 100% and npv was 40% determination of lg subtype was of limited value 25% (6) of patients were seronegatlve of whom 50% (3) had histologically proven disease 2 of these latter 3 cases were pcr negative the dye test was of poor predictive value this review confirms the need to combine all parameters in making a diagnosis of toxoplasmosis in lmmunocompromlsed hosts. the performance of the newly developed, rapid and fully automated m~croparticle enzyme immunoassay abbott imx hiv-1/hiv-2 3rd generation plus assay for the detection of antibody to hiv-1 and hiv-2 including subtype o m human serum or plasma was assessed the assay was evaluated by testmg specimens from blood donors, diagnostic populations and hospitalized patients, hiv seroconverslon panels confirmed hiv-positive specimens, and potentially interfering specimens. the abbott imx hiv-1/hiv-2 3rd generation plus assay showed an overall apparent specificity of 99.97% (lower limit of 95% ci 99.84%) in the tested blood donor populations (n=347t). this comparable to the specificity found for the abbott imx hiv-i/hiv-2 3rd generation plus eia (99 93%) and the axsym h1v-1/hiv-2 assay (99 90%). the apparent sensitivity of the abbott imx hiv-i/hiv-2 3rd. generation plus assay is at least equivalent to that of the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-i/hiv-2 assay. of 21 hiv-i seroconversion panels tested, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected seroconverslon earlier on up to 6 panels, depending on the comparison assay. among 785 specimens from asymptomatic and symptomatic hiv patients, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected 100 (785) including 7 specimens characterized as hiv-1 subtype o. the abbott imx hiv-1/hiv-2 3rd generation plus assay is an extremely sensltlve and highly specific assay for the early detection of antibody to hiv-1/hiv-2 and shows at least an equivalent performance to the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-1/hiv-2 assay. the fully automated imx instrument system offers ease of use and rapid results on a widely accepted and reliable platform. streptokinase (sk), a 47kd protein produced by group c b hemolytic streptococci, is a widely used thrombolytic agent. anti-sk antibodies arise either as a result of therapeutic administration of sk or following natural mfection with streptococci although the clinical significance of antl-sk antibodies is not clear, there is evidence that some anti-sk antibodies arising from natural infections can interfere with sk activity tn vtvo, resulting in thrombolytlc failure. to facilitate further investigations of these antibodies, we have developed and validated a highly sensitive functional assay, which measures sk neutralisatlon activity of serum independently of other circulating inhibitory factors in the sample, and a rapid and convenient enzymeimmunoassay for the detection of anti-sk antibodies. analysis of over 200 random serum samples from the local blood bank with the enzymeimmunoassay showed the prevalence of antl-sk antibodies to be approximately 13%. all the positive samples and an equal number of the negative samples randomly selected were analysed by the functional assay the agreement between the results of the two assays was excellent indicating that our enzymelmmunoassay was a convement method for detection of anti-sk antibodies which could neutrahse sk activity m vitro irish journal of medical science tuberculosis drug therapy, isolation precautions and prophylaxis. conventional methods of detection such as microscopy and culture either lack sensitivity and specificity or are timeconsuming. in this study we investigated the use of a pcr based diagnostic assay for the detection of m. tuberculosis in sputum samples this assay has been developed by bioresearch ireland (bri) and raggio italgene. sputum samples were lysed and pcr amplified using an m. tuberculosis complex-specific primers. the results obtained using the bri/c-trak tm technology were initially compared to the amplicor system (hoffman la roche). both probe detection methods represent fast and reliable methods for the detection of m. tuberculosts in clinical samples. this test is designed to eliminate the possibility of obtaining false negatives. strongyloldes stercoralis infection in humans ts endemic in the tropics. as travel is becoming more common, it will be seen more frequently. two cases of this infection in irish people are described. case i. a 21 year old women had travelled and worked in poor rural areas of mexico for one month, three years before presentation. two and a half years later she developed abdominal discomfort, anorexia and sore throat. myalgia, arthralgta and a transient skin rash began to appear in the next month. eosinophilia, mild anaemia and raised liver blood tests were noted. elisa test for strongyloides was positive but parasites were not seeri in the faeces. ivermectin was given and the patient feels better. case ii. a 31 year old nurse had arthralgia, fatigue and some weight loss for 18 months'. on two occasions in the last four years, she had been travelling extensively in s.e. asia for a total of four months. she was admitted to hospital because of acute fever and loin pain. a urinary tract infection was diagnosed. absolute eosinophil count was i'aised 0.63x10^9/1. esr was 17mm/hr. strongyloides elisa was positive and treatment administered as above. strongylotdes is the most important nematode in the returned tropical traveller. it can multiply and persist within the body for long periods of time and it can cause hypertnfection syndrome, a protean fulminating infection of bowel, lungs, blood stream and brain, in those who are lmmunocompromised. diagnosis can be difficult by stool microscopy. thlabendazole has side effects but ivermectin is safe and effective. june 1995, there was an outbreak of c.difficde-associated diarrhoea (cdad) at st. james's hospital. the aims of this study were to determine the incidence and outcome of cdad in hiv positive and negative patients we prospectively reviewed all patients with diarrhoea, a positive c.difftcile cytotoxin assay, and in whom no other infectious cause for diarrhoea was identified demographic data, history of diarrhoeal episodes, risk factors and outcome were recorded. the incidence of cdad in hiv negative patients was 1.2 per 100 hospital admissions, compared to 1 per 100 admissions m hiv positive patients. the average number of courses of antibtotlcs received, in hiv negative patients prior to the onset of symptoms was 2.3, and 76% of this group were exposed to third generation cephalosporins. hiv positive patients received an average of 3.4 courses of antibiotics and no patients received third generation cephalosporins. there were no deaths due to cdad in hiv positive patients however 4 hiv negative patients died from severe pseudomembranous colitis in conclusion we documented a unexpectedly low incidence and complication rate of cdad in hiv positive patients this is surprising considering their multiple hospital admissions and exposure to ant~microbial and chemotherapeutic agents. the number of new positive hiv specimens detected at the virus reference laboratory has risen from a cumulative total of 638 m july of 1987 to 1597 in september of 1995. we examined our data to determine the proportional make up of these positives by major risk group. in august 1987 64.7% of positive specimens were from intra venous drug abusers (ivda) by september 1995 ivda made up 49% of the total positive hiv specimens. positive specimens from homosexual individuals rose fro.m l0 9% of total positives in august 1987 to 20.6% in september 1995 there were no recorded positive specimens from heterosexual exposure in august 1987 but in september 1995 8.9% of positive specimens recorded heterosexual exposure. a further category which includes blood donors, haemophiliacs, transplant patients and organ donors made up 25% of total positives in august 1987 and in september 1995 made up 22.4% of total positives. we further examined our data in order to show when these changes occurred by ascertaining how many new positive patients have been discovered per year in each of the main risk groups. see in the united kingdom echovlrus type 22 (echo-22) is regularly isolated, with nearly 200 reports annually to the central public health laboratory. reports increased during 1986-1988 and overall it is the second most commonly reported echovlrus in the u.k. echo-22 epldemiology is different to that of other enteroviruses; over 90% of patients with echo-22 tsolated are less than 2 years of age echo-22 shows distinctive and unique cytopathogenic features in tissue culture, and based on sequence analyses, it seems to belong to a separate subgroup of picornaviruses. echo-22 has been associated with respiratory symptoms in premature infants, myocarditis and severe encephalitis. in 1989 an outbreak of acute flaccid paralysis associated with echo-22 was described in jamaica in six patients, four of whom died. we describe three cases of sudden death in infants associated with echovirus type 22 infection case i: s d. born 20/7/94; birth asphyxia and death at two days of age; echo-22 isolated on 2217194 from spleen. this study assessed the antibiotic sensitivity of organisms causing urinary tract infections (uti) among genito-urinary medicine (gum) clinic attenders in order to determine whether it is worthwhile giving tetracycline for dipstick (nitrite) positivity, even in the absence of clinical features of uti. we looked retrospectively at 100 laboratory confirmed uti's diagnosed among gum clinic attenders over a period of eight months. we assessed antibiotic sensitivities of the organisms involved, and determined how many dipstick positive urines which were left untreated turned out to be real uti's. 86% of uti's were due to coliforms and 66% of these were sensitive to tetracycline. 4% of uti's were due to staphylococcus saprophyticus, 3% due to beta haemolytic streptococcus group b, 2% due to enterococcus, 2% due to proteus species and 2% due to coagulase negative staphylococci. 25% of nephur positive urines were left untreated. 32% of these were nitrite positive. failure to treat a positive urine dipstick which turned out to be a uti necessitated a further clinic visit for adequate treatment. nitrite positive urines should be treated as a uti, even in the absence of clinical features of uti, either with trimethoprim or tetracycline. the number of untreated uti's and unnecessary extra visits to gum clinics would have been reduced with the use of judicious antibiotic therapy for nitrite positive urines. strains of enterococci resistant vancomycin have been reported with increasing frequency. in 1995, we investigated an increase in the frequency of vancomycin-resistant enterococcus faecium (vref) among patients in the haematology/oncology unit using pulse-field gel electrophoresis (pfge) to genotype these isolates and to assist in establishing the source of these vref. eighteen clinical isolates of vref from blood, urine sputum and-faeces and two environmental isolates were collected from separate patients between march and july 1995. minimum inhibitory concentrations (mics) to several antibiotics including teicoplanin and vancomycin were determined by agar dilution. pfge were performed following smal restriction endonuclease digestion. antimicrobial susceptibility testing revealed high level resistance to vancomycin and teicoplanin; mics >128 mg/ l and >32 mg/l respectively. this antibiogram is consistent with the van a phenotype. pfge of all 20 isolates revealed identical patterns indicating clonal spread of vree subsequent implementation or infection control measures reduced the frequency of vref isolation. pfge proved useful in demonstrating clonal spread of vref and.in emphasising the need for infection control measures. a prospective audit of baeteraemia in our 600 bed teaching hospital was carried out from february 1991 to march 1993. clinical and microbiological data were collected on 520 episodes of bacteraemia in 78 patients. of these 230 (62%) were hospital acquired and 200 (38%) community acquired. urinary tract and respiratory tract sources were implicated in 30% and 14% of community acquired episodes, making e. coli and s. pneumoniae the commonest community acquired isolates (41% and 16% respectively). other gram negative bacilli accounted for 13% and s. aureus for 11%. coagulase negative staphylococci were the commonest hospital acquired isolate (24%) followed by s. aureus (22%), e. coli (i5%) and enterococcus spp. (10%). enterobacter spp. were the second commonest gram negative isolate (5%). central venous cannulae were implicated in 43% of hospital acquired cases. urinary tract infections accounted for 20%. 63% of which were catheter related. invasive diagnostic procedures (angiography, prostate and liver biopsies, sinography) were implicated in t0 episodes. gentamicin resistance was found in 9% of hospital acquired aerobic gram negative bacilli and mrsa accounted for 13% of hospital acquired s. aureus. these figures are higher than expected but may be explained by outbreak of mrsa and gentamicin resistant entercobacter spp. which occurred during the study period. the past severalyears have seen a significant increase in the recognition of moraxella (branhamella) catarrhalis as a respiratory pathogen (~). the pathogenic mechanisms employed by the organism are largely unknown, but adherence may play a role ~2~. in our investigation the haemagglutinating ' activity of 40 isolates of m. catarrhalis was determined by a microtitre method. no isolate agglutinated horse, chick or sheep red blood cells (rbc). seventeen isolates agglutinated human rbc, x~hile 7 of these 17 isolates also agglutinated rabbit red. blood cells. haemagglutination of human and rabbit red blood cells was inhibited by porcine mucin. galactose inhibited the haemagglutiriating activity of the 7 isolates which agglutinate both human and rabbit rbc and yet bad no effect on the haemagglutinating activity of the isolates which haemagglutinate human rbc alone. .electron microscopy studies of the bacteria demonstrated a diffuse outer fibrillar layer on the surface of haemagglutinating positive isolates, thislayer was subsequently removed following trypsin treatment, as was the haemagglutinating activity. a 200 kda trypsin sensitive protein appears to be associated wfth haemagglutinating properties. mrsa is an increasingly important cause of morbidity, and is spreading from large hospitals to smaller community-based facilities and nursing homes. the objective of this survey was to obtain an indication of the size of the mrsa problem in ireland prior to introducing national mrsa control guidelines. a survey of all microbiology laboratories in ireland was carried out over two weeks in spring 1995. for patients from whom mrsa was isolated during the study period standard demographic and clinical data were requested and period prevalence/1000 discharges was calculated. all 45 microbiology laboratories surveyed responded. mrsa was isolated from 448 patients during the 2 week period. the period prevalence of mrsa/1,000 discharges was 16.5. males aged 65+ had the highest rate of infection (50/1000 discharges). half of all isolates were from patients in surgical or medical wards, but 4% were from community-based sources e.g. gps, nursing homes, hospices. thirty-two percent of mrsa patients were infected rather than colonised. mrsa is clearly a substantial problem in ireland. while it is largely a hospital problem at present, the increasing trend for day procedures and shorter stays means that infection will increase in the community. a survey in a university hospital in the usa revealed 41% of mrsa cases to be communityacquired. tonsil core specimens were cultured for bacteria including mycoplasma, chlamydia and ureaplasma urealyticum in 70 children undergoing tonsillectomy for recurrent acute tonsillitis. serology for chlamydia and mycoplasma pneumoniae was obtained in 55 of the children. the polymerase chain reaction (pcr) was used to investigate the presence of chlamydia pneumoniae in core tonsil tissue. ureaplasma urealyticum was cultured in three children (4.3%) and mycoplasma salivarium in two children (2.8%). culture was negative for chlamydia pneumoniae and mycoplasma pneumoniae. the complement fixation test for chlamydia species was positive in 16/55 children (29%) indicating previous infection. specific immunofluorescence testing for c. pneumoniae was positive for lgg (titre> 16) in 10/55 (18%). igm antibody to c. pneumoniae and antibodies to c. trachomatis and c. psittaci were not detected. ninechildren (16%) had titres > 32 to m. pneumoniae. pcr failed to demonstrate c. pneumoniae. aerobic and anaerobic bacteria were cultured from all specimens. the culture of ureaplasma urealyticum in 4.3% of our patients indicates a higher rate of colonisation then previously thought. this study 49 irish journal of medical science demonstrates past infection with c. pneumoniae in 18% and with m. pneumoniae in 16% of children with recurrent tonsillitis. however c. pneumoniae and m. hominis do not play a significant role in childhood recurrent tonsillitis. multiply resistant enterococci are increasingly common causes of serious infection in hospitalized patients. high level gentamicin resistance (mic > 1000 mga) in enterococci further compromises the therapy of such infections. we have identified seven clinical isolates of enterococcus hirae demonstrating high-level gentamicin resistance (hlgr: mic > i000 mg/1). to our knowledge this is the first report of hlgr for this enterococcus species. plasmid analysis has demonstrated the presence of a single, large plasmid in all seven isolates, as well as several smaller plasmids in some of the isolates. filter mating experiments have revealed that in all seven cases, hlgr was transferred to a laboratory recipient e. faecalis jh-22 by conjugation. plasmid analysis of transconjugant strains confirmed transfer of the large plasmid in all cases. based on restriction enzyme profiles, two distinct conjugative plasmids were identified for the e. hirae isolates investigated. at present we are using southern blot techniques with oligonucleotide probes designed to hybridise to the hlgr determinant found in other species of enterococcus. the results will confirm whether or not the same resistance determinant is responsible for the dissemination of hlgr in the genus enterococcus. dublin 8. aminoglycosides remain commonly used in the treatment of severe gram negative infection and have conventionally been given on a twice or thrice daily basis. single daily dosing offers advantages with respect to less nephrotoxicity, better bactericidal activity, convenience, nursing time, cost and should avoid subtherapeutic dosing which has a significant impact on outcome. we reviewed serum gentamicin assays from january to december 1995 to assess potential toxicity and subtherapeutic dosing in patients who received once daily gentamicin and those who received multiple daily dosing. 4346 assays were performed in the study period. 520 of those were random assays and not included. there was a trend towards significantly less potentially toxic levels in the once daily group compared to the multiple daily group (p<0.1). once daily dosing produced significantly less subtherapeutic dosing (p<0.001). over 98% of peak assays in the once daily group were in the recommended range. we conclude that current practice of multiple daily dosing of gentamicin leads to significant underdosing and more potentially toxic trough levels. measurement of trough assays only in patients who are treated with once daily aminoglycosides is sufficient and will have considerable cost savings. respiratory, syncytial virus (rs virus) is a major respiratory pathogen of infants less than 1 year old. it occurs in annual epidemics during the winter and early spring in temperate climates. during rs virus epidemics a significant number of infants less than 6 months old are hospitalised with symptoms of bronchiolitis and pneumonia. rs virus exists in two antigenically distinct subgroups, a and b which are known to cocirculate in the same community during the same rs virus season. there is much debate regarding the virulence of one strain over the other. using a panel of monoclonal antibodies specifically directed against the two rs virus strains, 167 rs virus isolates from specimens sent to the virus reference laboratory, university college dublin, over seven consecutive rs virus seasons (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) were typed and the rs virus subgroup predominance monitored. subgroup a was the most predominant of the rs virus isolates accounting for 67.7% of the total and was found to be the predominant rs virus strain in six out of the seven rs virus seasons studied. subgroup b predominated in a season in which the number of rs virus detections peaked much later than normal. treatment of fungal infections in patients in intensive care unit (icu.) is usually empiric. the aim of this study was to identify candida species isolated from i.c.u. patients and to test their susceptibility to antifungal agents to enable more directed therapy. forty candida sp. from 23 patients in i.c.u. were isolated from the following sites, blood culture (5), central venous catheter (7), chest drain fluid (5), wounds (8), catheter urine (8), bronchial lavage (3), sputum (4). strains were identified by standard procedures. minimum inhibitory concentrations of amphotoeracin b, 5-flucytosine and fluconazole were obtained by agar dilution test and e test. the isolates were identified as c.albicans (22), c.glabrata (8), c.krusei (5), c.tropicalis (2), c.parapsilosis (2), c.kefyr (i). all the candida species were sensitive to amphoteracin b (mic = 1-<025mg/l), and 5 flucytosine (mi c= <025mg/l). c.albicans and c.parapsilosis were fluconazole sensitive (mic = 16-15mg/l). four of eight c.glabrata were fluconazole resistant (mic = >256mg/l). c.krusei, c.tropicalis and c.kefyr were also resistant (mic = >256 mg/l). wbilst there were no major discrepancies between the agar dilution test and the e test, the agar dilution test was laborious and required a high degree of skill. the e test was easier to read and more reliable results were obtained provided the inoculum was carefully standardised. this study shows that azole antifungals should not be ganglion is probably the commonest tumour encountered in the hand and the wrist. it often arises from tendon sheath or lining of a joint capsul. the treatment can be surgical or nonsurgical, the latter includes aspiration with or without injection of steroids. surgical treatment of ganglion can pose a difficult situation to deal with. it requires hand surgeon to deal with one such problem, we present a 46 year old man with tender mass in the hypothenar eminence. during surgical exploration it was obvious that the ganglion was infiltrating the wall of the ulnar artery, and the histology proved this later. the clinical features, management arid the outcome of this unusual case are discussed. ischaemic preconditioning (ipc) of the myocardium with repeated brief periods of ischaemia and reperfusion (i-r) prior to prolonged ischaemia significantly reduces subsequent infarction. following ipc two "windows of opportunity" (early and late) exist during which prolonged ischaemia can occur with reduced myocardial infarction, we investigated if ipc of skeletal muscle prior to flap creation improved subsequent flap survival and perfusion in either early or late windows. the latissimus dorsi muscles (ldm) of sprague-dawley rats were used. group 1: (control, n=12). the ldm was elevated as a thoracodorsatly based island flap group 2: (early ipc, n=8). the ldm was preconditioned with two 30 minute episodes of normothermic global ischaemia with intervening 10 minute episodes of reperfusion prior to elevation. group 3: (late ipc, n=8). the ldm was elevated 24 hours after ipc ischaemia was created by occlusion of the thoracodorsal artery and vein and the intercostal perforators having previously isolated the muscle on these vessels. muscle perfusion was assessed by a laser doppler perfusion imager. one week after flap elevation the percentage of muscle necrosis was measured by computer-assisted planimetry ipc significantly reduced muscle flap necrosis (table) in both early and late windows. muscle flap perfusion was similar in all groups. this study compares the biochemical, serological and histopathological findings in 20 women with chronic hepatitis c virus (hcv) infection with 20 age-matched women with established autoimmune hepatitis (aih). there is increasing evidence of autoimmunity in hcv liver pathology. because of different treatment regimens for hcv (d-interferon) and aih (steroids, immunosuppression), clear distinction between the two diseases is desirable. liver enzymes (alt, ast, alkaline phosphatase), antinuclear factor (anf), anti-smooth muscle (asm) and antimitochondrial (ama) antibodies are compared for both groups of patients. liver biopsies from all women were compared using the grading and staging system of ishak et al (1995) . the results show that while some women in both groups show elevated liver enzymes, positive anf and asm autoantibodies, the values are much higher in aih. similarly aih patients show overall more severe disease on liver histology. both groups demonstrate poor correlation between histological features and autoantibody titre. we conclude that distinction between hcv and a!h is usually possible with liver histology and serology. some women with chronic hcv and positive autoantibodies however," demonstrate a histological and serological picture suggesting that chronic hcv may be mediated by an immunopathogenic mechanism. irish journal of medical science terminal changes only, 5 showed lymphocytic meningitis and 4 of these also had perivascular lymphocytic inflammation (cd3 positive) in the subependymal regions, brainstem and choroid plexus. two brains showed purulent meningitis and one case had central pontine myelinolysis probably related to profound metabolic disturbance. basal ganglia mineralization, hiv encephalitis (hive) or hiv leucoencephalopathy (hivl) were not present. these findings differ considerably from those described in us cases, in whom the majority have evidence of hiv within the cns. relatively early death from systemic infection may account for the lack of hive/hivl in these cases. the lymphocytic meningitis and perivascular inflammation may represent an immuno-allergic reaction, previously reported as "early" changes, regarded as important in inducing vascular damage which allows subsequent entry of h1v into the brain. the aim of this study is to assess apoptosis in areas of interface hepatitis and spotty necrosis in hepatitis c virus (hcv) infected liver biopsies, and to correlate the degree of apoptosis with severity of histological activity. 25 liver biopsies were randomly selected from a group of type lb hcv positive women. these patients were diagnosed by recombinant immunoblot assay (riba) test and the presence of hcv rna was confirmed using the polymerase chain reaction (pcr). apoptosis was demonstrated in the biopsies by the oncor apoptag in-situ hybridisation technique. the average number of apoptotic hepatocytes per portal tract, and within the parenchyma per 10x objective, was determined. the modified histological activity index (h.a.i.) was used to score each biopsy. comparison of the results shows that increasing numbers of apoptotic hepatocytes are consistently associated with increasing scores for interface hepatitis and spotty necrosis. it is concluded that apoptosis occurs in hcv infected livers and that it correlates with increasing histological activity .indicating a significant role for apoptosis in the pathogenesis of hcv liver disease. and university of edinburgh, scotland. paediatric aids represents only 5% of cases worldwide, in most published series parental iv drug use was the main risk factor, cns pathology was a late feature of the disease and antiretroviral treatment had been given. we studied eleven brains from romanian children with probable postnatal hiv infection using standard neuropathological stains and immunostains for lymphocyte markers and hiv p24 antigen. death was due to systemic infection, mostly pneumonia or gastroenteritis; antiretroviral treatment had not been given. three cases showed we studied the role of the p53 and bcl-2 genes in the pathogenesis of post-transplant lymphoproliferative disease (ptl). ten cases were examined by immunohistochemical and molecular methods. immunohistochemistry was performed using standard and antigen retrieval methods, with the p53 do-7 and the bcl-2 oncoprotein clone 124 antibodies. dna was extracted from paraffin blocks and subjected to pcr, and single-strand conformation polymorphism (sscp) analysis searching for mutated p53 genes. samples showing any evidence of aberrant migrations were further analysed by direct sequencing. pcr was also used to detect bcl-2 gene rearrangements. we employed a technique called representational difference analysis to search for previously undescribed translocations or deletions which may be involved in breast carcinogenesis. dna was isolated from both invasive ductal carcinoma of the breast and normal tissue from the same patient. following restriction enzyme digestion and tigation of oligonucleotide linkers, pcr was carried out on both tumour and normal dna using oligonucleotide specific primers to obtain a representation of each genome (amplicons). digestion with the same restriction enzyme removed the original linkers, and a second set of oligonucleotides were ligated to normal amplicons only. the tumour derived amplicons were subtractively hybridised to the normal and subsequent pcr was used to isolate fragments unique to the normal dna (difference products). this was possible since oligonucleotides were ligated to normal dna only. following a series of further subtractive hybridisations and subsequent amplifications, purified difference product was obtained. difference products in the size range 500-1500 bp were obtained. following further rounds of subtractive hybridisation and amplification, purified difference product will be sequenced and characterised by comparison with known gene sequences. the chromosomal location of the affected gene will be established by in-situ hybridisation and somatic ceil hybridisation, using the difference product as probe. protein s is secreted by osteoblasts and case reports of reduced bone mineral density in patients with total protein s deficiency have lead to the hypothesis that this inherited disorder is associated with generalised osteoblast dysfunction predisposing to osteoporosis ~. we have assessed bone formation in 8 patients (2 male and 6 female) with total protein s deficiency and 4 controls (2 male and 2 female) using two recently available sensitive markers; serum osteocalcin (oc) and serum procollagen 1 carboxyterminal peptide (p1cp), both secreted by the osteoblast. the mean total protein s level amongst the patients was 40 _+ 12% (ref range 62-135%), mean oc was 14.2 ng/ml (ref ~'ange 5.2-59 ng/ml) and the mean picp was 81.5 + 15 uga (ref range 38-02 ug/1). in the control group, mean oc was 12.6 _+ 5 ng/ml and the picp was 124 + 37 ug/1. there was no statistical difference between both groups using either marker. in conclusion bone formation as assessed by serum osteocalcin and p1cp appears to be normal in patients with total protein s deficiency. hereditary spastic paraparesis (hsp) is a variably expressed neurodegenerative disorder which exhibits clinical and genetic heterogeneity. hsp can be inherited in an autosomal dominant (ad), autosomal recessive (ar) or x-linked manner. ad-hsp has been linked to a number of loci. we have ruled out linkage to these loci in a large irish family affected with ad-hsp. the aim of this study was to determine whether ad-hsp is linked to spinocerebellar ataxia loci (sca), sca-i & sca-ii. ad-hsp can be clinically similar to sca. dna was extracted from blood taken from 45 co-operating family members. microsatellite markers spanning the sca-i and sca-ii loci were amplified by pcr. individuals were genotyped and linkage analysis was carried out using the linkage set of programs. significantly negative lod scores were obtained for both sca-i and sca-ii loci. d6s274 gave maximum exclusion of 18cm on either side of the sca-i locus with a lod of -2.11 at a recombination fraction of 0.18. d12s105 gave maximum exclusion of 13cm on either side of the sca-ii locus with a lod of -2.08 at a recombination fraction of0.13. other markers examined also outruled linkage to these loci. we conclude that the gene for ad-hsp is unlinked to the major sca loci. serum vitamin b12 is frequently measured in the investigation of anaemia, and in screening neurological and other disorders. frequently, patients are found with low serum vitamin b12 level with a normal hb and without clinical abnormalities relevant to vitamin b 12 deficiency. this study was carried out to determine the significance of a low serum vitamin b 12 level. 959 vitamin b12 measurements were carried out over an 8 month period using a chemiluminescence method (abbott imx). clinical data was obtained retrospectively. of the 959 samples 81 (8.4%) representing 65 patients had a low serum vitamin b12 level (>180 pg/ml) with a mean of 152 pg/ml (39-179). data was available on 44 patients. 20 (45%) had a hb below the normal range with median serum vitamin b12 level of 158 pg/ ml (75-184). 19 (45%) had a normal hb, mcv and mchc with a median serum vitamin b12 of 152 pg/ml (52-210), and 5 had a normal hb with an abnormal mcv or mch. lft's, autoantibodies, schillings test and bone marrow examination data will be presented. in conclusion in patients with a low serum vitamin b 12 level, there was no significant difference in the b12 levels in those patients with a normal or a low hb concentration. it would appear that serum vitamin b12 is a poor discriminatory test but that changing the normal range may not help in screening. low serum vitamin b12 on its own may not appear to provide adequate grounds for lifelong replacement therapy. of 1 in 133 for males and 1 in 172 for females. as expected the overall incidence of hodgkin's was lower with one third of male and one quarter of female lymphoma cases affected by the disease. a distinct age specific pattern is evident depending on lesion type. marked variation in incidence levels were noted throughout the study region. an extremely varied pattern is evident in the survival rates for lymphoma patients. the cork and kerry rates for malignant lymphoma are relatively low when compared with international levelstzl obstetric complications and schizophrenia: methodology and mechanisms perinatal complications and clinical outcome within the schizophrenia spectrum 96) negative symptoms, cognitive impairment and duration of initially untreated psychosis in schizophrenia davnet's hospital, monaghan, and royal college of surgeons in ireland retinal pathology in kearns sayre syndrome mitochondrial dna and disease mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling phenytoin induced pseudolymphoma. a report of a case and review of the literature cutaneous reactions in head injured patients receiving phenytoin for seizure prophylazis hydantoin induced pseudopseudolymphoma branhamella catarrhalis: an organism gaining respect as a pathogen correlation between branhamella catarrhalis adherence to oropharyngeal cells and seasonal incidence of lower respiratory tract infections 206) epidem1ology and survival rates for all 324 lymphoma patients registered in cork and kerry over the eight year period an indepth review of all 324 lymphoma (icd -o code 196) patients registered by the sourthern tumour registry during the eight year period 1983/1990~l annual age adjusted rates of 7.6. and 5.4 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk references 1. cancer, the irish experience cancer incidence in five continents volume v immunohistochemistry for bcl-2 oncoprotein without antigen retrieval gave negative results, but with antigen retrieval, showed positive staining in 6 out of 8 cases. no bcl-2 rearrangements were detected by pcr. the combination of sscp and sequencing confirmed only wild type dna in all cases, p53 immunohistochemistry by standard methods revealed positive staining in only one out of nine samples analysed. when the antigen retrieval method was employed for this antibody, positive staining was seen in > 10% of tumour cells in four further cases.our results suggest that p53 does not play major role in ptld. bcl-2 overexpression but not rearrangement may contribute to the development of ptld. transplant arteriopathy (ta) is the major cause of death in cardiac allograft recipients. the pathogenesis is unclear. we have previously shown a plasma cell predominance in the infiltrate of ta, leading us to hypothesise a role for epstein-barr virus (ebv) infection in its pathogenesis. an association between cytomegalovirus (cmv) and ta has previously been suggested. the aim of the study was to investigate the role of epstein-barr virus (ebv) and cytomegalovirus (cmv) in the pathogenesis of ta.we performed pcr for cmv and ebv dna and protein (lmp) in seven cases of ta, involving cardiac allografts. restriction mapping was used to confirm that pcr products were either cmv or ebv dna respectively.cmv dna was found in four cases. ebv dna was found in six of the seven cases and ebv lmp staining was present in six cases. ebv was detected in all cases by either pcr or ihc.our results suggest that ebv infection may play a pathogenic role in transplant arteriopathy. the evidence for a similar role for cmv ~s less strong. st. vincent's hospital. hereditary spastic paraplegia (hsp) is a neurodegenerative disorder characterised by progressive spasticity, primarily of the lower limbs. it can be inherited in an autosomal dominant (ad), autosomal recessive or x-linked manner~ we have identified a large irish family (family a) affected with ad hsp that cosegregates with dementia. three. loci have previously been identified that are linked to ad hsp in families of different ethnic origin. the locus on chromosome 2 is reported to be the major hsp locus. the aim of the present study was to examine family a for linkage to the chromosome 2 hsp locus.dna has been extracted from blood taken from all 45 co-operating family members for genotyping. polymorphic microsatellite markers from chromosomal region 2p24-21 have been amplified by pcr, electrophoresed on a denaturing polyacrylamide gel and detected by silver staining. linkage analysis was carried out using the linkage series of programs. linkage analysis excluded the hsp gene from the chromosome 2p locusl the most significant marker was d2s367, with a lod score of-2.12 for recombination fraction 0.19, thereby excluding approximately 19cm either side of this marker. negative lod scores were also obtained for the other markers chosen (d2s391, d2s392, d2s352, d2s 174) excluding 20cm, 8cm, 4cm and 8cm respectively.the current study has therefore successfully excluded linkage of ad hsp in family a to the major locus on chromosome 2p. further studies are underway to exclude linkage of hsp in this family from other candidate loci, prior to carrying out a genome wide search. the presence of dementia in this family in association with hsp suggests that a new and as yet unidentified gene is responsible. vincent's hospital. eye and ear hospital, dublin. ched is a corneal endothelial dystrophy characterised by diffuse bilateral corneal opacities resulting in impaired vision. both autosomal dominant and autosomal recessive modes of inheritance have been described. another endothelial dystrophy, posterior polymorphous dystrophy (ppmd) has been linked to 20qll.we have used homozygosity mapping to analyse a pedigree with autosomal recessive ched for linkage to 20ql.1. all affected individuals are offspring of consanguinous matings. homozygosity mapping is based on the principle that these offspring would be homozygous for genetic markers near the disease gene. homozygous regions would be random between different offspring of these matings, except at the di-sease locus shared by affected offspring.dna was extracted from blood taken from 22 family members, 14 of which have ched. allele frequencies were determined in pooled dna from affected individuals. pooled dna from unaffected individuals was used as a control. at the disease locus, a shift in allele frequencies towards a single homozygous allele would be observed in the affected dna pool. pooled dna was genotyped by pcr for 3 polymorphic microsatellite markers in the region of20qll. pcr products were separated on a polyacrylamide gel and visualised by silver staining. similar allele frequencies were observed at these loci in both dna pools demonstrating independent assortment of alleles. in addition, affected and unaffected family members were individually genotyped at these loci and no significant loss of heterogeneity in the affected individuals at these loci was observed. these data indicate exclusion of linkage of the ched gene to 20qll. key: cord-014712-5u4e00q6 authors: nan title: selected abstracts from the 100th j project meeting, antalya, turkey, march 12-14, 2014 date: 2014-08-02 journal: j clin immunol doi: 10.1007/s10875-014-0065-9 sha: doc_id: 14712 cord_uid: 5u4e00q6 nan hans d. ochs the identification of single gene defects involving genes that play crucial roles in adaptive or innate immunity is not only important for confirming a pid diagnosis, but may contribute to optimal therapy, and contribute to genetic counseling, carrier identification and pre-natal diagnosis. to accomplish this, the diagnostician has to consider the inheritance of these disorders: x-linked, autosomal recessive, autosomal dominant and the type of mutation: loss of function, hypomorphic, dominant negative or gain of function. conventional techniques to screen for single gene mutations include flow cytometry to measure disease-specific expression of proteins (cell surface, cytoplasmic or nuclear) or to analyze relevant signaling pathways (e.g. stat5b phosphorylation via the il-2r; pstat3 via the il-10 receptor); and sanger sequencing of mrna or genomic dna using dye-terminator sequencing. next generation sequencing ("by synthesis") has been refined, and is being used increasingly to study families with multiple affected members with an atypical pid phenotype, or to explore consanguineous families with one member affected. whole exome sequencing requires less data analysis, compared with whole genome sequencing, but may miss intronic or regulatory elements. the challenge of whole exome/genome sequencing is to confirm that the multiple variants identified by these techniques are causative for the clinical phenotype of the study patient. however, with increasing experience, next generation sequencing will become a standard procedure for the identification of genetic defects responsible for inherited diseases, including pid. stephen jolles immunodeficiency centre for wales, university hospital of wales, cardiff, uk. immunoglobulin (ig)-replacement therapy represents the mainstay of treatment for patients with primary antibody deficiency and is administered either intravenously (ivig) or subcutaneously (scig). recent developments using a high-purity recombinant human hyaluronidase have allowed the longer term repeated use of this enzyme to facilitate the delivery of immunoglobulin and other molecules including antibiotics, local anesthetics, insulin, morphine and fluid replacement into the subcutaneous space. hyaluronidase facilitated scig (fscig) has helped overcome the limitations on the volume which can be delivered into the subcutaneous tissues by enabling dispersion of scig and its absorption into lymphatics. the rate of facilitated scig infusion is equivalent to that of ivig, and the volume administered at a single site can be greater than 700 ml, an enormous increase over conventional scig, at 20-40 ml. the use of fscig avoids many of the systemic side effects of ivig, and has higher bioavailability than scig. over three years of safety data are now available for this approach though longer term safety data and information on anti-hylauronidase antibodies and their relevance will be required. fscig could aid several areas of patient management in both primary antibody deficiency and immunomodulatory indications. key factors influencing how it will be used in future are long-term safety data and cost-benefit analysis. date after the cd19 deficiency was firstly described in a turkish girl in 2006. the patients with cd19 deficiency had normal b-cell differentiation in bone marrow, normal absolute number of b cells in peripheral blood and normal bcr repertoire. also, the patients had normal stimulation via the bcr, normal proliferation response upon antigen stimulation but reduced memory-b-cell compartment in peripheral blood. the cd19 deficiency leads to hypogammaglobulinemia and impaired antigen-specific humoral immune responses after vaccination. we had described thirty carriers in relatives of our two patients with cd19 deficieny and also showed that the mfi value of cd19 and cd21 expressions were lower in the carriers than in controls. during the last five years, it was also showed that the mutations of the other coreceptors of b cells such as cd81 and cd21 caused antibody deficiency. in conclusion, the description of cd19 deficiency reminds the importance of the molecules on b cells and contribute to identify new genetic defects (cd81, cd21), and it was showed that coreceptors could affect the expressions and the functions of each other. ege university faculty of medicine, dept of pediatric immunology, izmir, turkey ig class switch recombination deficiencies are rare pids (1:500,000 births) with normal or elevated serum igm and low igg, iga and ige levels, defective or normal somatic hypermutation, defective t/b cooperation (50%), intrinsic b cell defect (50%), susceptibility to bacterial infections begining from the first year of age (impaired b cell immunity) and lack of germinal centres in secondary lymphoid organs. we present a cd40l defective case with clinical findings such as recurrent otitis media, recurrent upper and lower respiratory tract infections, sinusitis, arthritis, relapsing polychondritis , ebv-associated cervical lymphoproliferation, cmv infection, bronchiectasis, liver and spleen enlargement, multiple nodules in the liver, chronic diarrhea due to persistent cryptosporidium parvum, fungal pneumoniae, osteoporosis, and schwannoma. this case is remarkable with low igm levels and normal cd40l ezpression on activated t cells although he had a novel mutation in cd40l gene (a novel missense mutation in cd40lg (c. c139t), leading to an a. a. change from histidine to tyrosine at position 47 (h47y) at the start of the extracellular domain). in addition, we present two cases with cd40 deficiency with normal cd40 expression on b cells.both of these cases had homozygous-cd40-mutation leading to a longer protein due to deletion of stop-codon. in conclusion; cd40 molecules although non-functional in b cells, may be normally expressed on cell surface. these cd40 molecules are unable to trigger signal, because cd40l + il4 activation leads to complete lack of proliferation. evaluation of cd40 or cd40l expression by flow cytometry may lead false results. study of cd40l + cytokine (or cd40+ cytokine)induced b cell proliferation appears as a useful tool for these diagnosis. institute for immunology and physiology (ub ras). yekaterinburg, russia 2 regional children clinical hospital №1, yekaterinburg, russia the ural regional center of clinical immunology, which based on children clinical hospital number one (№1) in yekaterinburg, observe patients from different territories of ural region and neighboring areas. it consists of laboratory department, consultative department, vaccination and treatment rooms, beds and boxes in special departments in the regional children hospital. the close collaboration with j-project started in 2009. this is an example of such collaboration. in patient a. an international consilium was diagnosed a progressive neurodegenerative disease as a manifestation of primary immunodeficiency: x-linked agammaglobulinemia with b-cell deficiency. mri results: unspecified leukodystrophya rapidly progressive multifocal brain lesions with demyelinating, generalized cerebral atrophy, iii degree, signs of periventricular leukomalacia in the anterior horns of the lateral ventricles. the brain biopsy was recommended in order to clarify the nature of the defeat of the pathological process and define the role of the immune mechanisms of its development (held in the neurosurgical department with subsequent histological and immunohistochemical studies). histological and immunohistochemical study of the brain tissue of the right frontal lobe: a signs of productive meningoencephalitis in brain tissue with vasculitis, perivascular and focally moderate diffuse infiltration of mononuclear (accumulation of mononuclear cd45rb+, vimentin+), most of which are cd8 + lymphocytes with granules of granzymeb. around -dystrophy and necrobiosis neurons, intracellular edema, small focuses of gliosis -there are isolated myeloid cells (myeloperoxidase +) and plasma cells with cytoplasmic expression of immunoglobulin light chains lambda and kappa; cells and the extracellular matrix of brain tissue expressing cd56 antigen and s100 protein. virological and bacteriological studies of brain tissue and liquor: connection of progressive degenerative changes and infectious process weren't obtained verified acknowledgments of an infectious or autoimmune process has not been received. search for a genesis of cytotoxic process in the brain continues. center for chronic immunodeficiency, university medical center freiburg and university of freiburg, germany the essential role for igg replacement therapy (iggrt) for common variable immunodeficiency (cvid) has been demonstrated in many studies and metaanalyses. while patients with "infection only" reach a nearly normal life expectancy -though still not quality -under iggrt, cvid patients with additional manifestations like inflammatory lung, bowel or liver disease, lymphoproliferative and/or autoimmune disease often require additional immunosuppressive treatment. there is little consensus on the form of immunosuppressive regimen, once steroids have failed, with possibly the one exception of rituximab treatment for autoimmune cytopenia. additional studies are essential to guide therapeutic algorithms. some of these patients suffer from late onset combined immunodeficiency (locid). as in classic forms of combined immunodeficiency, iggrt can be only a part of the treatment strategy, which needs to additionally address the cellular immunodeficiency of the patients. therefore a retrospective survey was performed on patients diagnosed with cvid who underwent hematopoietic stem cell transplantation. the results of this study are currently in revision. in summary, iggrt is the baseline therapy for cvid but does not address sufficiently the immune dysregulation in a subgroup of patients. better predictive markers have to be identified for the selection of patients for additional, potentially even definite forms of treatment in order to prevent the morbidity and mortality associated with these secondary manifestations of cvid. the first department of primary immunodeficiencies in russia was established on the basis of the institute of immunology in 1980, when 3 patients with pid were registered. currently, 327 patients with pid are followed in the department of immunopathology in adults. 65% of pid adult patients have pid with immunoglobulin deficiency. analysis of this group of adult patients showed that the diagnosis of pid, on average has a delay of 10-15 years from the first symptoms. in 88% of cases, there is an infectious clinical phenotype, 32% -combined infectiouslymphoproliferative phenotype, 27% -infectious and enteropathy. the study of immunophenotyping of b-lymphocytes for the degree of maturation in this group of patients was begun. 13 patients are currently included in this study. 2 patients showed complete absence of b-lymphocytes, 11 -the reduction of b-cells, 2 patients of those have a normal amount switched memory b cells (mbc), 9 people -a decrease amount of switched mbc. 8 persons of the group with decreased amount of switched mbc had an expansion of transitional mbc. at present, a clear link of immunophenotypes with specific clinical phenotypes in not found, but this may be due to small sample of patients at the moment, the investigation continues. for the treatment of this category of patients only intravenous immunoglobulins are available in russia. we use drugs in various concentration of russian and foreign production. the availability of immunoglobulins for the adult patients unfortunately is not sufficient in russia, so the recommended pretransfusion level of igg is not achieved in about 70% of patients. our work presents the experiences of our center with the subcutaneous form of immunoglobulin therapy (scig). we have 64 patients on such therapy. the youngest child is 7 months old. the largest group consists of cvid patients, next-xla patients. we also substitute children diagnosed with the dgs and accompanying hypogammaglobulinemia and some children with subclasses deficiency as well as secondary hypogammaglobulinemia. in most cases we start therapy with intravenous preparates, but there have been some children to whom we proposed the subcutaneous form at the initial stage of the therapy. the main factors which made us change the mode of the drug application were adverse reactions to ivig, poor vein access and the parents`wish. the administration of scig is very rarely complicated by severe adverse reactions (the risk of their incidence amounts to about 0.3%). even patients with serious side effects to previous immunoglobulin therapy and/or blood transfusion can be safely treated with scig. the most common side effects are local reactions but their incidence decreases during following substitutions. we can observe swelling, redness, induration, soreness. but we should remember that more severe side effects are also possible, for example: the first cvid patient presented with fever, weakness, difficulties in breathing during the 3 following infusions. changing the brand of the drug turned out to be a sufficient method of getting rid of side effects. the second patient, also with cvid, suffered from nausea, headache, meningismus. we changed the drug brand, slowed down the infusion rate and introduced premedication with an antihistaminic drug. the third patienta girl with dgs and hypogammaglobulinemia, after having been operated on for hypoplastic left heart syndrome, responded to infusions with high fever, muscle and joint pain, skin changes (erythrodermia). we introduced premedication, changed the drug brand and slowed the infusion rate, yet without any positive effects. in two patients we observed adverse reactions after preparates at a concentration of twenty percent. there were: weakness, chills, fever, headache, and very intense pain in the place of injection. during the subcutaneous treatment of xla patients, we observe significant reduction in the number of infections and days of school absence. despite that, all our patients with xla suffer from chronic sinusitis. similar results occurred in cvid patients, but the severity of infections was the same. the use of scig results in more stable and higher igg through levels especially in xla patients. in our practice, we had only a few cases in which iv form appeared to be better than sc one. in the case of two boys with higm syndrome, we observed recurrent enthesitis of the first patient and progression of lung fibrosis of the other. ivig was better to control platelets levels in the girl with cvid and thrombocytopenia. it has also occurred that parents refuse to allow us to start subcutaneous therapy, giving two main reasons: they feel safer under frequent doctor`s control and they are afraid of making mistakes in procedures. as for the youngest children (below 7)their fear of needle is independent of its size, which is the third reason. in conclusion, we would like to emphasize that education programmes implemented by doctors and nurses are essential to make this form of therapy easier, safer and more satisfying for patients and their parents. despite intensive investigation into the nature of cvid, the exact molecular defect(s) and pathogenesis of disease remain unknown. our aim was to evaluate the role of t cells in the mechanisms of cvid development. additionally the impact of some innate and adaptive immunity related genes (hla, cytokine gene polymorphism, mbl genes) was investigated. based on previously observed by us constellation of shared immunogenetic profiles a comparison of t-cell phenotype of cvid patients, and elderly/young healthy individuals was performed. ten patients with cvid were enrolled (4 male, 6 female; average age -42,9 years) presented mainly with pulmonary infections, followed by bronchoectasis and splenomegaly. our study demonstrated multiple t-and b-cell abnormalities in cvid patients such as: decreased cd4+, increased cd8+ t cells and low cd4/cd8 ratio, loss of naïve and early differentiated t cells, expansion of terminal effectors (cd8 + cd45ra + cd62l-) t cells, memory/effectors (cd8 + cd28-cd27-) and terminally differentiated (cd8 + cd57+) t cells. excessive t-cell activation reflecting the prevalence of activated t cell phenotype was also detected, due perhaps to an antigen-driven process. the very low numbers of circulating mature (cd21 + cd24+) and class-switched memory (igm-igd-cd27+) b cells were pathognomic for our patients and could be used as an additional diagnostic criteria in the national guidelines. furthermore high level of nonclass switched (igm + igd + cd27+) b memory cells and suppressed nk cell count was observed. decreased responsiveness to polyclonal stimuli via cd3 and cd28 pathway correlated with the loss of cd28 expression which was more pronounced in the treatmentnaïve cvid patients. these findings were further discussed in the context of the similarities that exist along with markers for immune senescence (lack of cd28 or expression of cd57). increased frequency of ifn-γ polymorphisms associated with low expression level found could indicate genetically predisposition to high activation of th2 lymphocytes in cvid and consequently support the concept of impaired th1-type responses. in conclusion our study provided new insight into the pathogenesis of cvid. this work was partially granted by medical university sofia, grant#55 bcg vaccination at birth is the constant element of vaccination programmes in poland. high reactogenic bcg danish vaccine has been replaced in 1955 , by bcg moreau vaccine. frequency of disseminated bcg infection, in children with primary immunodeficiencies after bcg moreau vaccine manufactured by biomed, poland were estimated. one thousand five hundred sixty three cases of primary immunodeficiencies were diagnosed in the department of immunology, children's memorial health institute in warsaw between 1980 -2013. among patients with t cell predominant deficiency, group high risk of bcg infection, scid was recognized in 62 children. mendelian susceptibility to mycobactarial diseases (msmd) was detected in four patients: ifgr1 deficiency and il12 deficiency -equally in two patients, and nemo -in one. in the group of primary immunodeficiencies regarded to be less prone to mycobacterium infections, cgd was diagnosed in52, hies in 20 patients, and xl-higm in 4 patients. disseminated bcg infection was recognized in 10 scid patients, 2 of them died, because of bcg diseases. all 4 patients with msmd developed bcg infection, one with il-2 deficiency died. during nearly 30-year-follow-up, no case of tuberculosis or disseminated bcg infection have been diagnosed among cgd , hies and xl-higm patients. early anti -tb drug prophylaxis and usage of wide range of antibiotics in therapy is crucial for cleaning of bcg infection. peter čižnár 1 ; julia horáková 2 ; peter švec 2 ; ivana boďová 2 ; sabina šufliarska 2 ; linda libai veghová 1 ;, marieta hricová 1 1 1 st pediatric department, comenius university medical faculty, children`s university hospital, bratislava, slovakia. 2 transplantation unit, department of pediatric haematology and oncology, children`s university hospital, bratislava, slovakia. objectives: severe combined immunodeficiency (scid) is a group of disorders due to more than 15 genetic defects, characterized by increased susceptibility to severe infections and early life death. the diagnosis is supported by the demonstration of low absolute t lymphocyte count variably associated with numerical defects of b and nk cells. patients are very heterogeneous regarding clinical course, immune parameters and clinical outcome. bcg (bacillus calmette-guerin) vaccine, a life attenuated vaccine was the part of slovak immunization program, administered at birth until 2011. a comparison of clinical course of bcg exposed (bcg+) and non-exposed (bcg-) scid patients in slovakia in period of past 10 years are given. results: incidence rate of diagnosed and treated scid in slovakia was calculated to 1:87.000, meaning 1,5 cases per year. in total 10 cases represent 4 ada patients, 2 il2rg deficiencies, 1 case of complete del22q11 and in 3 cases genetic defect was not found by analysis of rag1/2, il2rg, artemis, il7ra, jak3 and ada genes. all patients were confirmed absent trec (t-cell receptor excision circles) copies in a retrospective neonatal guthrie card analysis. seven out of these 10 patients underwent hsct, in 5 the hsc source was a mud. favorite outcome was achieved in 6 of them. half of our patients have been exposed to a live bcg vaccine during neonatal period. patients vaccinated with bcg faced severe complications and organ damage due to generalized skin and organ abscess formation, requiring prolonged (up to 18 months) hospital care and complex antibiotic therapy with more than four types of anti-mycobacterium drugs, for more than 2 years. average length of hospital care for bcg exposed patient was 10,8 months vs. 2,7 months in non-exposed group (p < 0,05). no statistical difference was found between the time of recognized first symptoms, and time of diagnosis in bcg + and bcg-group. the clinical presentation of non-bcg vaccinated patient differs in the initial symptoms when failure to thrive and pneumonia at 4 months was the most common finding. post-transplantational recovery in bcg-group was less complicated. conclusions: two major improvements for the outcome for scid patients in slovakia have occurred in past 10 years. early life vaccination for tuberculosis has been retreated and improvements in diagnostics for severe t cell defects have been made, including flow cytometry phenotyping and genetic testing within the middle european countries cooperation, the j-project. bcg and late diagnosis prolongs time for hospital care, immune reconstitution and carries severe complications, consequently it increase the costs of health care and decrease the quality of patients' life. the perspective of newborn screening for scid would be the next major step in improving the outcome of scid patients. ahmed aziz bousfiha 1 ; leïla jeddane 1 ; nahla erwa 2 ; monika esser 3 ; shereen m. reda 4 in africa, primary immunodeficiencies are still largely undiagnosed, with no cases reported in 40 of 54 countries. though the african society for immunodeficiencies (asid) already organized 3 international meetings and 5 training schools, their impact outside the hosting country is still insufficient. at this time, only a few pid patients are reported in africa (less than 2000 patients), the majority of whom are in north africa and south africa. so, asid propose the a-project, a training program based on the j-project. some issues prevent effective training for pid in africa: diversity of languages, only a few are initiated to pid, lack of resources for travel expenses, difficulty to access to care and shading by the hiv pandemic. a-project is designed as a one-day training by an african pid expert in a small group of motivated caregivers. this project is adapted to the african context, as it only requests minimum funding and can reach more people. each a-project will be co-organized by asid and a local committee, and shall lead to some commitments to be realized by locals, in particular the establishment of a registry, a network between physicians and scientists and creation of a patient association. moreover, each a-project will be done in the medical language used in the country (english, french or portuguese). the first a-project was already done in benin, and five more are already planned for 2014. our goal is to reach all 40 countries where no patient were reported in 3 years. this clinical program will raise pid awareness in africa and can potentially discover new aspects of the immunity. till very recently primary immunodeficiency diseases (pids) were not being frequently recognized in india. however, the scenario has changed over the last 5 years or so. the indian society for primary immune deficiency (ispid) was founded in 2010-2011. over the last 4 years the ispid has organized 2 international conferences (at new delhi and mumbai), 2 national conferences (at chandigarh and varanasi) and 2 continuing medical education programmes (at new delhi and lucknow). these meetings have served to act as catalysts for the cause of pids and have resulted in increasing the awareness about these conditions amongst paediatricians and physicians in our country. several centres now have the clinical skills and the technical wherewithal to perform laboratory investigations for these patients. the repertoire of tests includes nephelometery, elisa based tests and flow cytometry. facilities for molecular diagnosis of pids are also being developed at some of these centres. a lot more, however, needs to be done. the clinical phenotype of several pids in india is likely to be different from that in the west 1 . further, the type of infections in these patients is also likely to be different. this is because of the differences in the micro-and macro-environment to which these patients exposed in developing countries. these differences have been well brought in the recent publication on chronic granulomatous disease from our centre 2 . further, the genetic background of the indian population is diverse and several new mutations are likely to be identified amongst these patients. the indian council of medical research has taken up the lead in this regard and is proposing to set up 2 centres for advanced research (cars) in pids -1 each at the post graduate institute of medical education and research, chandigarh and the institute of immunohematology, mumbai. the foundation for primary immunodeficiency (fpid), usa has also been closely involved in these efforts and has helped facilitate the development of these 2 cars. the field of pid research in india is wide open and we are likely to witness new and exciting scientific developments in the coming years. references: 1. gupta s, madkaikar m, singh s, sehgal s. primary immunodeficiencies in india: a perspective. annals of the new york academy of sciences 2012; 1250: 73-79. the prevention of pid's complications, the improvement of the health care of patients with pid, the creation of the registry of the patients with pid, the implementation of the finance regulations for detection and treatment of patients with pid diseases in public health facilities, the training and professional development of medical professionals in this field. objective: to improve the detection and diagnosis of pid diseases and the life quality of patients with pid in the republic of kazakhstan. undertaken activities for realization of the project: 1. professor. l. marodi visited kazakhstan in october 2012 and the meeting was held in ministry of health of the republic of kazakhstan, presentations were given at the international conference held in national research center for maternal and child health, the negotiations with heads of the national medical holding' clinics and scientific center of pediatrics and pediatric surgery were held. international islamic university, kuantan pahang, malaysia. two hundred and sixty three (263) suspected pid (primary immunodeficiencies) cases were referred to clinical immunologist led clinics in malaysia from 1986 -2013. patients referred were from all states of malaysia and seen at the institute of pediatric, hospital kuala lumpur and university associated hospitals in north and central peninsular malaysia the initial pid patients were seen by -1 pediatric immunologist between 1986-2006 followed by another 2, beginning in 2007 followed by the other in 2012. there were 168 (68.7 %) patients with at least 1 abnormality on the immune prameter recorded and regarded as probable pid. however 153 (62.7%) were recorded as pid based on existing criteria. (who scientific committee 1986, iuis scientific committee, primary immunodeficiency disease.1999) our population were mainly children, 83% below 10 years and 36 % below 1 year. only 3 were above 18 years. pid were classified as; predominant antibody deficiencies 34 %, combined immunodeficiencies 19.3 %, other cellular immunodeficiencies 14.7 %, phagogocytic defect 11.3%, immunodeficiency associated lymphoproliferative disorders 2 %. our data differed from most classification where predominant antibody deficiency is most frequent as high as 65 %, (steihm, 2004) . of the specific 153 pid recorded, x linked a gammaglobulinemia (xla) 19, hyper igm syndrome (higm) 11, common variable immunodeficiencis (cvid 7), selective iga deficiencies 7 severe combined immunodeficiencies (scid) 17, di george syndrome (dgs)12,chronic granlomatous disease (cgd) 14, hyper immunoglobulin e syndrome (hige ) 14, primary cd4 deficiencies 6, ataxia telangiectassi 3 % malaysia comprises of multi ethnic groups with a population of 29.6 million in 2013. pid amongst them showed, malays at 61.4%, chinese 11 %, indians 23.7 % and others 5.2 % whilst the male predominate over female at a ratio of 2.8:1. family history of affected sibling or in first degree relative, or early death with suspected infant dying of infection was positive in 53.3 % which is higher than in most reports eg egypt 23.4 % (reeda 2009). this could be due to high consanguinity in the population. alternatively the symptomatic sibling of affected patients is more likely to be referred to the clinical immunologist. scid records the most varied organism from the positive microbiological isolate viz bacteria 4, fungus 2, virus 1, parasite 2. chromobacterium violaceum was seen in 3 cgd patients in which 2 deteriorated with eventual death. as in many national registries diagnostic delays remains prominent. in our series the mean diagnostic delay was o 3.61 ± 4.82 years. in comparison thailand stands at 2.1 ± 2.6 years, while france, a median of 1.3 years. malaysia remains committed to provide better diagnostic services and improved care of the pid patients through research collaboration with foreign partners with a drive for creating subspecialty training. patient groups aligned to ipopi is now closer to its formation with the creation of its protem committee ensuring that patients' interest will always be guarded aknowledgement. the authors thank all who had contributed directly or indirectly, especially medical officers, nurses, consultant pediatricians especially dr kamarul azhar, institut of pediatrics hkl, laboratory scientists especially dr shanaz murad of the imr kuala lumpur university of manchester, uk although the concept of grouping mendelian disorders associated with an upregulation of type i interferon (ifn) has not been previously recognised in the medical literature, our past and current work argues that this concept has scientific validity and clinical utility. i will discuss the possibility that such conditions can usefully be considered to represent a novel set of inborn errors of immunity, and that the recognition of diseases as type i interferonopathies will have significance for the development of targeted therapies, as well as informing our understanding of viral and retroelement biology, and the pathogenesis of some forms of autoimmunity. 1 classic kaposi sarcoma (ks) is exceedingly rare in children from the mediterranean basin, despite the high prevalence of hhv-8 infection in this region. we hypothesized that rare single-gene inborn errors of immunity to hhv-8 might underlie classic ks in childhood. we report here autosomal recessive ox40 deficiency in an otherwise healthy adult with childhood-onset classic ks. ox40 is a costimulatory receptor expressed on activated t cells. its ligand is expressed on various cell types, including endothelial cells. the mutant ox40 protein was poorly expressed on the cell surface and failed to bind ox40 ligand, resulting in complete functional ox40 deficiency. the ox40-deficient patient had a low proportion of effector memory cd4 + t cells in the peripheral blood, consistent with impaired cd4 + t-cell responses to recall antigens in vitro. the proportion of effector memory cd8 + t cells was less diminished. the proportion of circulating memory b cells was low, but the antibody response in vivo was intact, including to a vaccine boost. together, these findings suggest that human ox40 is important for cd4 + t-cell memory, but redundant for immunity to most common pathogens, with the notable and surprising exception of hhv-8. the chronic mucocutaneous candidiasis disease (cmcd) is characterized by persistent or recurrent infection of skin, nails, oral, or genital mucosae with candida albicans. il-17-mediated immunity has been concerned in host defense against candida on body surfaces. we have investigated nine patients with chronic mucocutaneous candidiasis disease (cmcd) and signal transducer and activators of transcription 1 (stat1) mutations. the novel c.537c > a (n179k) and c.854a > g (q285r) mutations in the coiled-coil domain (ccd) and the c.1154c > t (t385m) mutation in the dna-binding domain (dbd) of stat1 are gain-of-function (gof) for γ-activated factor (gaf)-dependent cellular responses to stat1. low proportion of il-17a-and il-22-producing t cells, lower levels of intracellular il-17a and il-22 by t cells and impaired candidainduced secretion of il-17a and il-22 by leukocytes from cmc patients compared to that in healthy controls were found. the c.820c > t (r274w) mutation affecting the ccd and the c.1154c > t (t385m) mutation affecting the dbd of stat1 and resulted in gain-ofphosphorylation and gof. these mutant alleles enhanced the cellular responses to cytokines via stat1 signalling pathway. these data provide further insight into the mechanism of host defense against candida. heterozygous gain-of-function stat1 mutation is known as a major etiology of chronic mucocutaneus candidiasis. gof mutation affecting the stat1 coiled-coil domen (d165g) was initially discovered in 13-year boy with cmc. gof mutation of dna-binding domen (t385m) was found in the second our patient with cmc. both patients have early manifestation of recurrent or persistent infections of the skin, mucous membranes, and nails with candida albicans. they also have skin infections with dermatophytes. patient 1 presented from the first months of age with severe recurrent sinopulmonary infections. recurrent pneumonia and chronic bronchitis complicated by bronchiectasis, which resulted in cor pulmonale and congestive heart failure. patient 2 suffered from recurrent hsv infection, recurrent aphthous stomatitis, has several episodes of bacterial skin infections. he also has chronic bronchitis and several episodes of pneumonia, but does not have bronchiectasis. both boys developed esophageal stricture, patient 2 necessitating nissen fundoplication in the age of 5 years. the patients have mild autoimmune features: uveitis (p1) and alopecia (p2). immunological investigation revealed different impairment of immune system: more severe, similar to combined immunodeficiency in p1, which declines with age. the p2 does not have changes in lymphocyte number and immunoglobulin's, but impaired antibody production to pneumococcal antigens. western blotting performed with nuclear extracts of lymphocytes of both patients showed stronger stat1 phosphorylation after stimulation with cytokines ifnγ, ifnα, il-27. mononuclear blood cells from both patients released much smaller amounts of il-17a and il-22 than candida-exposed cells from healthy control. stat3 activation triggers transcription of interleukin (il)-17 which is crucial for mounting protective immune responses against fungi. several mutations affecting the stat3/il-17 pathway have been reported, resulting in selective susceptibility to fungal (candida) infection, a hallmark of chronic mucocutaneous candidiasis (cmc). in patients with autosomal-dominant (ad)-cmc we previously reported defective th17 responses and identified an underlying gain-of-function (gof) stat1 mutation leading to hyperphosphorylation of stat1. how this affects stat3 or leads to decreased il-17 remains to be determined. in patients with ad-cmc, we assessed how gof-stat1 mutations affect stat3 activation, dna-binding, gene expression, cytokine production and the effect of epigenetic modification. we show that stimulation of stat3 in the presence of gof-stat1 mutations leads to significantly reduced transcription of stat3-inducible genes (rorc/il-17/ il-22/il-10/c-fos/socs3/c-myc). this was not due to impaired stat3 phosphorylation, altered nuclear translocation nor sequestration of stat3 into stat1/stat3 heterodimers. dna binding to a stat-consensus binding site construct (hsie) was intact but binding to an endogenous stat3 dna target was impaired. the reduced stat3-dependent gene transcription could be normalized by inhibiting stat1 activation by fludarabine or enhancing acetylation with histone deacetylase (hdac) inhibitors trichostatin a or itf2357. silencing hdac1, hda2 and hdac3 indicated an important role for hdac1. impaired stat3-dependent gene transcription likely underlies decreased th-17 cytokine production, susceptibility to fungal infections and other pathology seen in ad-cmc patients and could be a new target for defining novel therapeutic approaches for this potentially lethal disease. autoimmune features have been long thought as association with immunodeficiency disorders, but are now viewed as a crucial component of some diseases attributed to the breakdown of self tolerance or defects of immune regulators. it had been previously established that a single gene defect of the foxp3 gene (foxp3 in humans) caused widespread autoimmunity in both humans and mice. the clinical syndromes observed in both scurfy mice and humans suffering from ipex are similar to those observed in experimental models in which treg are selectively depleted. in 2003, three groups demonstrated that these diseases were indeed the result of a regulatory cell deficiency. around one third of the patients with clinical manifestation closely resembling ipex syndrome, foxp3 is not mutated, these patients are referred to as ipex like. here we present a case; a female patient 13 years with multiple autoimmune manifestations; dm, coeliac disease and ulcerative colitis with marked decrease in the percent of cd4 + cd25 + foxp3+ cells. as she has a siblings suffering from dm; the whole family was investigated. the father and the mother had 0% cd4 + cd25 + foxp3+ cells. background: hids is an autosomal recessive disease, first recognized as a separated entity at 1984. in patients with hids, the activity of mevalonate kinase is reduced to 5-15% of normal levels. hids is caused by mutations in the mevalonate kinase gene (mvk), located on the long arm of chromosome 12 (12q24). it is manifested by cyclic attacks of fever initiated usually during first year of age. the frequency and severity of attacks tend to decrease later in life. materials and method: a retrospective analysis of medical history , clinical course and laboratory findings of two albanian children with periodic fever , diagnosed with hyper igd syndrome. results: case presentation 1: an 5-year-old boy admitted to the hospital because of periodic fever spikes, which occurred every 3-4 weeks and lasted 3-5 days, presented since the first year of life and coincided with the beginning of immunization. he had a tonsillectomy and adenoidectomy at the age of 3. the fever attacks were associated with chills, malaise, and abdominal pain without gastrointestinal signs. between attacks the patient was free of symptoms. from his family history, recurrent febrile episodes during childhood were reported to his father. physical examination showed normal findings, except for a cervical lymphadenopathy. laboratory: marked increase of erythrocyte sedimentation rate and crp. wbc-ranged from 5.710 to 12 000/mm 3. , high asto. serum igd was repeated several times and was always elevated (mean value: serum igd 122 iu/ml). the mutation v377i is found from the genetic examination done for gene mutations in chromosome 12 (12q24). he repeated attacks after initial treatment with corticosteroid ,than is suggested. the second case was a four-year-old girl hospitalized five times because of prolonged fever, and diagnosed as pneumonia, tonsillitis, acute otitis media and sinusitis, treated by antibiotics. her laboratory findings were not remarkable except for increased acute inflammatory responses. serum amyloid a (saa) 1100 μg/l (8 μg/l) and igd was extremely high 181.9 iu/ml. genetic examination for two mutations were negative, but reduced mevalonate kinase activity in white blood cells was demonstrated in more thorough investigations. treatment regime: colchicine conclusions: auto inflammatory syndromes always pose diagnostic and therapeutic challenges to the clinicians. the clinical description of the diversity of periodic fever syndromes is helpful in the assessment and management of these patients. although hids is predominantly identified in populations from northern european areas, it has to be considered in children with periodic fever. anastasiia bondarenko 1 ; liudmyla chernyshova 1 ; iryna sychova 2 1 shupik national medical academy of postgraduate education, kiev, ukraine. 2 dniepropetrovsk regional children's hospital, dniepropetrovsk, ukraine. background. aspergillus is an actual pathogen in chronic granulomatous disease responsible for about 20% of all infections. in 70-80% lungs are involved and in 5% -cns. case. we report a case of combined loci in 9-years old female patient with ar cgd. the child was born from iii pregnancy, ii delivery on 37 th week of gestation with body mass 2750g. she received bcg vaccination at 4 -th day of birth. at 3 months the local inflammation in site of bcg with regional lymphadenitis developed which was treated with isoniazid for 3 months. then bilateral purulent cervical lymphadenitis developed at 6, 9 and 11 months treated with wide spectrum antibiotics. culture from pus was negative. pcr for mycobacterium tuberculosis complex was negative. at18 months systemic infection without loci occurred with fever, lymphadenopathy, hepatosplenomegaly, loss of weight, progressive anemia, inflammatory changes in blood for almost 6 months. bacteriological cultures were negative. treatment with wide spectrum antibiotics was insufficient for 3 months. disseminated bcg infection was suspected and 4-compound amb treatment was started ex juvantibus with positive effect: the fever has stopped, the sizes of lymph nodes, liver and spleen have decreased, the weight of a body normalized. the child suffered from recurrent pyogenic infections, underwent disseminated salmonellosis. at the age of 5 years blood samples were tested at the laboratory of human genetics of infectious diseases, inserm (paris, france). an absence of p22 phox protein expression detected by western blot conferring a complete defect in cyba due to compound of geterozigous mutations in 16q24. at 6 years primary tuberculosis complex of right upper lobe (mbt -) was diagnosed. at the age of 7 during the unexplained fever multiple formations were identified in the liver, biopsy showed caseosis suspected the mycobacterial nature of lesions but mbt (-). at the age of 9 years because of shade in the left upper lobe and ineffective standard antibiotic treatment the tuberculosis again was suspected. due to ineffective antimycobacterial treatment for 8 months multi drug resistant tubercullosis was considered. anti-tb drugs ii line was appointed without clinical response. fever persisted. mri of brain revealed mass lesion in the left parietal lobe. because of suspected tumor the brain biopsy was done and the pus was obtained. microbiological studies revealed aspergillus fumigatus. at the same time subcutaneous tumor-like infiltrate 40 х 20 mm appeared on chest in a proection of lung lesions. the pus was obtained during thoracentesis. result of microbiological studies: aspergillus fumigatus. drainage of abscesses and intravenous voriconazolum led to dramatic clinical improvement and normalization of blood parameters. conclusion. features of our case is spread lesions of aspergillosis with relatively slow progression of infection. high incidence of tuberculosis in ukraine leads to a high suspicion regarding this infection. diagnosis of tb is mainly based on instrumental studies. radiological and histological differential diagnosis between tuberculosis and other infections with granulomas in cgd is difficult. high suspicion of tuberculosis led to late diagnosis of aspergillosis. great north children's hospital, newcastle upon tyne hospitals nhs foundation trust, and primary immunodeficiency group, institute of cellular medicine, newcastle university, newcastle upon tyne, uk even following the introduction of biologic disease modifying antirheumatic drugs (dmards), a small number of children suffering from severe, refractory autoimmune (ai), rheumatic and/or autoinflammatory disorders will not get into clinical remission (cr) and will potentially further suffer from multiple side-effects of combined and long-term immunosuppressive and anti-inflammatory therapies, in particular severe infections (marodi l, casanova jl. jaci 2010; abinun m. ped health 2010). whilst autologous t cell depleted hsct following the immunosuppressive conditioning regimen achieved complete clinical remission in majority of children with severe juvenile idiopathic arthritis (jia) (de kleer im et al. ann rheum dis 2004) , infection-related mortality remains significant (abinun m et al. mol immunol 2009) . therefore, following the success of allogeneic hsct in treating 6 children with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome (nademi z et al. bmt 2014) , we treated further 10 children with different severe ai (alps, autoimmune lymphoproliferative syndrome (n = 3); complex ai disorder (n = 1)), rheumatological (jsle, juvenile systemic lupus erythematosus (n = 1); jia (n = 2)) and autoinflammatory disorders (mkd, mevalonic kinase deficiency/ traps, tnf-receptor associated periodic fever syndrome (n = 1); eoc, early onset colitis (n = 2)). overall, 14 of the 16 children are alive (follow up 1-11 years), 12 in complete and 1 (complex ai disorder) in partial cr, original disease (alps) relapsed in 1, and 2 children died (1 each with alps and eoc). 2 children had significant, but transient acute (grade 3-4) and chronic (limited) graft vs. host disease (gvhd), 3 experienced multiple virus reactivation(s), and remarkably we saw significant secondary ai diseases post-hsct (transient nephritic syndrome (n = 2) and cytopaenias (n = 1); psoriasis, n = 1; and thyroid disorders (grave's thyrotoxicosis and hypothyroidism), n = 2). our data add to the positive experience and evidence acquired over the last 10-15 years (daikeler t et al. bmt 2009; snowden ja et al. bjh 2012) to propose the allogeneic hsct as a viable treatment option for the small group of children suffering from severe autoimmune disorders. the wiskott-aldrich syndrome (was) is an x-linked primary immune deficiency disorder characterized by thrombocytopenia, microthrombocytopenia, recurrent, mostly respiratory tract infections, eczema and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a 502-amino acid protein. wasp plays a critical role in actin cytoskeleton organization, signalling and different functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries, turkey, iran and azerbaijan. clinical and laboratory information of 116 affected males and 63 carrier females from 106 was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified 77 unique mutations including 23 novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes (20 missense and 14 nonsense mutations), 8 small insertions, 22 deletions, and 12 splice site defects. this study was financially supported by the tübi̇tak project 110s252 and bap project tda-2013-4075. background: chronic granulomatous disease (cgd) is a rare primary immunodeficiency disorder of phagocytes. resulting in impaired killing of bacteria and fungi. a mutation in one of the four genes encoding the components p22 phox , p47 phox , p67 phox and p40 phox of the leukocyte nadph oxidase leads to autosomal recessive (ar)-cgd. a mutation in the cybb gene encoding gp91 phox leads to x-linked recessive cgd. methods: we report here the results of genetically and functionally characterized 100 patients with cgd from 83 turkish families in turkey. results: most of the families (62%) have an ar genotype (%28 p22 phox , %20 p47 phox and %14 p67 phox ) and 38% have an x-linked genotype. patients with a22 0 , a67 0 and x91 0 phenotypes with oxidase null activity (dhr stimulation index of ≤1.5) were found in 73 patients. however, in p47 phox deficient cases and in 7 other ar cases with high residual oxidase activity (dhr stimulation index ≥3) were found in 27 patients. conclusions: residual oxidase activity is similarly lack in the x91 0 , a22 0 and a67 0 phenotype except 7 ar cases with missense mutation. in our cohort, the percentage of ar-cgd was different from european and usa registries (in comparison with %5, %5 and 30% of p22 phox , p67 phox and p47 phox deficient ar-cgd cases, respectively) with the higher percentage of patients with p22 phox (28%) and p67 phox -deficent (14%) phenotypes, and the lower percentage of patients with p47 phox -deficient (20%) phenotype. the basic difference in our results from those reported is the higher percentage of patients with ar-cgd (%62), which was lower than in the european and usa registries, probably because of the higher prevalence of consanguineous marriage in turkey. introduction: schnitzler syndrome is an autoinflammatory disorder of unknown etiology. at least some of its clinical presentation is mediated through an activation of inflammasome and release of il-1, as was repeatedly demonstrated by a prominent therapeutic effect of il-1 blockade. recent reports bring an evidence of an important role of mitochondria in inflammasome activation and in a pathogenesis of autoinflammatory diseases. we have therefore investigated mitochondrial function and structure in patients with schnitzler syndrome. materials and methods: activity and amount of oxidative phosphorylation complexes (oxphos) were analysed by spectrophotometry, histochemistry and imunoelectrophoretic methods in fibroblast cell lines derived from skin biopsies of three adult male patients with schnitzler syndrome. ultrastructure of mitochondria, mitochondrial network and reactive oxygen species (ros) were analysed by fluorescent and electron microscopy. results: the activities and amount of oxphos complexes i, iii and iv were decreased in patients with schnitzler syndrome. interindividual differences in the degree of impairment (from severe to moderate) in analyzed mitochondrial parameters were found. content of ros, previously suggested as main inducers of inflammasome, were not significantly increased in cells with schnitzler syndrome. we, however, did find consistent and prominent changes in mitochondrial structure of all three patients. disturbed mitochondrial network and mainly abnormal, partially swelling mitochondria with unusual and sparse cristae were characteristic for all patients. we did further notice marked accumulation of neutral lipids in all tested fibroblasts. conclusion: severe structural damage of mitochondria associated with milder functional changes represented a consistent feature found in all tested schnitzler syndrome patients. along with progress in basic and clinical immunology worldwide, the knowledge and activities in the field of primary immunodeficiencies (pids) have developed during last two decades. in 1997, a group of junior doctors and students joined seniors in this filed to establish iranian primary immunodeficiency registry (ipidr). several national and international research projects have been done so far which led to lots of publications, while improving the diagnosis of patients with pids, construction of iranian primary immunodeficiency association (ipia), and establishment of research center for immunodeficiencies were other activities which lead to better management of the patients. organizing annual meeting on clinical immunology and immunodeficiencies, celebrating pi week annually and active participation in the international congresses were all helped in to increase knowledge of physicians in the country. the overall activities in the field of pids led to an increased trend in recognition of more patients in the recent years, which was associated with decreased delay in diagnosis. based on recent report of the registry, published recently in the j clin immunol, more than 730 new patients with pid, in addition to previously 930 reported patients, were presented. predominantly antibody deficiencies were the most common form of disease, followed by combined immunodeficiencies, congenital defects of phagocytes, and other well-defined syndromes with immunodeficiency. the rapid progress in identification and registration of the patients with pids is important not only as of epidemiological aspect, but also as of timely diagnosis and appropriate treatment of the patients. the j project physician education and clinical research collaboration program was launched in 2004 in eastern and central europe (ece). in less than 10 years, it has achieved remarkable success. this project aims to increase knowledge in the field of primary immunodeficiency disorders (pid), and to improve the diagnosis and treatment of patients worldwide, particularly in countries with limited economic resources, which currently report fewer such patients than expected. in most ece countries, gene sequencing, which can provide a definitive diagnosis of pid, still remains unavailable. by contrast, such technology is used elsewhere to detect the more than 200 pid-causing genes that have been discovered in the last three decades. thus, pid awareness programs like the j project remain critically important, to improve diagnostic facilities and treatment and to promote clinical research collaboration. this paper highlights the achievements of the j project and the spread of its concepts and spirit to the countries of western asia. primary immunodeficiencis (pid ) are rare genetically determined diseases , occurring with an incidence of 10 per 100 000 inhabitants. it is heterogeneous group of disorders, from quite commonly found and usually asymptomatic iga deficiency (1 in 600), to a very rare diseases such as chediak -higashi (1 in 2 000 000 inhabitants). in 2005, within the framework of a government research project no. pbz-kbn-119/p05/ 2005 -" development , improvement and implementation of highly specialized diagnostic procedures for immune-mediated diseases", a network of cooperating national centers for diagnosis and treatment of pid, named polish working group on primary immunodeficiencies (pgr pno) was founded. as a result of joint efforts of the group as well as an implementation of three eu grants [euro-pid -nas qlrt -2001 -0274 ( 2001 -2004 , euro-policy -pid sp23 -ct-2005-006411 , euro-gene -scan (223, 293)] the number of centers actively working in the diagnosis and treatment of primary immunodeficiencies increased. up todate pgr pno includes 15 pediatric centers , and since 2012 -11 centers for adults. development and dissemination of new diagnostic and therapeutic standards contributed significantly to the increase in detection pid. with early diagnosis of the disease -the implementation of appropriate treatment, including gamma globulin replacement therapy together with quality of life has improved. in spite of all efforts recognition of pid in poland is very rare and currently is 1.44 to 100 000, what is almost 10 times smaller than in europe (www.esid.org). at the moment, a nationwide registry of children and adults with pid consists of 3 368 patients. in september 2013 pgr -pno summarized in the annual report the current status of the substitution therapy with intravenous and subcutaneous immunoglobulin therapy in children and adults with pid in poland. on the basis of available information, half of all patients (1 684 children and adults) were diagnosed to have antibody deficiency. these data are similar to the register of a european database esid , where the percentage of patients with pid with a predominance of antibody deficiency is more than 56% (www.esid.org) development and dissemination of new diagnostic and therapeutic standards as well as a national cooperation contribute significantly to the increased detection of pid. early diagnosis of the disease is followed by earlier implementation of appropriate treatment, including gammaglobulin replacement therapy together with improvement of quality of life. mutations of was were identified in 38 was boys and in 10 heterozygote mothers. frequently genetic damages occur in 1,2,7,10 exones and 6,8 intrones: deletions (11), splice-site mutation (9), missens (12), insertions (6). deletions and insertions lead to stop-codon in 7 cases. 31 nbs patients were homozygous for 657del5 mutation and oneheterozygote for 657del5 had 681delt x-cgd: deletions (10) and nonsens (6) mutations in different exons of cybb were observed most often. in 33 families was performed prenatal diagnosis: x-scid-4, higm1-4, xla-2, was-6, nbs-6, a-t-4, cgd-5, xlp-1, dnalig4-1. healthy children -31. recurrent severe complicated infections developed in 90% of pid patients. antibody deficiencies: bacterial infections -100%, enteroviral -17%, tuberculosis 2 cases, atypical mycobacteriosis -1 case. combined pid: bacterial infections -100%, fungal -40%, viral -37%, opportunistic (pneumocystis jiroveci) -48%, mycobacterial -70% (24 -complication of bcg vaccination, 5 -tuberculosis or atypical mycobacteriosis). cgd: bacterial -62% (staph.aureus, e.coli, b.cepatia, salmonella spp., klebsiella spp.), mycobacterial -75% (85% bcg origin, 15% -tuberculosis), aspergilosis -30% immune disregulation syndromes (xlp1 [10] and alps [41]): bacterial infections -20%, viral -8% (ebv). autoimmune violations were observed in 56% of all pid cases: 5% of combined pid, 35% of antibody deficiencies, 65% of other well defined syndromes, 100% of autoimmune syndromes. cytopenias developed in 68%, vasculitisin 15%, ulcer colitisin 14%, arthritisin 11%. oncology diseases developed in 4% of patients: mainly in nbs, a-t, was and cvid: t-and b-leukemia's, lymphomas, solid cancer. the study of primary immunodeficiency is a unique model for studying the molecular basis of immunity. question about intravital pid verification is still relevant. a regional center of clinical immunology was established in 1986 at the regional children's clinical hospital №1, yekaterinburg. the regional clinical hospital №1 joined the center in 2013. institute of immunology and physiology, ural branch of the russian academy of sciences carries scientific management of the center. the centre works closely with foreign counterparts in the international project j project and it has been one of the centers of jmf since 2013. over 28 years we formed a regional register of patients with primary immunodeficiency, comprising 309 patients: 224 children and 85 adults. analysis of the regional register allows to investigate the causes of deaths in patients with pid. infectious syndrome mortality -sepsis, generalized mycobacterial infection -22 patients, proliferative process -4 patients, dominate the mortality structure. creation of specialized immunological centers permit to raise the educational level of the medical staff, precisely identify nosological forms of pid among different groups of patients, to prevent the birth of children with pid, increase the length and quality of life such patients and take part in the collaboration with international experience in the pid. introduction: the aim of this retrospective study is to determine the frequency, and demographic, clinical and laboratory features of adult cvid patients referred to our clinic. materials and methods: we retrospectively evaluated 26 adult patients (9 female (%34,6), 17 male (%65,4); aged 18 to 67 years: median 27 years) who were diagnosed as cvid according to esid and pagid criteria during a 4 year period (january 2010-march 2014). results: the median current age of 26 patients was 27, and the median cvid diagnosis age was 22,5 years. the diagnostic delay in patients with cvid was 4,5 years (median). cvid patients presented lower levels of igm (12 patients, 46,1 %), iga (16 patients, 61,5 %) and igg (16 patients, 61,5 %). according to lymphocyte lmunophenotypes of cvid patients, cd4 (15 patients, 57,6 %), cd19 (13 patients, 50 %) and cd4/cd8 (13 patients, 50 %) values were observed the most lower ones. discussion: we found that both of the patients with bronchiectasis showed lower levels of immünoglobulins and lower imunophenotypes of b cell than the others that do not have bronchiectasis. in our patients cd4, cd19 and cd4/cd8 values have got enough priority to be mentioned about an immun deficiency. in conclusion, despite recent improvements in diagnostic tools, the diagnosis of mild or moderate cvid is often delayed. however, it seems that the diagnosis of cvid is delayed especially in adulthood on account of the fact that the lack of awareness of these illnesses among the medical professionals all over the world. primary immunodeficiency disease is important in turkey because of the high rate of consanguineous marriage. the lack of awereness about immunodeficiency can cause late-diagnosis and severe complications. the objective of this study was to assess pid awereness before and after clinical immunogy education among medical students. one hundred and thirty-two questionnaires 0 with 71 items (1) were distributed to seventh somestre medical students and 116 (88%) completed questionnaires were evaluated before (first) and after (second) their education about clinical immunology courses for 6 hours. questionnaire scores (qs) were detected as total correct answers. the mean of the first qs was 37.4 ± 3.7 and second qs was 42 ± 4.3 (p < 0.05). there was no statistically difference in gender (60 m and 56 f). of 69 questions, there were 39 related with pid directly. the correct responses rate less than 50% before education were 10 of 39 questions. all participants corrected their responses after education. the best improvement was detected in the responses of the clinical signs related with pid. it was remarkable that the participants have known the family history related with pid excellent before education. the majority of the participants (80%) believed that a lymphocyte count of 2500/mm3 was related to immunodeficiency. nbt and ch50 test were not found to be related with pid before education. it is also important to increase the awareness of pid among the physicians during their education in medical school and more comprehensive education in pid appears to be useful for medical students. chronic granulomatous disease (cgd) is a rare primary immunodeficiency with mutations in nadph oxidase enzyme complex which causes failure of phagocytic cells to produce superoxide and subsequent intracellular killing of microorganisms. we retrospectively analysed medical records of patients diagnosed with cgd in the last 35 years from immunological diagnostic centres from 9 central and eastern european countries (estonia, poland, belarus, ukraine, czech republic, slovakia, hungary, serbia and slovenia) and russia. genetic sequencing from patients' dna was performed in genetic centres in ljubljana, belarus and netherlands for mutations in known genes involved in cgd pathogenesis: cybb, cyba, ncf1, ncf2. we included 104 patients with cgd in our cohort, 7 were female. the mean age at presentation of the disease was 12 months and at diagnosis 3,9 years. lymphadenitis (43%), dermatitis (16%), enteritis (16%), pulmonary infections (13%), liver abscesses (4%) and septicaemia (6%) were the most common clinical presentation. complications of bcg vaccination (28%) were the most common presenting infection. in total 917,4 years of followup in our cohort, the patients suffered 834 different severe infectious episodes (0.9 per year). respiratory (25%), lymph node (25%) and gastrointestinal tract (18%) infections represented the most prevalent severe infections. we identified 87 different mutations out of 104 genes tested. in 78 patients we identified different mutations in cybb gene, 2 unrelated patients had the same mutation in cyba gene and in 5 patients had typical deletion in ncf1 gene. in our cohort we observed high incidence of bcg infections as a presenting symptom. apart from high bcg infections patients included in our study had similar frequencies of infections and infecting microorganisms as patients described in previous series. objectives: the aim of this study is to determine the carrier rate in healthy controls from central european and balkan region. methods: we screened more than 500 healthy subjects from 5 countries in the region. exon 2 and 10 was pcr amplified and subsequently sequenced with abi prism 310 genetic analyzer. results: heterozygous mutations were found in 4% of apparently healthy hungarians, 7% of slovenians, 8% of bosnians, 11% of serbians and in 16% of apparently healthy macedonians. mutations found in hungarian population were as follows: v726a (1), k695r (3). mutations found in slovenian population were: v726a (1), k695r (5) and e148q (1). mutations found in bosnian population were: v726a (1), k695r (6) and f756c (1). mutations found in serbian population were: e148q (6), k695r (5). mutations found in macedonian population were as follows: e148q (8), k695r (7) and m694v (1). conclusion: we found higher than expected carrier rate in screened populations, from 4% to 16%. it is interesting to note that more than half (60%) of detected carriers in all analyzed populations has k695r mutation. progress in the field of primary immunodeficiencies (pids) is reflected in national pid registries. data from slovenian pid registry were analyzed. patients' data were collected retrospectively before 2007 and prospectively afterward. patients were classified according to international classification and updated regularly. data of 193 patients with 45 different pids were analyzed. interestingly, complement deficiencies are the most common, accounting for 23% of all entries. second most common are antibody deficiencies with 19%, followed by well-defined syndromes (16%), immune dysregulation (13%), neutrophil defects (12%), combined deficiencies (9%), autoinflammatory disorders (6%) and defects of innate immunity (2%). prevalence of diagnosed pids in slovenia has changed in the last 5 years; less complement deficiencies and more antibody deficiencies were diagnosed in comparison to previous decades. the number of new pid cases has been gradually increasing, a more prominent increase has been noted in the last 10 years. the prevalence increased most for combined immunodeficiencies, cvid and autoinflammatory disorders. the spectrum of pid entities has also widened in the last decade. three patients with scid were diagnosed and successfully treated in the last three years (incidence -1:25.000 births). high prevalence of complement deficiencies reflects early implementation of good complement diagnostic facilities and awareness among infectologists. this group of patients was prospectively collected from 1987. combined immunodeficiencies, cvid and autoinflammatory syndromes were all probably underdiagnosed before due to lack of awareness among physicians. distribution of pid groups is more consistent with esid registry in the last five years. identification and successful treatment of scid patients in the last years is an important quality marker. bulgarian association for clinical immunology was set up in 2005 aiming to get together all specialists working in the field of clinical immunology. one of the important objectives of the association was to raise the public awareness and attract attention of specialists, national health system, government and other related societies in order to improve the diagnosis and access to treatment for children and adults with pid. efforts of immunologists led to the following results: 1. consensus on the diagnosis and treatment of the basic pid groups was created by the pid national working group that was established in 2010, and specific guidelines were disseminated as well. register for pid patients has been set up in bulgaria that allowed the collection of data on the incidence and prevalence of pid and the negative effect of these conditions on the population. 3. educational program to improve the qualification of the physicians and provide available resources to general practitioners and raise the public awareness were introduced. collaboration with patient's organizations was developed. 5. treatment of pid patients has been fully covered by the public health system since march 2013. all these steps made it possible to advance the diagnosis and management of pid in our country. pediatric pid patients care -single center experience g. petrova; p. perenovska; s. mihailova; e. naumova umhat "alexandrovska", sofia, bulgaria j-project in bulgaria started in 2005 and up to now we have elaborated programme with diagnostic criteria, well equipped laboratory, established some mutual connections with foreign colleagues, held regional meetings, conferences; created clinical standards for treatment and ensured an immunoglobulin treatment and replacement therapy. here are examples of some of the problems we face: 1. seven-years old boy with hypogamaglobulinaemia (normal number b-ly with abnormal function). ivig had some initial effect, but lately we noted very fast deceleration in the overall health status with possible need of lung transplantation. the case is posing a question what more could we do, could we have prevented this rapid worsening. 2. ten-month old girl with scid with severe bcg infection after first vaccination, referred relatively late to our center, but successfully transplanted. the case is posing a question about timing of bcg vaccination and of referral to specialized center. 3. nine-years old girl with unidentified immune deficiency, normal immunological follow up but clinical course as an immune deficiency with very favorable effect of ivig according the parents. the case is posing the question should we stop or should we continue ivig, despite failing to find immunological defect, based on the good clinical response. unfortunately pids are not very well recognized and sometimes the patients are referred late. sometimes poverty and lack of knowledge of patients leads to miscalculation and neglecting of their conditions by themselves, or refusal for specific tests for clarifying the diagnosis. background: although rare, primary immune deficiencies (pid) are manifested with high rate infections as well as with autoimmune and malignant disorders that are treated hardly and inefficiently. pid are not only immunological problem; they require close collaboration between immunologists, pediatricians, ent, lung and gut specialists, dermatologists, hematologists, oncologists, patients and administration. the aim of this study was to summarize the activity on registration and replacement therapy of pid patients in plovdiv region at the university hospital "st. george"-plovdiv for 1 year (03.2013-02.2014). methodology: children with pid of humoral immunity hospitalized at the clinic of pediatrics, and adults with hereditary angioedema (hae) were included in the study using immunological and other lab tests, clinical follow up and treatment: iv and sc ig for children with pid and c1 esterase inhibitor (ruconest, berinert) for hae patients. results: three national workshops and a national conference on pid were hosted and organized in plovdiv since 2005. well established university hospital immunological laboratory, detecting serum immunogloblins, blood lymphocyte populations and subpopulations and complement proteins; clinic of pediatric and genetic diseases and an information center for rare diseases and orphan drugs function in plovdiv. an expert center for diagnosis and treatment of pid was created at the university hospital. it is a team of two competent pediatricians, an immunologist and an allergist. the targets are hospitalized pid children, outpatient pid children and hae adults. the center introduced regular replacement therapy with iv and sc iv and c1 inhibitor, reimbursed by the national insurance. together with icrdod the experts provide education for patients and parents how to perform sc ig application as well as consultations of patients and relatives about pid.since march 2013 indicated reimbursed replacement therapy with iv ig -octagam, started regularly in 5 hospitalized pid children with bruton hypogammaglobulinemia, cvid, omenn syndrome or igg1 id. these patients, aged form 4 to 12 years, had 2 to 6 hospitalizations for one year. four outpatient children with omenn syndrome or igg1 subclass deficiency were subjected to sc ig -gammanorm, and 7 hae type 1 outpatients had good response for replacement c1 inhibitor therapy as follows: conestat alfa (ruconest)in 5 hae adults (150 iu/ml weekly), and berinert (20 u/kg b.w. weekley)in 2 hae adult patients. conclusions: the expert pid center in plovdiv university hospital provides competent diagnosis, therapy, education and consultations for pediatric and adult pid patients from plovdiv region. the recent introduction of reimbursed replacement therapy for pid patients (hospitalized or outpatients) allows regular immunological and clinical follow up of the diseases. background: intravenous immunoglobulins (ivigs) are scarce biological products used in a broad variety of disorders. tolerance to infusions is usually good but adverse events, including some serious ones, have been reported. methodology: a cohort study aimed for detection of adverse events that occur during and following intravenous immunoglobulin (ivig) infusions at cairo university children hospitals [patients were recruited at neonatal intensive care units (nicu), pediatric intensive care units (picu), general and specialized inpatient wards ] over a time period of six months, from april through september, 2013. the study included 62 transfusions for different disease conditions in 55 patients.three maltose-stabilized intravenous immunoglobulin products were administered to patients. assessments were done before, during and after the infusions. results: there were 22 symptoms and 26 laboratory changes of adverse events during ivig transfusions, with some patients experiencing more than one adverse reaction. adverse events were noted to occur most frequently within 1 to 6 h from onset of ivig infusion (n = 9, 39.1%). first hour after infusion onset was the most common timing for symptoms of adverse reactions (n = 5, 21.7%). patient characteristics of those with adverse reactions: adverse reactions occurred in 37.1 % of the infusions (n = 23) with the majority belonging to the 6-9 years old age group (n = 10, 43.5%), with variable diagnostic categories. ten patients observed during 14 infusions (60.9%) had one or more risk factors for complications, while 6 patients observed during 9 infusions (39.1%) had no risk factors. the commonest risk factor was administration of nephrotoxic drugs (n = 8, 34.7%), followed by presence of a suspected autoimmune disorder (n = 7, 30.4%) and preexisting renal insufficiency (n = 5, 21.7%). using regression analysis, the predicting variables for each complication were noted .for example ,fever and chills were related to infusion rate and dose whereas the predicting variables for pallor were infusion rate and presence of existing risk factors. conclusions: clinicians should be aware of the high need for special monitoring while infusing ivig to patients with primary immunodeficiency disorders, autoimmune hematological disorders and sepsis. certain diagnosis of a primary immunodeficiency disorders (pid) is most confirmedly performed by investigation of a gene defect, allowing genetic counseling and screening. molecular diagnosis helps both parents and index pid patient by carrier detection and pre-implantation testing for selecting appropriate reproductive decisions. furthermore, for confirmation of diagnosis and establishment of the inheritance pattern genetic analysis is necessary. this survey in all pid cohorts should be considered for long-term planning such as bone marrow transplantation of a pid infant at birth. the result of this testing also is important for screening of newborns and for those in specific family or ethnic groups. the prevalence of pids has been estimated to be more than 1/600 worldwide. based on the total population of iran reported in 2010 (76,424,000), the expected prevalence of pids in iran would be more than 127,300 individuals. however, because of the high rate of consanguineous marriages in iran and an increased risk for development of disorders with an autosomal recessive pattern of inheritance, this prediction is likely to be an underestimation. to date, 2247 clinically diagnosed patients of pids have been reported in iran, and a definite diagnosis, defined by mutation analysis, was made in 790 individuals. as a result, 1.76% of the expected pid patients have been identified, and among these, 35.2% have been diagnosed at a molecular level. the proportion of genetically definite diagnosis varied between 84.5 and 0% in the different disease categories. this wide spectrum might be due to unknown underlying genetic defects or modifying genes, especially in patients with predominantly antibody deficiency. on the other hand, the latter patients also had the lowest percentage of clinically diagnosed cases. croatia is a small country with 4 267 889 inhabitants . according to expected prevalence of pids we expected approximately four hundred patients with pid. for the past few years university hospital center zagreb is the reporting center for esid registry. it is also the national center for diagnosis and treatment of pids, including haematopoietic stem cell transplantation. current pid database is running manually, with the exception of patients reported to esid registry. most of them are of pediatric age, reffered to the hospital from all over the country. the pids patients are classified according to international classification ( iuis-international union of immunological societies ). there are 121 pids patients included in the database at the moment. the majority of them have antibody deficiency. combined immunodeficiency and well defined syndromes appear in equal distribution. other types of pids are reported in small numbers. although no consanguinity was reported, we noticed the geografic distribution of severe combined immunodeficiency patients (scid) mostly in 2 regions, istra and podravina. it can be explained with genetic isolation. the establishment of national online pid registry is in process. the aim is to improve the diagnosis od pid specially in adult patients, who are not included in present database, with exception of the patients diagnosed in childhood. primary immunodeficiency disorders are underdiagnosed in croatia, specially in adults. establishing national pid registry will improve the physicians awareness of pids, which is particularly important for adult patients. we expected the number of diagnosed pid patients will rise. this will give the opportunity to make progress in diagnosis and treatment, and the opportunity for further epidemiological and clinical studies. another 25 patients have well defined immunodeficiency syndromes from which: 11 are with di george anomaly; 2 with wiskott-aldrich syndrome; 4 with ataxia-teleangiectasia; 4 with hyper ige syndrome; 1 with schimke syndrome and 3 are with nijmegen breakage syndrome. in early nineties, two (2) of our patients had chronic mucocutaneus candidiasis. 3 patients are registered as x-linked scid and 1 with xlinked lymphoproliferative syndrome (xlp). 1 patient has chronic granulomatous disease; 3 have severe congenital neutropenia and 5 have hereditary angioedema. 2 patients are with whim syndrome. 6 patients are with anti-inflammatory disorders: 1 has hyper ig d syndrome (hids) and 5 have familial mediterranean fever. the j-project realization in the republic of kazakhstan allows to update the pid problem, to raise the availability of early diagnosis of pid, to improve the life quality of patients with pid by providing substitution therapy and as a result, the infant mortality and disability rate has been reduced. common variable immunodeficiency (cvid) is the most common primary immunodeficiency (pid) characterised by impaired immunglobulin production and immune dysregulation. chronic and recurrent infections and its results are typical manifestation of this disease. in addition there is a higher risk of autoimmune disorders, lymphoproliferative or granulomatous diseases and malignancies. successful management of cvid patients is based on prevention and consistent therapy of infections with sufficient immunoglobulin replacement and/or antibiotics, prevention and active screening of cvid related complications. in our study we analysed data of 41 patients gained from medical records. these data were also input into czech pid registry and pid registry organized by esid (european organisation for immunodeficiency). we aimed at the period before diagnosis-onset of the symptoms and their characters and the course of the disease-effect of therapy, occurence of the related complications. finally, we compared our data with similar performed studies. chronic and recurrent upper and lower respiratory infections were the most frequent first manifestation of our cvid patients, but developed chronic lung disease or autoimmune disorder as well. in all patients the intravenous or subcutaneous imunoglobulin replacement therapy, eventually combined therapy with antibiotic prophylaxis, was initiated. beside chronic lung disease the most common complications were autoimmunity disorders, especially autoimmune thyroiditis, evans syndrome, trombocytopenia (itp), autoimmune hemolytic anemia (aiha). on the contrary we revealed 2 patients with insuline dependent (type 1) diabetes mellitus and cvid. only few case reports have been published with such association. successful management of cvid patients is based on a prevention and a consistent treatment of infections with sufficient immunoglobulin replcement and/or antibiotic therapy, a prevention and an active screening of cvid related complications. such approach can significantly improve the prognosis of cvid patients and the quality of their life. laura zilinskaite 1 ; ieva bajoriuniene 1,2 ; raimundas sakalauskas 1 ; brigita sitkauskiene 1,2 1 department of pulmonology and immunology, lithuanian university of health sciences, kaunas, lithuania. background. primary immunodeficiency (pid) is considered to be a rare disease. despite that it is thought that six million people may be living with a pid worldwide. in the hospital of lithuanian university of health sciences (hluhs) patients with suspected immune disorders have been diagnosed and treated since 1998. we aimed to review the structure of pid diagnosed and treated in hluhs during the last five years. methods. data about patients with pid consulted in the hluhs was collected from the department of medical statistics. case histories of these patients were revised and patients' data was collected: onset of symptoms, type and duration of disorder, type of treatment. all patients with pid were divided into several groups according to the classification of international union of immunological societies primary immunodeficiency diseases classification committee (2011). results. there were 57 patients with pid diagnosed in the centre. antibody deficiency was diagnosed for 38 patients: 3 -bruton's disease, 14common variable immunodeficiency (cvid) and 21selective immunoglobulin (ig) a deficiency. complement deficiencies were diagnosed for 13 patients: 11 -c1 esterase inhibitor deficiency and 2 -c4 deficiency. another well-defined syndrome with immunodeficiency was found in six patients. the most prevalent symptom in patients with predominant igg deficiency was recurrent pneumonias which occurred at the age 11.9 ± 4.9 yrs. the mean time between the onset of symptoms and confirmation of the diagnosis was 14.6 ± 6.4 yrs. thirteen patients are on the replacement therapy with intravenous immunoglobulin. these patients had 2-6 infections/year before the treatment initiation and only 0-1 infections/year during the treatment. •pid center has successful sideproject as regional charitable public organization of invalids "society of patients with primary immunodeficiency diseases in st. petersburg" solovushka (nightingale) "". web site for patients (www.opidspb.ru) was opened due to mutual efforts of pid center staff and patients. pid center laboratory is based on spb pasteur institute central clinical diagnostics laboratory and laboratory of molecular immunology and seroepidemiology. main groups of tests perform for pid patients are: general clinical assays; flow cytometry (6-color assay on facs canto ii); humoral factors assays (ig levels, igg subclasses, post-vaccination igg levels, etc.); burst-test on flow cytometer; genetic analysis of btk, rag1, rag2,was, cybb genes. despite of relatively short story of spb pid center it has a variety of completely diagnosted and successfully cured cases of pid including agammaglobulinemia with b-cell in identical siblings, wiskott-aldrich syndrome, chronic granulomatous disease (cgd), etc. introduction: the primary immunodeficiency diseases are a group of disorders caused by basic defects in immune function that are intrinsic to, or inherent in, the cells and proteins of the immune system. there are more than 200 primary immunodeficiency diseases.. laboratory studies are necessary to determine the presence of a primary immunodeficiency diseases. the standard screening tests for antibody deficiency starts with measurement of immunoglobulin levels in the blood serum. these consist of igg, iga and igm levels. the results must be compared to agematched controls. additional studies used to evaluate patients with antibody deficiencies include measuring the different types of lymphocytes in the blood by marking those cells with molecules that can identify the different types. a commonly used test is called flow cytometry that can identify b-cells and t cells present in the circulation. methods: in 2008 in our laboratory of immunology is created the sector of examination for the pid and we measure the immunoglobulin levels with a beckman coulter immage immunochemistry system fully automated rate turbidometry and rate nephelometry method. we have determine the aged-matched levels of immunoglobulins in our laboratory. we have install also a new flow cytometer of beckman coulter company and we can determine the b cells by cd19 marker and t cell with cd3 , cd4 and cd8 marker. this examinations has been of great help in diagnosis of primary immunodeficiencies. results: in years 2010-2013 from the examination of children aged from 0-14 years old reported in our laboratory for immunological examination from the pediatric department of uhc mother teresa in tirana we have selected 41 cases with disorders regarding the primary immune deficiencies. we have 5 cases (3 cases reported in 2008-2009 and 2 new cases ) with nul b cell in cytoflorometry (b cd19 cell = 0 %). the level of immunoglobulins was indetectable for igg , iga and igm in the moment of diagnosis. they were boys and the age at diagnosis was 3 and 4 years and actually they are 4 and 6 years treated with ivig. we have classified them as bruton (xla). we have 12 case with low cd19 b-cell in circulation but only in 4 of them we have done the immunoglobulin level. the low level of cd19 b cell is accompanied with different kind of hypoglobulinemies: 1common variable immunodeficiency cvid, 1 with isolated iga deficiency, 1 with igg deficiency and 1 with both igg + igm deficiency. the immunoglobulins disorders : we have classified them according the antibody deficiencies (tab1) and we noted that the most frequent one is the deficiency of iga (12/19 or 63.2 % -from which 6 iga isolated, 5 accompanied with low igm and 1 with low igg ) with average age at diagnosis 5.09 years old. in children in the first years of live aged from 0.8 to 2 years old we found 8 case with transient hypogammaglobulinemia of infancy. according this survey we can report our 92 cases of primary immunodefiecies in uhc mother teresa of tirana with different classification conclusion: the finding are to be completed with other cases in albania and it is necessary to do the national register for pid in order to estimate exactly the prevalence of this disorders in our country. nyíregyháza-debrecen, hungary the stat3 mutation was confirmed in 2007 as the cause of autosomal dominant hyper-ige syndrome (ad-hies). the disease, which also mentioned in the literature as job's syndrome, is a rare primary immunodeficiency. this disease can be characterized by the following classic triad: recurrent purulent skin infections, cold abscessus which are formed in the ground of chronic ekcematoid dermatitis, pneumatocele, formation causing pneumonia and extremely high ige level. dental training interdependency as well as bone and connective tissue disorders frequently occur in the nonimmunological symptoms as multi-systemic disease. the stat 3 protein has an important role in the area of wound healing, immunity, tumor and neovascularization. we would like to show this in case of 3-years old kitti whom the perinatal medical history was eventless. there is a frequent hospitalization in kitti's case history since her infancy. due to the age of three months because of serious exsiccotoxicosis, 5 months old bilateral bronchopneumonia and pleurisy required icu care. bronchoscopy was made because of recurrent pneumonia, which excluded the bronchial malformation. lately rather otitis, mastoiditis and airway obstructive symptoms dominated the clinical picture. from serum immunoglobulins-igg is very low, whereas high levels of igewas indicated. on this basis, the diagnosis of job's syndrome was up, which is a negative stat3 mutations as the molecular genetic test performed demonstrated. the disease has a great clinical importance, since the risk of emergence of serious and often life-threatining complications are high. because of the prevention of disseminated infections, the early detection and the appropriate treatment are essential. in default of causal therapy, the treatment primarily concern to prevention of infections, or their aggressive antibiotic therapy. calcium and vitamin d and as well as histamine 1 on the case of itchy skin symptoms are reccurrended to use. laboratory of molecular immunogenetics, human genetics institute, cnrs and university montpellier 2, montpellier, france. we performed clinical, immunological and genetic studies of 12 hyper-ige syndrome (hies) patients from 4 hungarian, 2 lebanese, one russian, one polish, and one swedish families with autosomal dominant (ad) or sporadic forms of the disease to reveal cross-ethnicity of recurrent and novel mutations in the signal transducer and activator of transcription-3 gene (stat3). four patients from 3 hungarian families, and one russian, and one swedish patient carried the heterozygous r382w germline mutation at the dna-binding site of stat3. the recurrent v637m mutation affecting the src homology 2 (sh2) domain was detected in one lebanese and one polish family, and the v463del deletion located in the dna-binding domain was unveiled in another lebanese family. a novel h332y mutation affecting the dna-binding site of stat3 in three hungarian patients from a gypsy family was also found. the segregation of this mutation with hies, restriction fragment length polymorphism analysis of stat3 from patients and controls and the negligible production upon il-6 stimulation of monocyte chemotactic protein-1 by the patient's blood mononuclear cells suggested that the h332y mutation was disease-causing. these data suggest, that dominant negative mutations of the dna-binding and sh2 domains of stat3 cause ad and sporadic cases of hies in different ethnic groups with r382w as the predominant mutation found in 5 of the 9 families. functional and genetic data support that the novel h332y mutation may result in the loss of function of stat3 and leads to the hies phenotype. published in molecular immunology. 46:202-6. males with an expressed mutation in the sap (signaling lymphocyte activating molecule [slam]-associed protein) gene have an x-linked syndrome characterized by an increased vulnerability to infection with epstein-barr virus (ebv). we evaluated two related male patients with fatal infectious mononucleosis (fim) and mutation in the sap gene. sequence analysis revealed hemizygous g to a transition at nucleotide position 47 in exon 1 in one of the patients, and heterozygosity for this mutation in the genomic dna from his mother and maternal grandmother. this mutation resulted in asparagine instead of glycine in the sequence of the sap protein at amino acid position 16. to analyse the effect of this missense mutation on protein function cdna was generated by site-directed mutagenesis and cloned in pcmv-flag vector. we found that the mutant sap (sap/g16d) protein was defective in protein folding as manifested by the reduced half-life compared to that of wild type sap. furthermore, the sap/g16d protein was defective in binding to its philological ligands slam and 2b4. these results suggest that defects in protein folding and ligand binding collectively contribute to the loss of function of the sap protein in patients carrying g16d mutation. dedicator of cytokinesis 8 (dock8) deficiency is an innate error of adaptive immunity characterized by recurrent viral, bacterial and fungal infections, very high serum ige concentration and a progressive deterioration of t-and b cell-mediated immunity. traditional sanger sequencing may fail to identify mutations in dock8, due to overlapping large deletions in heterozygous patients. we studied the genetic and immunological features of two sisters (11 and 6 years of age) born to healthy hungarian parents. mutational analysis of genomic dna and cdna from the patients and parents by a combination of pcr and bidirectional targeted sequencing failed to identify the mutation. however, a multiple ligation-dependent probe amplification (mlpa) assay revealed two previously unknown large deletions, del1-14 exons and del8-18 exons, of dock8 in both patients. the children's mother was heterozygous for the del1-14 exons mutation, whereas the father carried the del8-18 exons deletion. immunoblot analysis showed an absence of dock8 protein from the peripheral blood lymphocytes of both patients. these data suggest that the new compound heterozygous del1-14 exons and del8-18 exons mutations result in a loss of dock8 protein function and a typical dock8 deficiency phenotype. our findings suggest that traditional sequencing technology may give misleading results in such cases and that mlpa may be indispensible for the definition of the large deletions frequently observed in patients with dock8 deficiency. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been docuthe wiskott-aldrich syndrome (was) is an x-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a 502-amino acid protein. wasp plays a critical role in actin cytoskeleton organization and signalling, and functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries and turkey. clinical and haematological information of 87 affected males and 48 carrier females from 77 was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified 62 unique mutations including 17 novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes (17 missense and 11 nonsense mutations), 7 small insertions, 18 deletions, and 9 splice site defects. genetic counselling and prenatal diagnosis were applied in four affected families. introduction: mhc-class ii deficiency is an autosomal recessively inherited combined immunodeficiency disorder characterized by less than 5% expression of hla-dr on b cells or monocytes. it is caused by mutation of genes (ciita, rfxank, rfx5, rfxap) regulating transcription factors which controls expression of mhc-class-ii molecules on cell surface. mhc-class ii molecules are expressed on thymic epithelial cells, antigen presenting cells (b lymphocytes, dendritic cells, monocytes and macrophages) and activated t cells. these molecules are of critical importance to immunity, cd4+ t cell development, antibody production, tolerance induction and inlammatory response. consequently, patients present with clinical findings related to combined immunodeficiency during infancy. material and methods: medical records of thirteen patients with mhc-class ii deficiency, followed-up in ankara university, medical school, division of pediatric immunology/allergy from 1999 to 2013, were evaluated retrospectively. findings: during study period, 6 male and 7 female patients were diagnosed with mhc-class ii deficiency. age of diagnosis were between 3 months and 4 years of age. consanguinity were present in eleven out of thirteen patients. most frequent clinical findings during initial diagnosis were failure to thrive, pneumonia and oral moniliasis. lymphopenia was absent in all of the patients, however, low serum igg level was present in all of them. except for the 15 yo female patient with a positive family history and whose hla-dr expression was 0,2%, hla-dr expression was 0% in the rest of the patients. eight patients underwent hematopoietic stem cell transplantation (hsct). two patients were lost soon after hsct due to complications and three patients died of opportunistic infections. four patients died of severe opportunistic infections without underwent hsct. results: mhc-class ii deficiency is a combined immunodeficiency and not considered a rare disease in our country. to date, only known treatment is hsct. since it has poor prognosis, hsct should be performed before development of chronic viral infections and sequelae related to infections in patients who have hla-matched sibling. fatih celmeli 1 ; giancarlo la marca 2 ; ines santisteban 3 ; michael s. hershfield 3 1 purine nucleoside phosphorylase deficiency (pnp deficiency) is a rare autosomal recessively inherited type of immunodeficiency. pnp deficiency constitutes about 1 to 2 percent of all combined immunodeficiencies. it is characterized by progressive combined immunodeficiency and neurologic findings which includes ataxia, developmental delay, and spasticity. the immunodeficiency is progressive, with normal immune functions at birth, but severe t cell deficiency with variable b cell functions presented by the age of 2 years . the only curative treatment is the hematopoietic stem cell transplantation (hsct). here, we present a 14 year-old girl with recurrent respiratory tract infections, short stature and spastic paraplegia. immunological, biochemically and genetics investigation revealed pnp deficiency with a117t mutation in pnp gene. case report: a 14 years-old girl was referred to our pediatric immunology clinic for recurrent sinopulmonary infections since 6 year of age. she was full term neonate and her parents were not consanguineous. she had a history of prolonged and resistant bronchopneumonia, and an attack of generalized chickenpox complicated with pneumonia. she had also severe zona infection resolved with ulceration in cornea, one year ago. she has been suffering from frequent infections like chronic sinusitis, oral moniliasis, recurrent pneumonia and sclerosing cholangitis. she has also nonprogressive cerebral palsy, spastic paraplegia, behavioral problems and limited motor and mental retardation. there was no family history of recurrent infections or immunological disorders. on her physical examination, there was failure to thrive, (her weight and height <3 rd p) oral moniliasis, bilateral crepitus ralls, and splenomegaly. she has also marked spasticity with brisk reflexes in lower extremities. laboratory tests revealed lymphopenia: hemoglobin 11 g/dl and platelets 158000/mm 3 wbc 4000/mm 3 , absolute lymphocyte 560/mm 3 . the laboratory results are shown in the table. lymphocyte proliferation is lower than normal limits in response to pha stimulation (wst1 assay). ppd response and hiv was negative. antihbs, and antihav were negative despite vaccination. uric acid level 1,8 mg/dl. direct coombs was negative, thyroid autoantibodies were within normal limits. genetic analysis revealed homozygous missense mutation (c.349g > a), which causes the a117t amino acid substitution in pnp gene, in exon 4, which has previously been reported. additionally, a homozygous g/a polymorphic site in ivs3 has been detected (c.285 + 10g (ivs3 + 10g)). discussion: pnp deficiency is caused by mutations in the pnp gene at 14q13.1. this gene encodes the protein purine nucleoside phosphorylase, one of the enzymes involved in the purine salvage pathway. adenosine deaminase (ada) deaminates adenosine to yield inosine, which is then converted to hypoxanthine by pnp. pnp also converts guanosine to guanine. a number of metabolites are elevated in the plasma and urine in pnp deficiency, including deoxyguanosine and deoxyinosine. there is an intracellular accumulation of their deoxy triphosphate compounds, particularly deoxyguanosine triphosphate (dgtp). the latter is toxic to t cells, a property similar to deoxyadenosine triphosphate in adenosine deaminase deficiency. in this report, we demonstrate the clinical characteristic of the patient with late diagnosis of pnp deficiency. pnp mutations likely lead to an intense alteration of the enzyme activity which in turn, cause severe and early onset of the clinical findings. however, in our case, the clinical onset of the disease is quite late (after 6 years) which can be explained by the residual activity of the pnp. in conclusion patients with pnp deficiency can be late onset. additionally, late diagnosis of this patient can cause severe comorbidity which limits the chance of bone marrow transplantation. di george syndrome, a disorder caused by a defect in chromosome 22 (22q11.2 deletion), results in the poor development of several body systems. clinical features include congenital heart defects, hypoparathyroidism and thymic hypoplasia or aplasia leading to t-cell immunodeficiency. the aim of our study is to screen and determine the incidence of di george syndrome within only one tube of blood in children with congenital heart anomalies in our population. children who were found to have a cardiac defect during routine visits in pediatric cardiology and neonatalogy departments were included into the study. cases with known genetic syndromes and newborns younger than 32 gestational weeks of age and small for gestational age (birth weight <2500 gr) were excluded. a total of 136 patients were included. there were 79 (58%) males and 57 (42%) females. age ranged between 0-80 months (10.2 ± 15.9 months). parental consanguinity was 17% (n = 23) in the study group. the majority of patients diagnosed after murmur was heard during the routine physical examination (n = 62, 46%). five patients (3%) diagnosed antenatally. remaining clinical signs on admission were as follows; respiratory distress (n = 33, 24%), tachycardia (n = 20, 15%) and central cyanosis (n = 16, 12%). echocardiagorafic examinations revealed ventricular septal defect (vsd) (n = 27), tetralogy of fallot (tof) (n = 17), vsd-asd (n = 13), aortic coarctation (n = 11), double outlet right venticle (dorv) (n = 10), transposition of the great arteries (n = 6), truncus arteriosus (n = 5) and pulmonary atresia (n = 6). pulmonary stenosis, endocardial cushion defects, total pulmonary venous return anomaly and hypoplastic left heart were the other defects. 22q11.2 deletion was ascertained in 7 (5.1%) patients; these patients were diagnosed to have tof (28.6%), truncus arteriosus (14.6%), dorv (14.3%), vsd (14.3%) and vsd-asd (14.3%). preliminary results of the study showed that the frequency of 22q11.2 deletion is 5% in patients with known cardiac defects. single tube of blood is enough for flow cytometric and genetic analyses. further studies involving higher number of patients is mandatory to give sufficient information about the exact incidence of the disease. di george syndrome -where do we stand now? małgorzata pac; małgorzata skomska; ewa bernatowska department of immunology, the children's memorial health institute, warsaw, poland di george syndrome (dgs) classically comprises t-cell deficiency (due to thymic hypoplasia), hypoparathyroidism, cardiac malformations,and facial abnormalities. deletions of the long arm of chromosome 22 at position q.11 are most commonly associated with dgs. syndrome is also found associated with other genetic abnormalities (10p deletions, char ge), certain teratogenic influences (retinoid acid, foetal alcoholic syndrome, maternal diabetes). the dgs phenotype is very heterogenous with variable expression of the different features including the immunodeficiency. the initial treatment emphasis is to control the hypoparathyroidism. correction of congenital heart defects (if present) is usually needed. the best treatment of the immune defects of dgs is still controversial. both hsct and transplant with fetal thymus are the option for complete dgs (cdgs). long term survival after hsct has been reported, though at a lower rate (41-48%) compared to survival after hsct for scid. survival in the subgroup receiving matched sibling donor transplants was better at over 60%. the use of post natal human thymus was pioneered by markert at duke university and has become established as the treatment of choice for cdgs, with the result of 43 out of 60 treated patients survived (72%). more recently this approach has also been used in london, at gosh. . under care of cmhi there are119 patients fulfilling esid criteria, 59 girls (49.6%) and 60 boys (50.4%), age 2/12 -23 y.o. in 70% of them 22q11 deletion in locus d22s75 was found. the vast majority children were diagnosed as partial dgs. none of them had significant hypogammaglobulinemia and no regular ivig therapy or antibiotic prophylaxis were required.. the mean number and percentage of cd3, cd8 and cd4 lymphocytes as well as lymphoproliferative answer to pha and cd3 in dgs patients were slightly diminished. in many improvement of cellular immunity was observed with age. about 86% presented with congenital heart disease, requiring surgery, while almost 50% had the symptoms of hypocalcemia and hypoparathyroidism, next 46% -speech and learning difficulties. one child was diagnosed as cdgs. scid is a rare, inherited condition, is caused by numerous molecular defects that lead to severe compromise in the number and function of t cells, b cells, and occasionally natural killer cells. seventeen patents with scid were registered during the period from 1994 till 2013 in the children's clinical university hospital. medical charts of these patients have been reviewed. there were 9 boys and 8 girls. positive family history was in 4 families. mean age at the onset of symptoms and scid diagnosis was 1.7 ± 1.23 and 4.03 ± 1.6 months, respectively. pneumonia (65%), candidacies (65%), bcg infection (47%), diarrhea (35%) were the most important infections. anemia and relative lymphopenia were in 76% cases , growth retardation, hypotrophy had 59% children . pathogens such as candida albicans (11), mycobacterium tuberculosis complex (8), cmv (3) and others have been identified. totally 15 patients died.two girls are alive (29 and 17 months post-transplant). autopsy was done in 8 patients. we saw different changes in thymus and lymphatic nodes. artemis deficiency (n = 1), t-b-scid (n = 4), t-b + scid (n = 5), γc deficiency (n = 1 mutation r224w in γ-chain of receptor for il-2), unspecified scid (n = 6) were detected. conclusion: generalized bcg infection had 47% of our scid patients. (incidence of tb is still high 36.2 / 100 000 population in latvia and newborns obligatory are vaccinated on the second to fifth day of life). due to possibility of absence for pid routine genetic identification in only few scid forms were identified precisely. children's hospital, university of freiburg, freiburg, germany. purpose: ipex (immunodysregulation, polyendocrinopathy, enteropathy, x-linked) is a rare x-linked recessive life-threatening disorder characterized by autoimmunity and early death. pulmonary complication related with ipex has not elucidated exactly. here, we report 4 i.e. patients, 3 of which died from severe pulmonary disease. methods: clinical data and laboratory findings included autoantibodies, immunoglobulin levels as well as number of t, b and nk cells were evaluated. foxp3 expression was performed by flow cytometry. genomic dna was isolated and all exons and exon-intron boundaries of the foxp3 gene were sequenced by sanger sequencing. results: patient i (pi) presented with nephrotic syndrome at 3 years of age and then developed autoimmune hepatitis without eczema, enteropathy or high ige and died at 9 years of age due to acute respiratory distress syndrome (ards). two cousins of pi had the same hypomorphic splice site mutation leading to normal foxp3 protein expression and suppressive capacity. however, they exhibited typical symptoms such as eczema, diabetes and enteropathy with eosinophilia at early age (pii, piii) and were transplanted in infancy. one of them had severe respiratory distress right after birth (piii). patient iv from another family presented with chronic diarrhea without autoimmune manifestations and died due to ards. conclusion: lung disease related to ipex syndrome has not been reported before and this entity could be a critical factor in disease outcome. severe combined immunodeficiencies (scids) are a group of primary immunodeficiencies that comprise a number of monogenic disorders characterized by a block in t cell differentiation with or without impairment of b cell and natural killer (nk) cells. without early diagnosis and treatment most children die in the first year of life. a lack or very low number of t-cell receptor excision circle (trec) detected by realtime quantitative polymerase chain reaction assay (qpcr) is consistent with t-cell lymphopenia and has repeatedly demonstrated clinical validity in population based newborn screening for scid. however, the impact of population screening will be less in communities with high consanguinity and family history of scid, in which targeted screening may be more appropriate. here, we screened 100 high risk neonates and infants (with one or more of the following: clinical presentation and/or family history suggestive of pid, failure to thrive otherwise unexplained, lymphopenia) at cairo university hospitals. their full history and clinical examination were recorded. immunoglobulin profile and immunophenotyping of peripheral lymphocytes were performed as confirmatory tests. sixteen classical scid cases were detected, as well as another 2 scid variants (omenn syndrome and major histocompatibility complex class ii deficiency) (totally 18.4% of all subjects screened). the rate of consanguinity in this group was 77.8%. secondary causes of low trecs, other than scid, in our series included: bacterial septicemia (4 preterm, 1full-term), prematurity (2 cases), one preterm with omphalocele and facial dysmorphism, one preterm with congenital adrenal hyperplasia, one full term with microvillus inclusion disease, and one full term with idiopathic tcell lymphopenia. this demonstrates that in populations with high consanguinity rates, as in egypt, targeted (non-population based) trecs assay may provide a more efficient screening strategy. case 1: a is a 4 years old female, 1 st kid of non consanguineous marriage presented with fever for 5 months. one week after the onset of the fever red patches appeared on the face, hands, abdomen, ll. mother sought medical advice and received antibiotics, antipyretics and oral steroids for about one month with no signs of improvements regarding fever. one week later the mother noticed pallor and sought medical advice and the baby was admitted to local hospital and received one bag of blood. and then she was referred to our hospital (zagazig university hospital) where she developed acute pallor again which needed recurrent blood transfusions. dark colored urine occurred in frequent attacks with abdominal enlargement and pain, and interestingly upto this time, no improvement regarding fever, bone pain or pallor. on examination she was underbuilt (all parameters are under 3rd centile), pallor, tinge of jaundice, generalized skin pigmentation, generalized lymphadenopathy, 2 nd degree clubbing and hsm investigations: cbc (pancytopenia with reticulocytopenia) -lft (increased ast with indirect hyperbilirubinemia)-kft (normal)-ldh (highly elevated 7238 u/l)-esr (105) -fibrinogen (0.336 gm/ l)serum triglycerides (315.7 mg/dl)-ebv-vcm igm positive and igg negative -c3 (1.3 normal) -rf and ana(-ve) -serum ferritin (32436 ng/ml) -cd 56 is low -bone marrow biobsy revealed dysplastic changes -l.n. biopsy revealed non specific inflammatory changes. treatment: hlh protocol 94 with no sct prognosis: patient passed initial phase with complete resolution and waiting for bmt case 2: m is 4 months old boy, 2 nd kid of non consanguineous marriage (the 1 st is healthy 3 years old female) presented with fever and difficult breathing since the age of 2 months. the fever was of gradual onset stationary course and not responding to treatment with antipyretics and antibiotics. one week later the mother noticed abdominal enlargement with red rash over the abdomen that was associated with pallor but there was no evidence of bleeding from any site, no change in the color of urine, no jaundice, no ecchymosis, no joint swelling then he is referred to our hospital (zagazig university hospital). this boy is delivered by nvd at term with no evidence of any problem either during pregnancy or delivery, he is exclusively breast fed and vaccinated as scheduled. examination: his weight was 6 kg, length 53 cm, and head circumference 35 cm all are average for age, he was pale with no jaundice or cyanosis he had hsm and other systemic examination was quite well. children with pid usually are admitting to the hospitals and intensive care units of infants' pathology regional children clinical hospital №1, but at a later date, in serious condition, after the development of clinical manifestations in the form of severe generalized infectious disease, or various complications including hematological. the screening technology approves children to be diagnosed in newborn period and to be observing by immunologist and hematologist for the next preventative therapy and bone marrow transplantation or hematopoietic stem cell transplantation. costs for the differential diagnosis and verification of the diagnosis before the manifestation and complications development of pid -51 245 usd. costs for the differential diagnosis and verification of diagnosis after manifestation and complications development of pid would be 115 208 usd in 2 months. economic loss prevention in pid -63 872 usd. economic loss prevention in t -lymphopenia -105 884 usd. we also should include into account the contribution to the state's economy, which will later be obtained, due to the presence of a healthy member of society. at is an autosomal recessive multisystem disorder characterized by progressive cerebellar ataxia, telangiectasia and increased susceptibility to infections and malignancies, particularly lymphomas and leukemia. laboratory immune investigations typically show decreased peripheral tcells, particularly of naïve t cells, with abnormal in vitro response to mitogens. most at patients have decreased serum iga and igg subclass concentrations. while about 10% of patients with at show raised serum igm concentrations during the course of the disease, it is unusual to find a high level of igm at onset. as cerebellar ataxia and oculocutaneous telangiectasia are not present at very young age, these patients are often erroneously diagnosed as hyper igm syndrome (higm). to prevent mistaking a-t patients for higm it is proposed to add dna repair disorders as a possible cause of higm. . diagnosis of at, suggested by elevated alfa-feto-protein and increased sensitivity of patients' cells to irradiation, can be confirmed by identifying a mutation in the atm gene.we report 3 female case of at that with diagnosis of hyper igm received ivig but later they had manifestation of ataxia in the course of their disease and then had telangiectasia of conjunctiva. first case was a 3 yrs girl that was suffered from itp and granulomatosis lesion of skin associated with hyper igm before at diagnosis. second case was a 6 years old girl with microcephaly, sever ftt , neurodevelopmental delay , abnormal faces and hypo and hyperpigmentation lesions and anemia. with respect to these manifestation and increased afp ,nijmegan breakage syndrome was suggested. third case was suffered from hyperigm before a t diagnosis for 2 years. we present a patient with a dock8 deficiency. mutations in the dedicator of cytokinesis 8 gene (dock8) cause a combined primary immunodeficiency syndrome that is characterized by elevated serum ige levels, depressed igm levels, eosinophilia, sinopulmonary infections, cutaneous viral infections, and lymphopenia. onset of the disease was observed at 1-month of age with severe eczema and recurrent respiratory infections (pneumonia, bronchitis, otitis). at 5 years of age neuroblastoma was diagnosed. from the age of 6 years he started with severe skin infection , subsequently recurrent mucocutaneous aspergillosis was established based on skin biopsy and bacteriological studies. immunological investigations revealed persistent leukocytosis, hypereosinophilia, low level of igm and increased ige up to 5 000 iu/ml. so hyper ige syndrome was diagnosed and genetically confirmed when a large deletion of the dock8-gene was identified. stem cell transplantation was performed in 2012 when the patient was 11 y.o. one year later progressive multifocal leukoencephalopathy secondary to infection by polyomavirus jc was diagnosed. but after immunosuppressive therapy with cyclosporine awas suspended our patient's condition improved: the load of polyomavirus jc on plasma showed a decrease; mri brain was essentially stable ; immunological tests showed an initial improvement of subpopulations and proliferative response to mitogens; donor chimerism 100% stable. özdemir öner 1 ; bozdoğan sıla 2 1 department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. background: bronchiolitis and infantile asthma are the most frequent causes for typical wheezing signs in infants. however, when a physician comes across patients with recurrent wheezing are resistant to β2-agonist and anti-cholinergic therapy, known as atypical wheezing cases; he should investigate for hypogammaglobulinemia in these patients. aim: here, three cases are reported to make pediatricians aware of hypogammaglobulinemia, which is one of the reasons causing recurrent and persistent wheezing attacks during infancy and beyond. case presentations: case 1: 24 month-old girl presented to us with complaining of coughing and persistent wheezing. she has been having wheezing and breathing difficulty for the last 2 months after she got upper respiratory tract infection. her symptoms persisted even though she was using religiously nebulized salbutamol + budesonid therapy. before this episode, she had had 9 other wheezing attacks in her past medical history beginning from 2 months of age. in her family history, her father has asthma. physical examination revealed her breathing difficulty. ronchi as well as rales were heard on the auscultation of her lungs. at the fourth day of admission, she was given ivig 500mg/kg/dose. later, her symptoms did improve and not recur for the last 5 months. laboratory findings showed normal routine biochemistry, complete blood count and sedimentation rate. chest x-ray showed normal findings. echography was normal. ph-metry for reflux investigation was normal. sweat test was normal. in the serological evaluation: low igg level for his age (358 mg/dl) was detected at two different times. igg subgroups, igm (156 mg/ dl), iga (34 mg/dl) and ige (68) levels were within normal. case 2: 8-month-old girl came to our outpatient clinic with complaints of coughing and wheezing. at 2 months of age, she had urinary and upper respiratory tract infections. despite antibiotic therapy, wheezing persisted for 2 months and wheezing severity increased and it did not respond to β2-agonist therapy. thereafter, she was admitted to the hospital for 7 days and symptoms resolved. however, she came back to hospital due to recurrence of her symptoms in 10 days. in her family history, grandmother and her cousins have asthma. physical examination showed breathing difficulty. ronchi as well as rales were heard on her lungs. although salbutamol, ipratropium, antibiotherapy (clarithromycin) and anti-reflux therapies were given, her symptoms did not improve for 2 weeks. at the 15th day of admission, she was given ivig 500mg/kg/dose. later, her respiratory system symptoms did not recur for the last several months. once she was evaluated for persistent wheezing attacks during admission, biochemistry, cbc, esr were normal. chest x-ray and echography were normal. in serological evaluation: low igg level for his age (304 mg/dl) was detected at two different times. igg subgroups, igm (106 mg/dl), iga (34 mg/dl) and ige (<5) levels were normal. case 3: 20 month-old boy was brought to us complaints of having frequent lower respiratory tract infections (bronchiolitis). he was experiencing recurrent wheezing attacks almost every other week for the last 6 months. in past medical history, he was diagnosed with trisomy 21 and hypothyroidism at the 3 months of age. he went thru an operation for atrio-venticular septal defect. physical exam revealed dyspnea, tachypnea and wheezing. crackles were heard on the chest auscultation. abdominal, cardio-vascular and the rest of the examination were normal. when he was evaluated for frequent wheezing attacks in our outpatient clinic, routine biochemistry, cbc and esr were normal. chest x-ray showed normal findings. in serological evaluation: low igg level for his age (300mg/dl) was detected twice. igg subgroups, igm, iga and ige levels were within normal. he was given ivig 400mg/kg/dose. for the last three months, he did not have any lower respiratory tract infection. conclusion: the awareness of immunodeficiency among pediatricians has been greatly improved. recurrent respiratory tract infections are major infections in these patients. thi is a relatively common condition associated with infant hypogammaglobulinemia. in patients with recurrent and/or persistent wheezing symptoms during infancy and beyond, especially resistant to therapy, hypogammaglobulinemia should be excluded from possible diagnoses. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: 15 month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at 5 months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at 7 months, family fed the patient with cow's milk but 3 hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at 3 weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was 4.420/mm3, with 21% neutrophils, 6% eosinophils and 69% lymphocytes. his hemoglobin was 8.2 g/ dl , platelet count was 280.000/mm3. total ige : <5 and immunocap specific ige against milk, grain and other classsic foods was <0.35. skin prick test results: saline: 0x0mm, histamine 4x4mm, fresh cow's milk:2x2mm, other food allergens (peanut, egg, fish, soybean, wheat): 0x0mm. conclusion: our patient seemed to have cow's milk allergy related to liver transplantation. laboratory investigations and clinical presentation of the patient did not look like typical ige-mediated food allergy, which is expected in tafa. patient history: 33 years old male patient, the first pneumonia at the age of 18 followed by 3 times pneumonia attacks/year, otitis media and sinusitis. hospitalization due to respiratory insufficiency caused by bronchiolitis obliterans and diagnosed with hypogammaglobulinemia at the age of 27. no consanguinity. patient findings at diagnosis (02/2008) had hepatospenomegaly, bilaterally cervical, supraclavicular axillar lap, osteoporosis, bronchiolitis obliterans organizing pneumonia, hyper igm, lower igg, iga, ige, low b-cell. no response to tetanus toxoid. i̇sohemagglutinin antib was ¼. cd40,cd40l, aid, taci, baffr, icos gene mutation were negative. protein electrophoresis revealed polyclonal igm increase, immunofixation was no clonality. lymph node biopsy result was available paracortical expantion . cd3(+) t and cd20 were positive. b lymphocyte distribution were normal. malignancy ruled out. no giant germinal centers. evaluation of bone marrow aspiration/ biopsy were abnormal localization of megakaryocytes, dismegakaryopoiesis, blasts in normal range. lymphocyte ratio 10% mostly consisting of cd3+ cells, cd20+ cells were rare, plasma cell ratio was 2%, amyloid negative. progression of patient, igm level and spnenomegaly were increased and patient had respiratory failure. splenectomy could not be done due to respiratory failure. than patient was treated with rituximab (375mg/kg/week). after rituximab therapy, lymph nodes and splenomegaly were (5 cm), regressed and igm level decreased (from 4390 mg/dl to 2040 mg/dl), increased effort capacity. after 7 month after rituximab therapy splenomegaly and igm level were progressed. splenoctomy was performed. pathological evaluation of the spleen malignancy ruled out. current igm level is 6300 mg/dl. conclusion: the most convinient scenerio for this patient would be a csr defect of unknown etiology presented as cvid. the recent litarature revealed genetic defects of some molecules operating in dna repair pathways such as msh2, msh4, msh5, msh6, mlh1, rad50, rad 52, nbs1 leading to csr abnormality and impaired antibody maturation. cvid is characterized by hypogammaglobulinemia, recurrent respiratory and gastrointestinal bacterial infections. good's syndrome(gs) is a thymoma-related immunodeficiency and characterized by hypogammaglobulinemia, decreased b cell and variable deficiencies in cell-mediated immunity. case: a 47-year old male patient presented with a palpable anterior chest wall mass. in 2005, he was diagnosed with rheumatoid arthritis. laboratory showed hypogammaglobulinemia and all autoantibodies were negative. cd19 + , cd20 + , cd22 + cells were <1%. bone marrow(bm) examination demonstrated low cd20 + b-lymphocyte and increase in cd3 + t cells. tuberculin skin test was positive. t-cell proliferative response was normal. immunizations with h.influenzae type-b and tetanus toxoid revealed no response. anti-b titer was low. taci, btk and icos mutattions were negative. ultrasonography showed hepatosplenomegaly. mild edema, mononuclear cell infiltration (suggested the early stages of extrahepatic biliary obstruction) were detected on liver biopsy. in the medical history, he had reported chronic sinusitis, otitis and bronchitis dating back to 3rd decade of life. at age 38, he underwent surgery for thymoma. in follow-up, the computed tomography showed soft tissue mass on the anterior chest wall and pathology was thymoma. discussion: frequent respiratory infections with encapsulated pathogens beginning at the age of 30, lack of opportunistic pathogen infections, presence of hepatosplenomegaly and rheumatoid arthritis, bm examination findings and successful management of infections with ivig therapy all indicated a diagnosis of cvid. the coexistence of cvid and thymoma has been reported in the literature. introduction: common variable immunodeficiency (cvid) is a primary immunodeficiency disorder characterized by impaired b cell differentiation with defective immunoglobulin production. it has heterogeneous clinical manifestations including recurrent infections, chronic lung disease, autoimmune disorders, gastrointestinal disease, and susceptibility to lymphoma. patients with this disorder have evidence of immune dysregulation leading to autoimmunity. autoimmune cytopenias are a more common presenting disorder in children and may be the initial manifestation of the disease. we want to present a patient presenting with autoimmunue hemolytic anemia and finally diagnosed as cvid. case: a 15 years old, previously healthy female patient applied to emergency clinic with complaint of paleness, light headedness and yellow discoloration of her scleras. her history was not compatible with blood loss. she denied having melena, hemotochezia or hematuria. her mensturation history was also normal. her hemoglobin level was 7.2 gr/dl, reticulocyte count was % 4.17, complete blood count was otherwise normal. direct coombs was (+++). for the differential diagnosis of immune hemolytic anemia, viral serology, ana and anti-dsdna were studied, but the results were normal. igg, igm and iga levels were lower than normal normal for her age. other causes of hypogammaglobulinemia were excluded. blood group was arh (+), blood isohemagglutinin were anti a(-) and anti b (-/1+). lymphocyte subsets were also studided. as the patient has reduced immunoglobulin levels with normal lymphocyte subset analysis, she presented after puberty and other defined immunodeficiency states were excluded, she was diagnosed as cvid and monthly immunoglobulin replacement therapy was planned. she had aseptic meningitis after her first ivig transfusion. the ivig preparation was changed with another trade and she did not have any problem during the following treatments. it has been one year since she was diagnosed and she did not have any other medical problems. conclusion: the diagnosis of cvid requires decreased igg, igm and iga levels are also reduced but are less valuable for diagnosis. ige level is checked to exclude other disorders. igg subclass determinations are indicated if antibody titers are decreased but immunoglobulin levels are near normal. hypogammaglobulinemia secondary to other disorders should be excluded. autoimmune conditions can be the presenting signs/symptoms in cvid. autoimmune hematologic disorders may precede, present at the time of diagnosis or develope during the course of cvid in approximately one-half of the patients with autoimmune problems. selective immunoglobuline deficiency is an uncommon dysgamaglobulinemia, in which immunoglobuline levels except igm level are normal. it can be primary or secondary to cancer, autoimmune diseases, gastrointestinal system diseases and immunosuppressive therapy. patients can be asymptomatic or have recurrent infections, asthma, angioedema, autoimmune diseases, celiac disease and bronchiectasis. allergic diatheses are the second commonest presentation of selective igm deficiency. in this presentation, we report a case with asthma and angioedema who has selective immunoglobuline m deficiency. a 16 year old male patient who has been diagnosed with asthma for 12 years with a well controlled asthma for 2 years presented with labial angioedema. he had labial angioedema daily without antihistamines. he did not have any suspected food or drug allergy. he did not have family history of angioedema. physical examination was normal under antihistamine therapy. laboratory evaluation revealed a 4.3% percentage of eosinophils. absolute lymphocyte count was 2700, absolute neutrophil count was 7000 cells/mm 3 . immunoglobuline e value was 260 ng/ml, levels of immunoglobuline g and a were within normal limits for age. immunoglobulin m value was 28.7 mg/dl (73-448). anti a was 1/8, anti b was ½ positive, antihbs was above 1000 mlu/ml. lymphocyte subsets were normal. because of the continuous usage of antihistamines, prick tests could not be done. levels of d1 was17.8, d2 was12.4 ku a /l. thyroid functions, antitpo, c3, c4 and c1 esterase inhibitor values were normal. antinuclear antibodies and antitransglutaminase lga was negative. immunoglobuline m value of his father was normal, immunoglobulin m value of his brother was 53.50 mg/dl (88-322). selective immunoglobuline deficiency is a rarely seen dysgamaglobulinemia. it was reported in children with asthma, but it was not reported in children with angioedema. it can have value in the clinical evaluation of patients with angioedema. cukurova university, department of pediatric allergy and immunology, adana, turkey a patient who is at age of 11 years at present and who has been followed up at the our clinic with xla diagnosis presented with pain in his ankles and wrists and swelling of his right knee. he also was suffering from skin tightening of the lower extremities. his physical examination revealed arthitis of the right knee and sclerotic changes were detected in the skin. skin biopsy was performed and it revealed morphea (localized scleroderma). after the diagnosis of morphea and arthritis, ivig 2 gm/kg and nsaid were applied. following the treatment skin findings and arthritis resolved, however approximately two months later liver enzymes were detected to be high in his routine control. liver biopsy performed to clarify the aetiology of elevated liver enzymes was reported as autoimmune hepatitis. in addition to ivig 2 gm/kg, budenofalk 6mg/day was started. after 3 months of treatment, his liver enzymes normalized. currently he is being treated with ivig monthly and ursofalk daily. patients with xla typically present with recurrent bacterial infections and it might be associated with some autoimmune diseases. there are not any reports indicating an associaton of autoimmune hepatitis and scleroderma with xla. bruton agammaglobulinemia is an inherited immunodeficiency disease caused by mutations in the gene coding for bruton tyrosine kinase (btk). the median age at the diagnosis of the antibody deficiency is about 4.6 years in turkey. here, we report a case of bruton disease presenting with recurrent cervical abscess at two months old infant. a 2-months-old boy was firstly hospitalized for the treatment of the right cervical abscess at 25 days old. after the recurrence of swelling on the cervical area, the patient was referred to the admitted to hospital secondly. there was no consanguinity between parents and, his family history was unremarkable except four of the mother's cousins, died because of unknown etiology in infancy. on his physical examination, his weight, height andhead circumference were normal range by age. there were no visible tonsils. there was a palpable, mobile 1x1 cm mass on right upper cervical area. the molecular analysis of the causal gene for bruton's tyrosine kinase (btk gene) revealed the mutation in exon 19. this mutation g.67662delg (c.1750 + 57del) leads to the changes of amino acid order in the protein with the subsequent changes in activity of btk (at the level of dna: substitution of glutamic acid (p.glu507*) causes non-sense mutation leading to the formation of stop codon with premature end of dna transcription to cause stop codon. after initiating the intravenous immunoglobulin with antibiotics, the cervical mass was getting smaller in a short period, and had not observed again. neutropenia was improved within the 3 months. this case is an important example to diagnose bruton disease in early life. it demonstrates that maintaining a high level of clinical suspicion is essential for the diagnosis of bruton disease in a child with recurrent cervical masses. elif azarsiz; neslihan edeer karaca; guzide aksu; necil kutukculer ege university faculty of medicine, department of pediatric immunology, izmir, turkey transient hypogammaglobulinemia of infancy (thi) is characterized by recurrent infections and reduced serum immunoglobulin levels. typically, thi patients recover spontaneously, mostly within 30-40 months of age, but sometimes recovery may be delayed until 5-6 years. the use of intravenous immunoglobulin (ivig) as an alternative to antibiotic prophylaxis remains contraversial also in symptomatic patients. some authors believe that ivig therapy may cause a delay in the maturation of the humoral immune system because of the interference from passively transfered antibodies. the aim of this study was to investigate the effect of ivig replacement on recovery from immunodeficiency in these patients. 43 patients (65%) received ivig therapy while 23 patients (34.8%) showed spontaneous normalization without ivig. the percentages of patients who had more than six times the number of febrile infections in a year decreased from 91% to 21% in the group receiving ivig treatment. at admission, before being recruited to ivig therapy, serum immunoglobulin g (igg) levels and anti-hemophilus b (hib) antibody titers were found to be significantly low in cases who were selected for replacement. the percentages of patients who did not have protective levels of anti-hib, anti-rubella or anti-rubeola-igg were also significantly high in ivig cases. there was no statistically significant difference in the age at which igg levels normalized between both groups. patients in the ivig group and non-ivig group reached normal igg levels at the age of 42.9 ± 22.0 and 40.7 ± 19.8 months, respectively. in conclusion, ivig infusions do not cause a delay in the maturation of the immune system in thi patients. the very low and non-protective specific antibody responses against previously applied vaccines are important factors to consider when selecting patients for ivig therapy. zoltán ellenes-jakabffy 1 ; ibolya kovács 1 ; mihaela bătăneanţ 2 ; maria cucuruz 2 ; margit şerban 2 ; lászló maródi 3 1 department of pediatrics, clinical city hospital oradea, romania. objective: to study the amplitude of the chronic inflammatory phenomena: atopic and autoimmune diseases, as well as their associations in pediatric sigad patients and to study the sigad patients' family history (1 st degree relatives) for pids (primary immunodeficiencies). methods: retrospective analysis of the clinical and laboratory records of pediatric sigad patients diagnosed between 1998 and 2013 at the departments for pediatric immunology of the medical universities of debrecen (hungary), oradea and timişoara (romania). results: out of 148 patients, we found out 74 (50%) with atopic diseases, mostly with respiratory localizations (asthma and allergic rhinitis), 16 (10,81%) patients with autoimmune diseases (jia, psoriasis, celiac disease, thyroiditis etc.) and other 15 patients without clinical symptoms of autoimmunity but constantly elevated autoantibody levels. there were 7 patients with coexistent atopic and autoimmune diseases. regarding the family history, we identified 8 families with multiple cases of pids : 6 with multiple sigad, 1 with sigad and cvid, 1 with sigad and higms (hyper igm syndrome). conclusions: the chronic inflammatory phenomena are present in the majority of the studied sigad patients: symptomatic atopic diseases in 50%, symptomatic autoimmune diseases in 10,8%, that means a cumulative 61%. there are comorbid associations within the atopic and autoimmune disease groups and also between the two groups. sigad is the most common primary immunodeficiency. it's prevalence is 1/400-1/800. aim was to assess the prevalence of co-morbidity in patients with sigad in latvian pediatric population and the analysis of some immunological abnormalities in these patients. the study included 120 patients 4 -18 years old. medical charts have been reviewed. into account were taken the data, which were made at time of diagnosis. patients were divided into 4 groups: 1 st -patients with allergic disease, 2 nd -patients with autoimmune diseases, 3 rd -patients with infectious diseases, 4 th -asymptomatic patients or patients with sigad unrelated diseases. patients with multiple co-morbidities of various disease groups were not placed in any of these groups. each patient group was divided by age: 4 -6 years, 7 -13 years 14 -18 years. results.58.4% were boys and 41.6% girls. sigad in 53.9% of the cases were diagnosed before 7 years of age (inclusive). 85% of the patients had co-morbidities: allergic (30%), autoimmune (37.5%) or infectious diseases (17.5%). patients 4 -6 years old: children with infectious and autoimmune diseases have 1.3 times greater igg than healthy children or children with allergic diseases (p < 0,05); children with autoimmune diseases has 1.9 times more cd8 cells than children with allergic diseases (p < 0,05).; children with infectious diseases have 2.1 times lower absolute number of cd25 cells than children with autoimmune diseases (p < 0,05); patients 7 -13 years old: children with infectious diseases have 1.5 times more absolute number cd25 cells than children with allergic diseases (p < 0,05). patients 14 -18 years old: children with autoimmune disease have 1.6 times higher cd4/cd8 index than children with infectious diseases (p < 0,05) karakina m. l. 1,2,3 , tuzankina i. a. 1,2 , vlasova e. v. 2 1 introduction: antiepileptic drugs are known to cause immunosupression in some cases. levetiracetam is an anticonvulsant medication used to treat epilepsy in the posttraumatic seizures. we report a rare case of hypogammaglobulinemia and b cell aplasia associated with levetiracetam treatment. case report: a 45-year-old female was operated for pituitary tumor with transnasal surgery and required second operation for postoperative rhinorrhea. after operation, menengitis developed and antibiotic treatment was administrated. however, there was a poor response to this treatment after one month and craniotomy was performed due to the diagnosis of "shimic menengitis". her seizures occurred as a postoperative complication and levetiracetam was initiated. after the 9-month follow-up, the findings of menengitis could not be controlled with antibiotherapy. she was referred to our immunology department for chronic menengitis with fever, headache and high cerebrospinal white blood cell count. results: in her clinical evaluation, it was learned that she had previously healthy. laboratory examination showed that decreased levels of igg 778 mg/dl (normal: 913-1884) and iga 97 mg/dl (n: 139-378). peripheral blood flow cytometric analysis revealed the absence of b cells (cd19+ b cells; <1%). t cell subsets and natural killer cell numbers were normal. neutrophil function, chemotaxis, phagocytosis and oxidative burst activity were found to be normal. isohemaglutinin titer, levels of pneumococal and tetanus specific igg antibodies were also normal. antiepileptic drug was discontinued after epileptic seizure was controlled. b cells gradually increased three weeks later and returned to normal within two months (cd19+ b cells: 9.8%). conclusion: patients requiring levetiracetam should have serum immunoglobulins measured and lymphocyte subsets analysis performed if they experience recurrent or persistent infections. mustafa gulec 1 ; fevzi demirel 1 ; ugur musabak 1 ; ozgur kartal 1 ; sait yesillik 1 ; abdullah baysan 1 ; ergun ucar 2 ; osman sener 1 1 gulhane medical school, division of immunology allergic diseases, ankara, turkey. 2 gulhane medical school, department of chest diseases, ankara, turkey. introduction: common variable immune deficiency (cvid) may present with several clinical manifestations involved in different organs and tissues in adults. we present a case with a history of chronic cough for more than twenty years and further diagnosed as cvid. case: a 49-year-old male who works in a chemistry lab admitted to our clinic with a history of frequent upper respiratory tract infections for more than 20 years. he also had intermittent diarrhea symptoms and his respiratory symptoms have been worsened since 2009. he had been hospitalized due to pneumonia and empyema several times. he had undergone left lower lobectomy due to bronchiectasis in 2012. he had been admitted to intensive care unit due to worsening of his medical condition. he was further diagnosed with cvid and ivig treatment was initiated. he is currently under remission with monthly ivig treatment and without any respiratory or gastrointestinal symptoms. discussion and conclusion: cvid is a clinical disorder in which the humoral part of the immune system is affected. most frequent presenting symptoms belong to respiratory, gastrointestinal systems and skin. however, due to the organ specific physical examination and lack of awareness, the diagnosis is frequently overlooked. in our case, frequent upper respiratory infection, loss of weight, diarrhea, bronchiectasis and empyema with unknown etiology are the most informative clinical signs. medical history is the most important part of patient evaluation. immunoglobulin replacement therapy is a basic treatment in primary immunoglobulin deficiency disorders. immunoglobulin substitution can be given intravenously (ivig) or subcutaneously (scig) for patients with antibody deficiency. both of these treatments are effective in prevention and cure of infections, although differences in advers events profile and patients' quality of life can be seen. the authors describe here their experiences in switching patients from ivig treatment to scig and a few years observation of scig therapy of patients with antibody deficiencies. sirje velbri 1 , mirja varik 2 1 tallinn children's hospital, tallinn, estonia. 2 north-estonian regional hospital, tallinn, estonia. j clin immunol (2014) 34:696-747 antibody deficiencies are the most common group of primary immunodeficiencies. the main hallmark of antibody deficiencies are recurrent infections but the patients have also higher risk of autoimmune and allergic diseases. we analysed retrospectively the frequency and character of autoimmune diseases in 159 patients (98 children and 61 adult patients) with primary antibody deficiencies. there were ana-lysed 2 patients with xla, 24 patients with cvid, 65 patients with selective iga deficiency, 26 pa-tients with iga/igg subclass deficiency and 42 patients with isolated igg subclass deficiency. autoimmune diseases were found in 39 patients (24,5%) besides in 17 children (17%) and in 22 adult patients (36%). in two boys with xla there was not found autoimmune diseases but in patients with other forms of antibody deficiencies in 12-57% of cases. autoiimmune diseases were found more often in iga/igg subclass defi-ciency (57%) than in other forms of antibody deficiencies (0-25%). the spectrum of auto-immune diseases differed in adults and child-ren and in different forms of antibody de-ficiencies. immune thrombocytopenia was found in adult patients with cvid or igg subclass deficien-cy, autoimmune connective tissue disorders in iga and iga/igg subclass deficiency. in children there was found mainly thyroiditis, diabetes i type and juvenile arthritis. rostov state medical university. research institute of clinical immunology, rostov-on-don, russia. primary immunodeficiency (pi) is currently one of most important genetically determined immunological clinical pathology which is hard to manage . among different types of pis almost 60 % of cases occurs due to a deficiency in antibodies production. injections of immunoglobulin are current standard in the management of pi. however this treatment is expensive, often is hard for patients, and frequently has limited effectiveness. substitution of immune proteins frequently is inefficient for treatment of severe infectious conditions in pi patients. we investigated maturation, activation and differentiation of immune t-cells in the dynamics of ivig replacement therapy. we observed patients with with cvid (7) and xla (5 people) over a year of regular replacement therapy. we have found that recovery of the humoral component does not affect the maturation and the differentiation of t-cells, but can reestablish activation and regulatory properties. these changes are more evident among patients with cvid, which immuneregulatory and functional potential reestablish faster. obviously, the effects tlymphocytes increase the effectiveness of replacement therapy in patients with xla, cvid common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies. the diagnosis is based on significantly decreased levels of immunoglobulins, with poor or absent response to vaccines and by excluding other defined causes of hypogammaglobulinaemia. as suboptimal antibody production is mainly due to b cell defects, therefore, we aimed to study lymphocyte subgroups of cvid patients and to compare the patterns with the clinical presentation in these patients. six adult patients with cvid diagnosis were studied. lymphocyte subpopulations were determined by flow cytometry. for b-cells subgroups cd3, cd27, igm and igd reagents w e r e u s e d . a l l l y m p h o c y t e s w e r e g a t e d f o r f i n d i n g cd19 + cd27 + memory b cells and from this population switched (cd19 + cd27 + igd -igm -) and non-switched (cd19 + cd27 + igd + igm + ) memory b cells were counted. all patients had normal levels of total lymphocyte count and absolute counts of cd4 + , cd8 + and cd16 + /cd56 + cells were also normal in all patients. only one patient showed low levels of cd19 + cells levels. according to paris classification scheme the patients could divided into two subgroups: mb0 and mb1. although almost all our cvid patients had normal number of total b cells, most of them showed reduced number of memory b cells and/or switched memory b cells. all of our patients had very low or absent level of class-switched memory b cells, therefore can be possible associated with a higher risk of granulomatous disease and splenomegaly. detailed investigation of b-cell phenotypes can better characterise cvid patients and can provide more information about possible clinical outcome. background: common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies, often related to spectrum of infectious and autoimmune diseases. in cvid patients wide spectrum of gastrointestinal disorders, including infections, are frequently seen. inflammatory bowel disease, helicobacter pylori infection, giardia lamblia infection, campylobacter or salmonella infection have been reported. however, abnormal liver function test and liver disease are found in approximately 10% of cvid patients. case history: a 47-year old female patient was admitted to infectious disease department due to recurrent pneumonia and purulent rhinosinusitis. blood analysis confirmed panhypogammaglobulinaemia with impaired responses to vaccinations, elevated liver function tests and anti-hcv positivity, interpreted as old and passed infection. due to cvid intravenous immunoglobulin substitution was started. however, liver function tests remained elevated and with hcv-rna analysis high hepatitis c viral load was detected. chronic hepatitis c virus infection was diagnosed and treatment with peginterferon α-2a and ribavirin was started. conclusion: our case emphasizes the need for hcv-rna and hbv-dna analysis in patients with hypogammaglobulinaemia, as the serological detection is impaired and prognosis for chronic hepatitis in immunodeficiency patients is poor. outpaitent clinic of clinical immunology and allergology, east tallinn central hospital, tallinn, estonia background: primary antibody deficiencies are the most frequent primary immunodeficiencies. recurrent respiratory tract infections may result in permanent lung damage in 20-40% of patients, most commonly presenting with the development of bronchiectasis. we aimed to evaluate the lung function and radiographic pulmonary changes in our patients with primary antibody deficiency. material and methods: we reviewed the records of adult patients with a confirmed diagnosis of primary antibody deficiency at our clinic. 10 patients were included in this analysis in whom ct scan was performed during the last 24 months, comprising 8 patients with cvid and two with igg subclass deficiency (median age 33 years; range 19-68 years; 60% females). all patients were on regular immunoglobulin replacement and one of the patients was on prophylactic antibiotic at the time of analysis. mean trough levels of igg were calculated based on the results measured during the last 12 months prior to ct scan. the spirometry was performed according to published protocols. results: all patients demonstrated normal spirometry data based on fev1 and fvc. two patients had slightly lower mmef rates, however, the changes were not associated with higher rate of infection nor changes in ct scan. none of our patients had bronchiectasis or atelectasis. among parenchymal changes fibrotic lines were most frequently detected. in two patients ground glass due to fibrosis was noted. mean immunoglobulin trough levels in our patients were between 4.9-8.6g/l, with the median of mean trough levels of all our patients 6.9g/l. when comparing the trough levels to lung function and ct scan results, no significant associations were seen. conclusion: no remarkable changes in lung function or chest ct scan in our patients with primary antibody deficiency were noted. as regular immunoglobulin replacement therapy could have prevented the development of permanent lung damage. rationale: patients with primary immunodeficiencies (pi) (n = 83) were treated subcutaneously (sc) with immunoglobulin g (ig) preceded by recombinant human hyaluronidase (ighy) at 3 or 4 week intervals based on their previous intravenous ig (igiv) dose. we report data for a subset of 59 patients aged ≥18 years from the final efficacy, safety and tolerability data of a pivotal phase 3 trial of ighy. methods: patients received igiv for 3 months at prestudy doses and frequencies. subsequently, ig 10% was administered sc, at 108% of the weekly equivalent of the iv dose, following rhuph20 infused through the same sc needle at a dose of 75 u/g igg. after a ramp up from a 1-to a 3-or 4-week dose interval, 59 patients received ighy every 3 4 weeks for 12 months. the primary efficacy endpoint was the mean rate of validated acute serious bacterial infections (sbis) per patient-year during the efficacy period. results: fifty-nine patients received 985 ighy infusions; 97.8% were completed without administration changes due to tolerability concerns or adverse events (aes). median infusion sites/month was 1.13. the temporally associated systemic ae rate was 0.20/infusion (ighy) vs. 0.33/infusion (igiv). the local adverse drug reaction rate was 0.286/infusion. the annual sbi rate was 0.00 and 3.10/patientyear for all infections. conclusion: in adults with pi, ighy was effective in preventing infections. the majority of patients received full 3-to 4-weekly doses of ig using a single sc site with good local and systemic tolerability. rationale: in a pivotal phase 3 trial of facilitated-subcutaneous (sc) infusion of human immunoglobulin g (igg), 10%, and recombinant human hyaluronidase (rhuph20) (ighy) in patients with pi, rhuph20 permitted most patients to have a single-site infusion (every 3-4-week igg dosing) with bioavailability and infusion rates comparable to intravenously administered igg (igiv). we report the final analysis of the long-term extension of the initial phase 3 study, with a duration of up to 3 years of treatment with ighy plus additional follow-up. methods: sixty-six patients who completed the initial phase 3 study enrolled in the extension study. patients continued their pre-study ighy dose/frequency every 3-4 weeks. after 3 months, some patients switched to 2-week dosing to evaluate effects of shorter ighy interval on trough igg levels. from the final analysis, tolerability and safety after up to 3 years of treatment were evaluated. the ighy part of the extension study was followed by a 24-48 week observation period during which patients received igg 10% administered iv, or sc weekly without rhuph20. results: in the extension study, 66 patients were treated with ighy and 14 discontinued prior to safety follow-up. following discontinuation of rhuph20, 48 patients switched to follow-up. no patients withdrew due to ighy-related reactions (adrs). no serious adrs related to ighy were reported. the maximum ighy exposure for the initial and extension studies combined was 3 years (total exposure = 187.69 patient-years; n = 2959 ighy infusions); during this time, there were no clinically observable long-term changes in the skin or sc tissue. the rate of temporally related systemic adverse events (aes), excluding infections, was 0.159/ infusion. the rate of all local aes was 0.103/infusion. of the 1600 ighy infusions administered in the extension study, 97.9% had no administration changes (rate reduction, interruption or discontinuation) due to tolerability concerns or adverse events. the annual rate of all infections under ighy treatment was 2.86/patient-year. reducing the dosing interval from 4 to 2 weeks (same monthly dose) resulted in a 13% increase in trough igg levels. thirteen patients had at least 1 non-neutralizing anti-rhuph20 antibody titer of ≥1:160 with no associated aes; no patients had neutralizing anti-rhuph20 antibodies. conclusions: in the extension study, ighy was well tolerated and effective, with no serious adrs for treatment periods up to 2 years. over a maximum 3-year ighy exposure (for an individual patient) in the initial phase 3 and extension studies combined, no long-term changes in skin or sc tissue were observed. the rates of infections and adverse reactions were stable or decreased over the course of the two studies, suggesting no increased risk with continued exposure to ighy. rationale: we report interim analysis of safety, tolerability and pharmacokinetics (pk) of igsc 20% in patients with primary immunodeficiencies (pi) aged ≥2 years in europe. methods: epoch 1: igsc 16% or intravenous ig 10% (igiv) administered at pre-study doses every 3 months. epoch 2: igsc 20% administered 1 time per week for 12 months at epoch 1 doses. serum igg trough levels are maintained at >5 g/l. the primary endpoint is validated acute serious bacterial infection (sbi) rate. results: at the interim analysis in october 2013, 49 patients started the study. during igsc 20% treatment (n = 48), 1 acute sbi episode (pneumonia, moderate in severity) was reported. the infection rate per patientyear was 4.3 (igsc 20%). there were no serious adverse events considered related to any treatment. the rate of local adverse drug reactions (adrs) was 0.055/infusion and all were mild in severity; no severe systemic adrs were reported with igsc 20%. of 1812 igsc 20% infusions, only 0.4% required slowing or interrupting the administration rate. mean serum igg trough levels were 9.2 g/l (igsc 20%, n = 46), 9.1 g/l (igiv 3-week interval, n = 4) and 7.7 g/l (ivig 4-week interval, n = 25). conclusion: igsc 20% provided an effective and well-tolerated therapy, with no dose adjustments needed from pre-study ig dose. this study is ongoing to confirm results over 12 months. it is well known, that intravenous immunoglobulin (ivig) is the main therapeutic modality in b-cell primary immunodeficiencies (pid), it decreases mortality and morbidity in these patients dramatically. yet, it is also well known that all ivig products are very expensive, especially considering life-time use and almost normal life expectancy in these patients, if treated correctly. irregular treatment and problems with insurance/state coverage of ivig in some countries stems from this. goal: the goal of our study was to compare medical and other costs, related to the disease in patients with humoral pids with or without ivig treatment. patients: 21 patient with b-cell deficiencies (73% x-linked agammaglobulinemia, all genetically confirmed, 27% common variable immunodeficiency). the age of patients varied from 2 to 16 years. methods: we analyzed medical and other disease-related costs during 2 years preceding the diagnosis (without ivig therapy) retrospectively and during 2 years on regular ivig therapy. the costs included those incurred by the state (hospitalization, home visits, emergency calls) and by the parents (costs of drugs, private consults, etc). the state costs caused by parents missing work were also considered. for standardization purpose for calculation we used the prices for the end of 2013. results: the patients analyzed fell into 3 different categories: 1. the 1st group -(76% of patients studied) -patients with several severe infections before therapy, some chronic conditions as a result of those. age of diagnosis varied from 2 years to 16 years (with average time to diagnosis 5 years). in this group costs of ivig treatment were 2,5 times higher than before the diagnosis (fig. 1 ). 2 nd group(10% of all). patients with late diagnosis (average time to diagnosis 7 years), who had multiple severe infections before the ivig treatment and acquired serious chronic lung complications due to it. in this group the costs before the treatment were higher, than on ivig treatment. 3. very early diagnosis -within the first year of life (mostly because of preceding family history) (14%). the comparative analysis was not possible, but it was noted that these patients had no history of serious infections before of while on ivig therapy. the only additional costs, besides ivig, were related to bad venous access, requiring occasionally 1 day in-patient hospitalization for ivig infusion. as expected, in all 3 groups, the number of infectious episodes, the number of hospitalizations (fig. 2) and doctor visits (fig. 3) after beginning of regular ivig treatment dropped dramatically. we also followed 2 patients with xla, who did not receive ivig therapy because of social aspects. both patients died from severe infections. we evaluated this fact in economical prospective: in russia one worker, who works continuously and retires at 60 years of age brings about 130 mln roubles into the state budget (when costs for schooling and routine medical care are subtracted). if one supposes that an xla patient have been diagnosed very early in life, did not form complications prior to therapy and was on regular ivig therapy for life, this sum equals to 160 years on ivig. conclusions: regular ivig therapy not only leads to reduction of infectious episodes, hospitalizations, and as a result improved quality of life. in some cases it even brings down the disease-related costs, incurred by the medical system and the family, and is economically advantageous for the state. introduction: replacement of immunoglobulins is a standard therapy for patients with primary immunodeficiency disease (pidd) characterized by primary antibody deficiency (pad). this poster represents our clinical experiences of initiation of home-based treatment with subcutaneous immunoglobulin (scig) with the patients diagnosed with primary variable immunodeficiency (cvid). case report: the patient (age 30) has been treated at our immuno-allergy outpatient clinic since 2005 with the diagnosis of hypogammaglobulinemia (igg, iga, igm) with normal b cell count, withsusp. cvid. with the repeated administration of intramuscular and intravenous immunoglobulins (ivig, imig) repeatedly occurred serious adverse reactions, which resulted in discontinuation of the replacement therapy. in february 2012 the health condition of the patient worsened due to recurrent bacterial respiratory infections. there was a progressive decrease of serum concentrations of immunoglobulins (igg 2,1 g/l, igm 0,25 g/l, iga 0,23 g/l). the patient was admitted to the intensive care unit of the 1 st internal department, university hospital bratislava, for a subcutaneous immunoglobulin replacement trial. despite serious adverse reactions with previous administration of several types of immunoglobulins, there have not occurred any clinically relevant side effects. conclusion: compared with im or iv formulations and administration, for selected patients, scig is better tolerated, clinically efficacious, safe, and appreciated by the patients. background: common variable immunodeficiency (cvid) is primary immunodeficiency (pid) classically viewed as antibody deficit. although, cvid is considerd to be a humoral immunodeficiency, approximately 40% of cvid patients have low t-cell counts or abnormal tcell function. despite adequate immunoglobulin replacement patient morbidity and mortality is variable and a number of complications are not those typically seen in pure antibody pid e.g. xla. so, t-cell rather than b-cell phenotype could determine outcome in patients with cvid. many patients with cvid have clinical history suggestive of allergic respiratory disease, but prevalence of asthma and role of atopy have not been well established. apart from recurrent infections and their sequelae, cvid patients suffer from other disease-related complications in up to 45% of the cases. about 25% have onset before the age of 15 years. aims: 1) to present one more case of tadolescent with cvid and allergic asthma, 2) emphasise ultimate need of collaborative network of primary immunodeficiency centers. case report: parents of 12,5 y boy were sure that "something was wrong" with their son and were seaking for problem solution for many years. since age 3, child had frequent respiratory infections. adenoidectomy and tonsillectomy were performed at age 6. sinuitis was diagnosed several times. boy was complaining of fatique for a long time. last several years, his main problems were fever (max 41 c) usually lasting 5 days till 2 weeks, accompanying running nose, coughing, conjunctival problems; intermittently headache (lasting for a few hours till all day). oral aphtae were present almost every two weeks. he was incompletely vaccinated (bcg, and once diteper). morbilli and varicellae infections passed without complications. in jan 2012 he was diagnosed as allergic asthma in sarajevo (allergy to pollen, soya, nuts and antibiotics ("ceclor" and "pancef"). in february 2012 was admitted at children's hospital sarajevo for suspected primary mmunodeficiency. he had slightly lower levels of igg and iga (twice measured), normal ige and decreased number of t helper ly. due to suspected pid, boy was checked up in two nearest regional pid centers : hypogammaglobulinemia was confirmed (igg 4,7, iga 0,24, igm 0,46, as well as deficiency of igg1, igg2 and igg3). flow cytometry showed slightly raised concentration of lymphocites b (cd8), slightly raised number of nondifferentiated b cells. cvid was suspected but not proved. in july 2012 child visited center for primary immunodeficiencies in munchen, germany, were he was diagnosed ad probable cvid on the basis of hypogammaglobulinemia, lower levels od switch memory b cells, normal number of t-cells, positive antibodies to vaccinations and overcome infection (morbilli, varicellae). allergic asthma was additionaly confirmed in specialised pediatric pulmology hospital in germany (abnormal spirometry, normal ige, positive skin prick test, abnormal fractional exaled nitric oxid test, incipient brochiectasies due to asthma confirmed by high -resolution lung ct scan). low human immunoglobulin replacement was started (200 mg/kgbw) as well as antiasthmatic therapy (inhalatory steroids, antihistaminics). excellent therapeutical response were achieved : after one 1,5 y follow up, we can confirm patient has excellent general condition, no subjective symptoms, no tiredness, no severe infections. conclusion: diagnosing cvid is challenging task and quite often could be "per aspera ad astra". there is ultimate need of collaborative work of primary immunodeficiency network aimed of diagnosing patients on time. cvid patients with history suggestive of allergic asthma, are negativne on traditional tests, additional test designed to identify allergic asthma might be conducted. common variable immune deficiency (cvid) is a heterogeneous syndrome characterized by hypogammaglobulinemia, recurrent infections, immune disregularity (autoimmunity, autoinflamation) and propensity to malignancies. in the us report, 8.2% of cvid patients had a lymphoid malignancy, and cancers of other sorts developed in 7% of patients. it is not clear why cvid patients have higher risk of malignancy but chronic antigenic stimulation, chronic inflammation and increased chromosomal radiosensitivity may be the cause. cvid patients with higher igm level, reduced or absent b cell numbers, cd4 t cells lower than 200 and pli phenotype have higher prevalence of malignancy. allergy and clinical immunology department of rasool e akram hospital has registered 47 cvid patients. mean age of the onset of cvid symptoms was 11.2 years. mean diagnosis age was 20.2 years with mean diagnostic delay of 9 years. mean follow up time was 7 years (minimum 0.5-maximum 23 y). malignancy occurred in the follow up of 6 patients (13%). one patient had two different malignancies (breast cancer and gi adenocarcinoma). malignancy risk per case was 15%. hodgkin's lymphoma was the most common type (43% of cancers common variable immune deficiency is a heterogeneous syndrome characterized by hypogammaglobulinemia , recurrent infections , auto immunity and auto-inflammation . more than 25 % of cvid patients have auto immune complications and among them, auto immune cytopenia is the most common. cvid patients with higher igm levelhigher 21 low b cellslower t reg levelslower cd4/cd8 ratio and lower class switched memory b cells have higher prevalence of autoimmunity. allergy and clinical immunology department of this hospital has registered 47 cvid patients. mean age of onset of cvid symptoms was 11.2 years. mean diagnosis age was 20.2 years with mean diagnostic delay of 9 years. mean follow up time was 7 years (minimum 0.5 maximum 23 y). autoimmunity was detected in 23 cases (49 %) and 12 cases (26 %) had more than one autoimmunity. autoimmunity was the first symptom of cvid in 15 percent of cases. autoimmune disorders should be considered in the follow up of cvid patients. igg is major immunoglobulin and classified subgroups as igg1, igg2, igg3 and igg4. igg1 and igg3 subclasses are rich in antibodies aganist proteins such as the toxins produced by the diphtheria and tetanus, as well as antibodies aganist viral proteins. recurrent ear infections, sinusitis, bronchitis and pneumonia are common in ig g subclass deficiency. ig g1 is the major subclass of ig g. igg1 subclass deficiency is very rare. chronic eosinophilic pneumonia is one of the eosinophilic lung disease and is seen rarely. in the presence of peripheral eosinophilia and radiological pulmonary infiltrates diseases suspected. when increase in the number of eosinophils in bronchoalveolar lavage fluids and/or presence of eosinophils in lung tissue diagnosis is confirmed. according to different recording systems chronic eosinophilic pneumonia is %0-2 of the interstitial lung disase. there is not any criteria for diagnosis but also diagnoses is confirmed with suspected findings. symptoms inludes that: 1)in the presence of respiratory symptoms for two weeks long 2)eosinophilia at alveolar lavage and\or peripheral blood ( bal fluid cytological examination > %25, blood >1500/mm 3 ) 3)radiological imaging of the lung peripheral infiltration 4)exclusion of the other causes of eosinophlic lung disease there is eosinophilia over 1000/mm 3 nearly all patients. one of third or half of patients have diagnosed as asthma. disease begin with systemic symptoms such as night sweats, weight loss, anorexia and pulmoner symptoms such as cough, shortness of breath, wheezing. patients have restrictive or obstructive findings in pft. one of third patients, especially with history of asthma, have obstruction in pft. i̇n the pathologic biopsy findings include; thickening of alveolar walls and accumolation of eosinophils and lymphocytes. long time used corticosteroids treatment is recomended. relaps is common when treatment is discontinued. we present the patient who has ig g1 deficiency, chronic eosinophlia and %42 eosinophils in bronchoalveolar lavage fluid. the patient improved long time used oral steroid then inhaled steroids. this was presented in terms of clinical association. case: a 12 years old female patient who were followed due to asthma in the other center for two years, although use of combination inhaled fluticasone and salmeterol, patient was admitted with cough and sputum production. in thorax ct there were, bronchiectasis at right lower lobe, pneumonic consolidation in the right lower lobe and ground glass opacities. we detected as igg 924 mg/dl (835-2094mg/dl), iga 157 mg/dl ( 67-433 mg/dl), igm180 mg/dl (47-484 mg/dl), ige 108. 6 mg/dl, igg1 4896 mg/dl (5990-15600 mg/dl), igg2 2354 mg/dl (1110-5150mg/dl), igg3 1024 mg/dl( 290-2000mg/dl), igg4 616 mg/dl (40-1600 mg/dl). because eosinophilia (717 cells) and symptoms continued, bronchoscopy was performed. left main bronchus was normal, right bronchus were seen dilated. purulent secretion was aspirated on rigt bronchus with flexible bronchoscopy ..in cytological examination % 42 eosinophils was detected in bal. bronchoalveolar lavage cultures was negative. the patient was diagnosed chronic eosinophilic pneumonia and 1 mg/ kg oral steroid was began. ivig was given up to patient; because of frequently recurrent sinopulmonary infection and patient had igg1 subclass deficiency. in the third month of oral steroid therapy physical examination findings and pft were improved and started inhaled steroid. conclusion: immunodeficiencies often can be seen alone. although, the pathogenesis of immundeficiencies with lung disease is not well understood and associated with interstitial lung disease. therefore, investigations must be include bronchoscopy and bronchoalveolar lavage. scid is a congenital heterogeneous group of diseases characterized by severe impairment of t,b, nk cell development and function. the hematopoietic stem cell transplanted 93 patients with scid in the time period from 1994 to 2014 were evaluated. male/female ratio is 59/34. median follow-up time is 5 years (6 months-20 years). parental consanguinity ratio was 81,7%. mean age of onset of the symptoms was 3 ± 2,7 months. most common clinical findings on admission were; pneumonia (75,3%), moniliasis (62,4%), diarrhea (39,8%), dermatitis (19.4%). classification according to t, b, and nk cell counts; (figure 1 ). molecular genetic defects were determined in 60 patients (figure 2 ) are given. there is no difference between age groups according to occurrence of acute and chronic gvhd. death ratio increases with the increasing age. acute and chronic gvhd and number of deaths were significantly higher in peripheral stem cell transplanted patients. there is no statistically significant difference in occurrence of acute/chronic gvhd between b-and b + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. there is no statistically significant difference in occurrence of acute/ chronic gvhd between nk-and nk + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. death ratio was similar in groups. bcg dissemination in bcg vaccinated patients is significantly higher in the nk + group. the rate of complications due to severe infections is high and increases with age in patients with scid. hsct is curative, should be considered as early as possible. trec analysis for neonatal screening will give chance for early diagnosis and treatment of the patients. the hyper-ige syndromes are rare pids and are characterized by atopic dermatitis, skin abscesses recurrent pneumonias, elevated serum ige levels and sometimes mucocutaneous candidiasis. we have been evaluating and following up a group of patients with features suggestive for autosomal recessive hyper ige syndrome (ar-hies). homozygous dock8 mutations were identified in several of these patients. however, two siblings from a consanguineous family in this group of patients showed homozygous block in chromosome 20p1.2 in homozygosity mapping which includes stk4 gene. sanger sequencing was performed for stk4 deficiency and showed a novel c.57-60delgata mutation in stk4 gene causing a premature stop codon. clinical manifestations of stk4 deficiency, also known as a macrophage stimulating 1 (mst1) deficiency, stk4 deficiency comprise recurrent and severe viral skin infections including molluscum contagiosum and warts, fungal and bacterial infections and autoimmunity which are also the features of ar-hies. our patients's main features include autoimmune cytopenias (aiha and itp), cutaneous viral (molluscum contagiosum and mild perioral herpetic lesion), mild atopic dermatitis, seborrheic dermatitis, lymphopenia (particularly cd4 lymphopenia), and intermittent mild neutropenia. serum ige level was mildly high in these patients. our results indicates that patients that show clinical phenotype of ar-hies needs to be also evaluated for stk4 deficiency. determination of the underlying defect and reporting the patients are required for the description of the phenotypic spectrum of pids and the frequency of these diseases as well as for genetic counselling. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il-12rβ1 deficiency, most common form of msmd, is caused by mutations in the il12rb gene. 37 patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il-12rb1 was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il12rβ1 expression was found to be less than %1. prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il12rβ1 on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd19, increased cd45ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. özdemir öner 1 ; bozdoğan sıla 2 1 department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. 2 department of pediatrics, sakarya university, medical faculty, adapazarı, türkiye. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: 15 month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at 5 months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at 7 months, family fed the patient with cow's milk but 3 hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at 3 weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was 4.420/mm 3 , with 21% neutrophils, 6% eosinophils and 69% lymphocytes. his hemoglobin was 8.2 g/ dl, platelet count was 280.000/mm 3 . background: except for antibody deficiency and complement defects, hematopoietic stem cell transplantation (hsct) is the single best curative treatment defined for primary immunodeficiency (pid) so far. in the current study, we aimed to assess the role of pid type, donor type and clinical status on hsct success rates. materials and methods: we retrospectively reviewed the records of a total of 91 hscts procedures performed in 68 patients diagnosed with pid between 1997 and 2013, in ankara university pediatric allergy and immunology department. results: of the 68 patients, 40 had severe combined immunodeficiency (scid) and 28 had non-scid. the survival rates following hsct, in both scid and non-scid patients were 75%. when classified according to the source of donor, patients who had a hla-matched sibling donor (msd) in the scid group had 90.9% survival rate post transplantation, those who had a matched related donor (mrd) had 85.7% and those who received a haploidentical donor had 63.6% survival rates. in the non-scid group there were 3 patients with haploidentical transplants (2 omenn syndrome and 1 mhc class ii deficiency) and all patients died. we assessed several potential risk factors associated with survival in scid patients. patients diagnosed over 6 months of age with a pre-existing pulmonary infection, requiring intensive care and/or mechanical ventilation had significantly lower survival rates. conclusion: hsct is the best curative treatment for pid. our results demonstrated that hsct performed from matched family donors or even haploidentical parents is a lifesaving treatment in various types of pid's, especially in scid. introduction: in case of donor availability allogeneic hematopoietic stem cell transplantation (hsct) can be regarded as a definitive therapy for a variety of primary immunodeficiency syndromes (pids), including severe combined immunodeficiency (scid) and non-scid pids. study period: we retrospectively reviewed the hospital records of 32 consecutive children with pid, who had 41 allogeneic hsct in the last 22 years, between january 1991 and january 2014. our median follow-up time is 2,1 years (2 months -22,1 years) patients and methods: the median age of children at hsct was 10,5 months (3,5 months-18 years). 23 boys/9 girls diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. conditioning regimens included busulphan + cyclophosphamide, busulphan + cyclophosphamide + atg, fludarabine + melphalan or fludarabine + anti-cd52 in b-, t-, nk-scid cases conditioning was not used. indications for transplantation: patients' diagnoses were severe combined immunodeficiency (n = 16), wiskott-aldrich syndrome (was, n = 6), chronic granulomatous disease (cgd, n = 3), xlinked lymphoproliferative disease (xlp, n = 3), whim-syndrome (n = 1), hyper igm syndrome (cd40 ligand deficiency, n = 1), leukocyte adhesion deficiency (lad, n = 1), dock8 mutation (n = 1) transplantations: 41 hscts for 32 children were performed. 6 patients were retransplanted (4 pt once, 1 pt twice, 1 pt 3 times), because of rejections. at the first hscts in 5/32 cases sibling bone marrow, 1/32 sibling peripheral blood, 1/32 sibling cord blood, 4/32 unrelated cord blood, 9/32 unrelated bone marrow, 2/32 unrelated peripheral blood, 7/32 haploidentical donors were used median cd34 count was 2,52x10 6 /kg (0,13x10 6 -26,95x10 6 /kg) patients engrafted on median 27 ± 8 day (anc > 0,5 g/l) acute gvhd occured in 17/32 cases ( pathomorphological findes: distortion of the structure of lymph nodes due to enlargement of paracortical zones and follicules was found in all patients. presumed atypical cells (makrolymphoblastes and berezovsky-sternberg-reed cells), were detected in 2 patients overwise during follow-up for 2-5 years in these patients revealed no specific infectious and malignansy. accumulation of proliferating dn cells (a lot of mitoses) was characteristic fiture in lymph nodes and spleen of alps patients. setting of plasma cells in follicular zones in the lymph nodes was reveald in 5 cases, and eosinophilesin 2, pronounced immunoblast proliferation in 2, sinus histiocytosisin 3. multiple hyperplastic follicles, extended sinuses with many phagocytizing macrophages, lymphocytic infiltration was revealed by histological examination of the spleen in all cases. immunohistochemistry findes: t-and b-cells proliferation (ki-67 expression), atypical location of lymphocyte populations, settings of plasma cells in all lymph node zones. in paracortical zones was found cd5+, cd7+, cd3 + cd4-, cd3 + cd8-cells in 5 patients, cd38+, cd68+ cells in 1 patient; and in restricted follicular zones -cd19+, cd22+, hla-dr + cells in 4 patients, cd3+, cd15+ and cd68+ in 2 patients. the presentation of a case of kimura disease in a patient with was. the was was diagnosed in 1 year 2months, based on infectious syndrome, atopic dermatitis, hematological syndrome -skin hemorrhages and thrombocytopenia -25*10 9 /l. after he performed often sars. regularly received ivig at 400mg/kg, antibiotic therapy with a positive effect -infectious syndrome stopped, controlled hemorrhagic syndrome. since 2010 there was a herpes virus infection with frequent exacerbations on the face. patient received acyclovir, valacyclovir, famvir with a temporary effect. in 08.2011 on the left side of the face -periorbital region, eyelids, malar region appeared the site of soft tissue hyperproliferation 10 -12 cm in diameter with a thickness of 3-4 cm, with moderate moist, painful on palpation, the left eye was closed, the lid margin ciliated dramatically thickened, deformed. in october-november 2011 was admitted in diagnostic department, massive antibiotic, antimycotic, antiviral therapy, ivig therapy. the histological study showed the angiolimphoid hyperplasia with eosinophilia without immunomorphological signs of malignant tumor growth. based on histological, immunohistochemical studies of the skin, these clinical and laboratory findings, diagnosis: mass lesion of the left face -kimura disease. patient helded 6 courses of chemotherapy with positive dynamics: mass lesion decreased, erosive surfaces disappeared and now receives romiplostimum, acyclovir, biseptolum, ivig 0.5 g/kg 1 time per month. under the observation there is a family k., which is unique in the deletion variants of the same region 22q.11.2 and the presence of an adult patient with the di george syndrome. clinical manifestations of the syndrome in family members is differ. methods: clinical, laboratory, instrumental and genetic ( dna was isolated using a kit «qiaamp dna mini kit» and dna from dried blood spots was isolated using a commercial kit "dna-sorb-b». mlpaanalysis set salsa mlpa probemix p250-b2 digeorge, genetic analyzer applied biosystems 3500). mother (34 years), had few incidents of pneumonia before 10 years. she was diagnosed ullrich-turner syndrome at age 10 years. all childbirths by caesarean section. she has not any laboratory and instrumental findings of immunodeficiency, endocrinopathy, cardiac and thyroid abnormalities. she has deletion in the starting area of the di george region (lcr22-a), including genes cltcl1, hira, cdc45, cldn5, gp1bb, tbx1, txnrd2, dgcr8. father (36 years), healthy, current smoker. the dna microstructural violations in di george region haven't been identified. son was from i pregnancy, heart defect was set prenatally, marked growth retardation. childbirth at 38 weeks. he had unstable reduced cell parameters and hypogammaglobulinemia, the size reduction of the thymus and congenital heart disease: truncus arteriosus, dc 2b stage by lang. after 42 days of life operated for congenital heart disease. during the surgical intervention the thymus wasn't found in a typical place. the postoperative period was complicated by sepsis, heart and respiratory failure. received ivig, antibiotic and antifungal therapy. he suffered from sars, severe course, the degree of respiratory failure 2 (8 months) -death. in mlpa-dna defined microdeletion disorders starting region di george (lcr22-a) -from the 4-year-old dry blood sample (postmortem study a 3 year old boy with bleeding, eczema and recurrent pyogenic infection was admitted to our department. the child who received treatment in hematology accidentally a few times, but has not had the effect of treatment . laboratory parameters -cd3-cells-59% (5550), cd4-cells -22% (1221), cd8-cells -37% (2053), cd19-cells -29% (2728), cd16 / 56-cells -15% (1411), cd4 / cd8-0.59, hla-dr-22, nbt-73. igg-14.6 q /l, iga-3.3 q / l, igm -4.3 q / l, ige-328 u / ml. hb-40 g / l, erythrocytes-1.75x 10 12 / l, leukosytes -15.6x10 9 / l, neutrophils-31%, lymphocytes -56% (9408), eosinophils-2%, monocytes-2%, metamielosytes:-9%, plateletsrare was observed.normal bone marrow cells, the separation of platelets from meqakariosyt has become weak. the advantage of erythroid unordered have been observed. bone marrow puncture the mielokariosytes -87.0 x10 9 / l, meqakariosytes -0.01x10 9 / l, blasts-1.2%, myelosytes -7.4%, metamyelosytes -9.2%, seqments -14.2% neutrophils-% 51.6, eosinophils-1.4%, lymphocytes-5.8%, monocytes-0.4% erythroblasts-2.0%, pronormoblasts-2.8%, normoblasts-40.0% meqakariosytes -0.4%, plasmatic cells-0.2%.his younger brother with eczema, pyogenic infections (furunculosis) was admitted to our department. cd3-38% (1761) ataxia telangiectasia (a-t), is a genetic disorder caused by the homozygous mutation of the atm gene and, frequently associates with variable degrees of cellular and humoral immunodeficiency. however, the immune defects in patients with a-t are not well characterized. to our knowledge, there is no work on major lymphocyte subpopulations and recent thymic emigrants of a-t patients comparing to age-matched healthy controls. according to esid criteria, 17 patients diagnosed as a-t and 12 agematched healthy children were assigned to the study. both patients and healthy controls were grouped as 0-5, 6-10, 7-15 years and older than 16 years. using flow cytometer, major lymphocyte subpopulations and cd4 + cd45ra + cd31+ recent thymic emigrants (rte) were determined as per cent and absolute cell numbers and compared. no significant differences regarding all lymphocyte subpopulations were observed between age groups of a-t. comparing to the healthy controls, there were a decrease (in t cells, effector memory t4 cells, b cells, naïve b cells, switched b cells and rte) and there were an increase (in active t8 cells, naïve t4 cells and nonswitched b cells) in the absolute number and percent of some cell populations in the a-t group. findings of this study showed effector functions in some cell lymphocyte populations were decreased and could be thought that bone marrow of patients should be tried to increase the cells numbers. however, the study has an important limitation about patient and healthy population. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd19, increased cd45ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. nijmegen breakage syndrome(nbs) is a rare autosomal recessive disease usually presenting at birth with microcephaly. here we present a case of nbs diagnosed at the age of twenty seven who admitted to our outpatient clinic with malaise, loss of appetite, weight loss and dyspnea on effort . she was hospitalized in another centre 2 years before because of pneumonia, pancytopenia, generalized lymphadenopathy, hepatosplenomegaly and hypogammaglobulinemia (very low igg and iga) where she couldn't have any definite diagnosis. her past medical history was remarkable for primary amenorrhea and basal cell carcinoma excision from preaericular region when she was 23. she had no severe infection history before 25 except frequent upper respiratory tract infections. her parents were consanginous and she had a brother died at 6 months of age. microcephaly together with short stature, multiple palpable lymphadenopathies , splenomegaly and absent secondary sexual characteristics were prominent features in physical examination. direct fluorescence sequencing of the nbn gene showed a homozygous mutation in exon 6 (c.657_661delacaaa) which confirmed the diagnosis of nbs in our patient. nbs is mostly diagnosed in childhood. the delay in diagnosis was partly due to the lack of severe infections in her past medical history until she was 25. another factor that lead to the delay is that it is an unknown disease among physicians in turkey. the longest known survival is 53 years in a patient who had no clinical features of nbs other than primary amenorrhea. as a result, our patient is one of the oldest patients reported in the literature presenting nearly all of the classical features of the disease and carrying the most common pathologic mutation. wiscott-aldrich syndrome (was) is a rare x-linked recessive immunodeficiency disorder characterized by thrombocytopenia, small platelets, eczama, recurrent infections and an increased risk of autoimunity and malignancy. the gene responsible for was is located in chromosome xp11.22-p11-23, which consists of 12 exons, and encodes a 502-amino acid protein. in this study, we aimed to screen was gene mutations and analyze the effects of determined mutations in 2 boys with non-classical was phenotype. ivs10 + 1g > a gene alteration in intron 10 of was gene was identified in case 1, previously. so, rna isolation and then cdna synthesized was carried out. in case 2, after amplification of 12 exons of was gene by pcr, the amplicons were sequenced. in this patient, g > c alteration was detected in the first base of intron 9. afterwards, cdna synthesized for detecting the splicing effect. based on the gel image results, cdna found to be 400 base pairs smaller than the normal in case 1. in case 2, g > c alteration was detected in the first base of intron 9 and then we determined multiple splicing products. two different splicing mutations (ivs10 + 1g > a and ivs9 + 1g > c) were detected in two cases with non-classical phenotype. ivs9 + 1g > a splicing mutation was stated in the literature, previously, but ivs9 + 1g > c mutation was first time identified in this study. whim is rare (<1 / 1 000 000), heterozygous, autosomal dominantly inherited pid, caused by mutations in the gene encoding for the chemokine receptor cxcr4, mapped on 2q21 locus. the altered cxcr4/ cxcl12 interaction impairs cellular homeostasis and trafficking, resulting in immunological dysfunctions with abnormal retention of mature neutrophils in the bone marrow (myelokathexis) and consecutive severe neutropenia, variable degree of lymphopenia and hypogammaglobulinemia. whim patients suffer from recurrent bacterial infections since early childhood and later on manifest a specific susceptibility to hpv infections with developing widespread warts. because of rarity of the disease, heterogeneity in clinical presentation and usually incomplete phenotype, the diagnosis is often delayed and whim syndrome is not suspected. a 13 year old boy who is suffering from recurrent bacterial infections, often complicated with bronchopneumonia, with severe neutropenia, but also, with lower level of lymphocytes and still, normal serum immunoglobulin level is presented. he has no developed warts; neither his parents nor relatives have warts. at the age of 10 years was unveiled the diseasecausing mutation in the cxcr4 gene (c.1000c > t; p.r334x; heterozygote). severe congenital neutropenia accompanied with lymphopenia and findings of mature neutrophils in bone marrow, might be an easy approach for getting closer to the clinical diagnosis of whim syndrome. early identification is important for clinical and therapeutic management, allowing a more comprehensive follow-up and administration of appropriate therapy. we report two cases of congenital heart disease (tetralogia fallot and interruption aortic arch) with confirmed microdeletion chromosome 22q11.2 by karyotype and fluorescence in situ hybridization analysis (fish). children underwent surgical correction of congenital heart defects with good postoperative outcome, although were complex. the phenotype of these patients can be extremely variable, frequently leading to clinical confusion, diagnostic delay, excess morbidity, early mortality. identification of these patients is essential for their adequate management and genetic counseling. a multidisciplinary approach is fundamental to ensure that the patient will be able to attain his or her maximal potential. the underlying cause of the juvenile periodentitis is not well understood but is now thought to be related to an abnormal immune system and to invading bacteria in the cementum of the teeth. instead painful fissures and recurrent pyogenic infections of the skin seem to be the most common medical complications. however, a number of pls patients with abscesses or pseudotumors of the liver have been described. there have been reports of pls patients with other stigmata such as growth retardation, non-symptomatic intracranial calcifications and mental retardation furthermore, coinheritance of pls and albinism type 1 has been reported. case presentation: a 13 yr old girl admitted to our hospital with chief compliant of skin lesions since early months of birth. in the past medical history; she had skin abscess and failure to thrive. on admission she had erythematous, shiny skin with generalized dry scaly predominantly palmoplantar hyperkeratosis and loss of teeth except four or five molar teeth. she informed that she had malformed teeth since childhood which fell off one by one. she had also poor oral hygiene non-pitting edema on lower extremities. no hepatosplenomegaly detected. family history was negative. she investigated for probable immune-deficiency with regard to skin lesions, history of skin abscess and ftt the lab finding are as following: cbc diff = normal ,crp = neg, esr = 6, ast = 31, alt = 15, alph = 1656, alb = 3.8, total pr = 6.2 , urea = 16, cr = 0.4 igg = 1620, igm = 230, iga = 43, ige = 10.8, cd19 = 17%, cd 8 = 20%, cd3 = 55%, cd4 = 34%, cd8 = 20%, cd 56 = 15% conclusion: according to nearly normal lab values and presenting signs such as: generalized pyogenic periodentitis, palmoplantar hyper keratosis and negative family history were attributed to a very rare autosomal recessive disorder with ectodermal dysplasia known as papillon-lefevresyndrome manifesting with palmoplantar hyperkeratosis and severe early onset of destructive periodontal leading to pre-mature loss of both primary and permanent dentitions. nijmegen breakage syndrome (nbs) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. the disease is caused by mutations in the nbs1 gene, which encodes nibrin, a component of the hmre11-rad50-p95 complex involved in cellular response to dna doublestrand breaks. the aim of the present case report was to discuss two siblings with immunologically and clinically different phenotypes of disease presentation and to make an attempt to explain possible genotype-phenotype relation. the two patients and their non-consanguineous parents were clinically, laboratory and genetically investigated. for mother and father we observed no clinical presentation and no immunological abnormalities. sibling no.1 (6 years old boy) had no history of recurrent infections and no deviation in immunological tests. sibling no.2. (6 month old girl) had recurrent infections since birth and iga, igg2 and igg4 deficiency as well as t-and b-cells deficiency. cytogenetic analysis revealed variable percent of spontaneous chromosomal instability which was more severe (in 50% of chromosomes analyzed) in sibling no.2. additionally we sequenced bi-directionally (26 amplicons) the dna samples from all family members to survey the germline genetic variation in the nbs1gene. the 657del5 (exon 6) was detected in both siblings in homozygous and in both parents in heterozygous feature. in order to explain different clinical and immunological presentation of two siblings the rest of the nbs exones were analyzed for genetic heterogeneity. no additional changes were observed. in conclusion patients with the same nbs1 genotype may show different phenotypes. other gene/epigenetic factors seem to play a role in phenotype modulation. omenn syndrome [mendelian inheritance (omim 603554)] is an autosomal recessive form characterized by the presence of fatal generalized severe erythroderma, lymphoadenopathy, eosinophilia and profound immunodeficiency. objective: we studied clinical and immunological presentation of the disease manifestation and frequency c.368-369delaa (p.k86vfs118) in rag1 gene among eastern slavs population. results: we collected clinical and immunological data of 9 patients (1 from belarus, 5 -ukraine, 3 -russia) 6 females, 3 males. age of omenn syndrome manifestation varied from 1 st day of life to 1 yr 1 month. age of diagnosis -20 days to 1 year 10 months. 8 in 9 patients had classical immunological phenotype t(+/-)b-nk+, 1 pt had tlowb + nk + with cd3 + tcrgd + expansion. 6 in 9 pts had mutation in rag1 gene, 4 in 6 had c.368-369delaa (p.k86vfs118) in one or two alleles. at present moment 4 in 9 pts are alive, 3 were transplanted, 1 pt is prepared to bmt. conclusion: this study demonstrates that the most popular genetic abnormalities in eastern slavs children with omenn syndrome is c.368-369delaa (p.k86vfs118) in rag1 gene. this information may be useful for rapid diagnostic of omenn syndrome in laboratories used sscp (single strand conformation polymorphism) before sequencing. under examination the patient particularly bright phenotype attracted attention: microcephaly, "birdlike" facial features (sloping forehead, nape, hypoplasia of brow ridges, broad nasal bridge and protruding midface, hypoplasia of the mandible). in addition, besides specific anomaly of the facial bones we noted: big ears, sparse hair and clinodactyly of the fifth fingers. clinical and immunological characteristics: the feature of the case is pancytopenia syndrome we have diagnosed at the early stages of observation and which is continued throughout the period of observation. erc -2.9-3.6 x 10 12 /l hb -71-105 g/l leuk -0.5-3.0 x 10 9 /l neu -10-26 % (300-780 cells/mcl) plt -40-60 x 10 9 /l data of immunological examination: iga -0,06 g / l igm -0,6 g / l igg -0,75 g / l (other results of immunological examination are without features) the deep insufficiency of antibody production in our patient was the cause of serious, recurrent, and subsequently chronic bacterial sinopulmonary infections after 3 years old. the results of clinical laboratory and immunological examination without significant features: erc -4,0-5,4 x 10 12 /l hb -121-133 g/l leuk -11,5-6,2 x 10 9 /l neu -40-70% (2640-11680 cells/ mcl) plt -512 x 10 9 /l iga -0,85 g/l -1.45 g / l igm -1,33 g/l -0.66 g / l igg -11,3 g/l -11.8 g / l (other results of immunological examination are without features) the x-linked chronic granulomatous disease (cgd) is a primary phagocytic cell deficiency characterized by severe bacterial and fungal infections of various organs. we report of a 19 years of a male patient with xlinked cgd who presented with recurrent hepatic abscesses as the sole manifestation of the disease. phagocytic and bactericidal activities of granulocytes were studied by using microbiological assays. generation of superoxide anion by blood granulocytes was measured by the ferricytochrome c reduction test. cgd is an immunodeficiency caused by mutations in genes encoding subunits of the nadph oxidase complex. normally, assembly of the nadph oxidase complex in phagosomes of phagocytic cells leads to a "respiratory burst" essential for the clearance of microorganisms. cgd patients lack this mechanism, which results in life-threatening infections and granuloma formations. the leading cause of death are pneumonia and pulmonary abscess, septicemia and brain abscess. in neurogical manifestations various pathogens have been involved including aspergillus spp., s. prolificans, a. infectoria, salmonella and staphylococcus spp. there are only some several reports on fungal brain and spinal cord infection, aspergillus abscess resembling brain tumor, meningitis due to streptococcus and candida spp. in the past 20 years we treated 7 children with cgd. we present the infectological challenge of an x-linked cgd patient with brain abscess. in spite of our effort we were unable to identify its causative pathogen. empiric therapy sometimes resembles polypragmasia in cgd. to decrease mortality and morbidity from fungal infections in cgd the prophylactic use of itraconazole or voriconazole is widely recommended. a relatively new azole, posaconazole is active in pulmonary and cerebral fungal manifestations , indeed may be effective against fungi with inherent resistance to ampb or voriconazole. there was no etiological diagnosis in our case that did not respond to conventional antifungal and antibacterial treatment. based upon the findings and literature data we presume the causative agent might be some kind of moulds. we suppose the use of echinocandin and posaconazole as salvage ("prophylactic") therapy has resulted significant regression of the brain abscess. the diagnosis of chronic granulomatous disease (cgd) was verified in 7 children during the past few years in the department of clinical immunology of regional children's hospital of chelyabinsk (russia). in our opinionin 3 casesthe diagnosis was establishedearly enough. michael w. transferred to the neonatal pathology unit of our clinic at the age of 5 days with vesiculopustules. take into consideration our own experience of observing children with this form of primary immunodeficiency in previous years, the child was conducted immunological examination, in particular, the test of restoration nitroblue tetrazolium by superoxide anionformed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test). this decision is caused by the fact that previously observed children with chronic granulomatous disease, had vesiculopustules in 90 % at birth. the survey has revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test, which allowed us to suggest the diagnosis of cgd. the baby was banned vaccination against tuberculosis, and after reliever vesiculopustules the boy was discharged home. however, at the age of 1 month the baby suffered from glandular abscess and right-segmental pneumonia. in cbc there is expressed anemia (hb 60 g/l, erythrocytes -1.98 x 10 12 /l), leukocytosis (22x 10 9 /l), accelerated esr (36-55 mm/h). at the age of 3 months in the university of debrecen, medical and health science center debrecen, hungary, molecular genetic studywas conducted by prof. dr. laszlo marodi. it was revealed a mutation in c. 374 g > a in exon of 6 gene cybb (encoding subunit gp91-phox), after that the diagnosis of cgd was finally verified. it was assigned a basic preventive antimicrobial therapy by trimethoprimsulfamethoxazole, and itraconazoleto prevent fungal infections. despite this since 6 months the child has repeatedly and consistently suffered from bilateral groin lymphadenitis during the year, acute hematogenous osteomyelitis of the left ulna, 2 pneumonias, purulent mesadenitis, endoperitonitis. the fact that the child aged 8 months was diagnosed sepsisis evidence of the severity of infectious complications. clinical and biochemical blood tests were distinguished by consistently high levels of esr and the presence of leukocytosis, and also severe anemia. now the child is 2 years 11 months, he is undergoing treatment at the department of clinical immunology, russian children's clinical hospital (moscow) after bone marrow transplantation. case history № 2, 3 christina n. from the early history we know that the girl's newborn period was uneventful. she was vaccinated against tuberculosis in the nursing home (no reaction). it was noted the formation of abscesses after vaccination against whooping cough, diphtheria and tetanus in 3 and 4.5 months. in 2 years 8 months she suffered from mezootit. for the first time the girl came under our observation in the children's hospital in chelyabinsk (russia) at the age of 3 years with right segmental pneumonia, complicated by the destruction. after further examination it was diagnosed tuberculosis of intrathoracic lymph nodes to the right with upper lobe bronchopulmonary defeat. to take into consideration given above, it was conducted immunological test that revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. on the basis of the history, clinical manifestations and results of immunological examination chronic granulomatous disease was diagnosed. after fourmonth period treatment a recovery came from tuberculosis and child was transferred to outpatient monitoring. in 2 years in this family a second daughter was born -arina n. taking into account revealed immunodeficiency of her sister, the child was not vaccinated against tuberculosis on our recommendations. a six-month-old child was conducted immunological examination, which, like her older sister, also shows a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. during the molecular genetic studies of both sisters it was identified identical mutation c. 73-74 del gt gene ncf1 (encoding subunit p47phox). now the older girl is 8 and her sister is 3 years old. observing them in the dynamics neither of them do not show any life-threatening infections. thus, the recovery test of nitrobluetetrazolium by superoxide anion formed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test) is a fairly reliable method of early diagnosis of chronic granulomatous disease. chronic granolomatous disease (cgd) is due to defective phagocyte superoxide production leading to impaired microbial killing. it is comprised of a group of five genotypes with a common phenotype, chracterized by recurrent severe bacterial and fungal infections and tissue granuloma formation. patients with cgd often present with pneumonia, liver abscess, skin infections, lymphadenitis, osteomyelitis. a five month old boy was referred to pediatric infection unit by lymphadenitis. on the medical history, he had taken antibiotic therapy for lymphadenopathy when he was 2 months old. the abnormal eye movement was noticed by the family and on the eye examination peripheric chorioretinal hypopigmented lesions were determined when he was 4 months old. i̇n his labratory examination, viral serology were negative. his parents were not consanguineous. his mother's brother had died at 3 years old. he had history of skin infections and osteomyelitis. his grandmother had been diagnosed as having tuberculosis lymphadenitis one year ago. on his physical examination, his growth was normal, patological findings were horizontal nistagmus, 2/6 degree systolic murmur, fistulized lymph tissue on the left submandibuler region. on the laboratory findings; he had anemia and neutropenia. immunglobulin levels and lymphocyte subtypes were normal. his respiratory burst activity was very low. chronic granulamatous disease was thougth in the patient by the clinical and laboratory findings. i̇t was detected gp91phox mutation in the genetical analysis. he was diagnosed as having x-linked cgd. antibiotic prophylaxis (tmpsmx, flucanazol) and interferon gamma were started. he is 14 months old now and he is on the list of match unrelated donor screening. patiens who has history of lympadenitis, skin infections and chorioretinal findings should be evaulated for the x-cgd. leucocyte adhesion deficiencies (lads) are rare autosomal recessive inherited disorders. three different forms of lads have been described so far. in lad-i, the most common leucocyte adhesion deficiency, the function of β2 integrin cd18 is lost. . while one of the first signs of the disease consist in delayed separation of the umblical cord, severe infections already start early during infancy. another feature of lad-i includes impaired wound healing. therefore, mortality during infancy is high. a fifty day old boy was referred to the hospital due to diarrhea and leukocytosis. the patient was delivered following an uncomplicated full term pregnancy. the parents were first degree cousins. father's brother and mother's uncle had died during infancy period. his umblical cord had separated on the 19th day. patient had applied because of diarrhea by starting in the first days of life and leukocytosis was detected (61.000/ mm 3 ). on the physical examination, his body weight was 5200 gr (10th to 25th percentile) and his height was 56cm (10th percentile). there was a granuloma on the umbliculus. other systems were normal. in the laboratory examination, leukocyte count was high, immunoglobulins, respiratory burst activity, gaita analysing and culture were normal. in the lymphocyte subtype analysing, cd19+ b cells ratio was mildly low. cd18, cd11a, cd11b, cd11c levels were found to be very low. in the genetic analysing, it was detected two deleterious mutations in the itgb2 gene. patient had been diagnosed as lad-1. he treated by antibiotics and then started prophylactic antibiotherapy. family screened for tissue match. his older sister was found full matched. he was referred to transplantation unit. it was applied bone marrow transplantation when he was 3 months old. presence of delayed umblical cord separation and leucocytosis should be considered in the diagnosis of lad but these patients might have different symptoms such as diarrea. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il-12rβ1 deficiency, most common form of msmd, is caused by mutations in the il12rb gene. 37 patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il-12rb1 was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il12rβ1 expression was found to be less than %1. prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il12rβ1 on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. introduction: mendelian susceptibility to mycobacterial disease (msmd, online mendelian inheritance inman 209950) is a rare immunodeficiency characterized by predisposition to infections caused by weakly virulent mycobacteria, such as mycobacterium bovis bacille calmette-gue´rin (bcg), environmental nontuberculous mycobacteria (ntm), and salmonella strains in otherwise healthy individuals. il-12rb1 deficiency is the most common form of msmd and is characterized by childhoodonset mycobacteriosis with frequent recurrence. it has been found that patients with il-12rb1 deficiencies are also prone to developing infections with nontyphoidal salmonella species with bacteremia and lymphadenopathy. here we present a girl with recurrent cutaneous leukocytoclastic vasculitis (clv) with salmonella enteritidis due to il-12rb1 deficiency. case report: a four year old girl that had been diagnosed serologically with recurrent salmonella infections, associated with lymphadenopathy and skin eruption was admitted as having henoch-schönlein purpura. she had been vaccinated with bcg and developed left axillary lymphadenitis which spontaneously drained and had recurrent oral monilia plaque. edema and purpuric eruptions were present on the upper and lower extremities and the abdomen.multiple mobile, painful,enlarged submandibular lymph nodes of about 2x2 cm in diameter were palpable. skin biopsy showed a dense inflammatory site with eosinophils, neutrophils and fibrin in the upper dermis and dermal vessel wall,compatible with leukocytoclastic vasculitis. serological studies to assess diagnostic markers for vasculitis and infectious agents were all negative. immune work-up were unremarkable other than hypergammaglobulinemia. salmonella enteritidis was identified in blood culture. she responded dramatically to ceftriaxone treatment within a few days and lesions cleared completely. extended immunological and molecular genetic examination of the patient was carried out for il-12/ifn-γ pathway defects. on the facs analysis of t cells for cell surface expression of the cytokine receptor chains, she did not express any il-12rβ1. discussion: in thepresent report, we describe a child with clv with salmonella enteritidis due to il-12rb1 defi-ciency.in a large cohort of patients with il-12rb1 deficiency, ntm, m. tuberculosis, disseminated bcg infection after inoculation with the vaccine, and salmonella infection have been described. sporadic cases with other infectious agents have also been reported. salmonella infections reported in these patients were due to extraintestinal, or septicemic, recurring infections caused by nontyphoidal salmonella species. only two il-12rb1-deficient patients have been identified with vasculitis due to salmonella strains; both came from turkey, where consanguineous marriages are common. kutukculer and colleagues reported the first case of s. enteritidis-associated clv. sanal and colleagues reported a clv case associated with group d salmonella infection. leukocytoclastic vasculitis is an immune complex mediated disease predominantly involving small vessels of the skin and can be associated with drugs or can be found as a component of other disease, such as infections, connective tissue disorders, and malignancies. infectious agents can cause vasculitis directly or clinically mimic primary vasculitis .multiple infectious agents have been suspected as triggering or contributory factors in the vasculitic process . several factors contribute to the primary vasculitis related to infections: a type 3 or immune-complex reaction, cell-mediated hypersensitivity, abnormal immune regulation, and direct endothelial cell invasion by infectious agents. in our case, extensive evaluation was performed to determine the underlying vasculitis process. clinical and laboratory examinations revealed no association between vasculitis and other infections or an underlying connective tissue disease or medication. she responded well to ceftriaxone treatment, and clinical manifestations gradually resolved within a few days, providing strong evidence that improvement of the vasculitic lesions was due to elimination of the salmonella with antibiotics conclusion: our patient has one of the exceptional forms of il-12rb1 deficiency, with recurrent clv due to salmonella enteritidis. although common presentations for salmonella infection in individuals with il-12rb1 deficiency are lymphadenopathy and bacteremia, it can be present clinically as clv. some infections such as salmonella may be responsible for different types of vasculitis even though they are not common . in this respect, clinicians should be aware of possible infectious causes of vasculitis, and children presenting with unusual recurrent infections caused by non typhoidal salmonella, bcg, or ntm should be investigated for ifn-γ ⁄ il-12 pathway defects. gülez n.; genel f. dr. behcet uz children hospital allergy-immunology department, izmir, turkiye the complement system is an important part of the innate immune defense and also plays a major role in shaping the adaptive immune response. these functions are required for a good defense against infections, especially bacteria. the c8 deficiency is a rare disease that is associated with recurrent neisserial infections, especially meningits caused by n. meningitidis. the patient, a seven years old girl was admitted to hospital with high fever and diffuse, purple-coloured skin lesions. her symptoms gave the diagnosise meningococcal meningitis. she had also earlier been diagnosed with the same disease when she was 5 years old. a sister to the patient had died from meningitis at 3 years of age. she has also one older and one younger sister. there is no consanguinity between her parents. the laboratory analyses of the classical pathway measured as complement hemolytic activity (ch50) and c8 concentration revealed no activity and absence of c8, respectively. analysis of serum from her younger sister showed the same results, while her older sister's ch50 and c8 levels were found normal. thus, our patient and her younger sister were diagnosed with hereditary c8 deficiency. the genetic analyses have not been completed yet. we here report the third and fourth cases of c8 deficiency in turkish patients. interferon-g receptor-1 (ifngr1) deficiency is caused by mutations in ifn-γ receptor-1 gene and is characterized mainly by susceptibility to mycobacterial disease. we report a boy with complete recessive ifngr1 deficiency, afflicted by recurrent mycobacterial diseases with m. bovis, m tuberculosis, m. avium intracellulare and m.fortuitum. genetic analysis showed a homozygous mutation (106inst) in ifngr1 gene leading to complete ifngr1 deficiency. in addition, he had atypical mycobacterial skin lesions caused by m.avium intracellulare and he developed scrotal and lower limb lymphedema secondary to compression of large and fixed inguinal lymphadenopathies. to our knowledge, the patient is the first case with interleukin-12/interferon − γ pathway defect and severe lymphedema. defects in the il-12/ ifn − γ pathway must be considered in patients with disseminated or recurrent mycobacterial infections and in patients with severe viral infections, especially in countries where bcg vaccination is part of the national health programme. it must be kept in mind that these patients may develop granuloma-like skin lesions and severe lymphedema. hsct must be applied at the earliest time before developing organ damages. t lymphocyte/nk cells. restricted defective molecules in the circuit and recently discovered cybb responsible for autophagocytic vacuole and proteolysis have been identified in around 60% of patients with the mendelian susceptibility to the mycobacterial disease (msmd) phenotype. primary defects in oxidase activity in chronic granulomatous disease (cgd) lead to severe, life-threatening infections. the role of phagocytic respiratory burst in host defense against mycobacterium tuberculosis was controversial. previous studied showed that the critical role at reactive oxidants is to serve as intracellular signals for activation of microbicidal enzymes, rather than excretions a microbicidal effect perse.the role of phagocytic respiratory burst in host defense against m. tb is further supported by recent studies discovered immunological defects secondarily affecting phagocyte respiratory burst function and resulting in primary immunodeficiencies with varied phenotypes, including susceptibilities to pyogenic or mycobacterial infections. the patients with severe pid's like scid have broader diverse infections susceptibility and mycobacterial infections as well, however, common variable immunodeficiency (cvid) mostly characterized by a deficiency of immunoglobulins and recurrent sinopulmonary infections. method: we overview the clinical rate of mycobacterial disease in our pid cases and evaluate the complex cases. results: two hundred pid cases were evaluate between 1996-2013 in our clinic, among 5% of them which diagnosed as msmd nearly all presented with mycobacterial infection.8% diagnosed as cgd and interestingly 60% of them have been experienced mycobacterial disease sometimes in their life, as disseminated bcg or late onset complications of bcg including osteomyelitis or mtb once or more than one episode through their life. also we have presented a cvid patient with disseminated tb and granulomatouse hepatitis, tb arthritis , peritonitis and a patient with lad and nontubercolouse mycobacterial infectiouse abcesses of her skin. conclusion: pid cases like cgd, msmd or cvid which are living in area's with high prevalence of mycobacterial infection could have quiet different presentations and the study of these complex cases has provided essential insights into the functioning of the immune system. despite the conventional view we have confirmed that the generation of rois by phagocytic respiratory burst may play a role in the defense of the host against m. tuberculosis by clinical evidence. goran ristic, srdjan pasic, bojana slavkovic division of clinical immunology, mother and child health care institute of serbia, belgrade chronic granulomatous disease (cgd) is a rare disease caused by mutation in any of the five components of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase in phagocytes, resulting in recurrent, life-threatening bacterial and fungal infections of the affected individuals. our proband male patient presented at age of 2 years with bilateral pneumonia and positive serology for aspergillus sp. the phorbol myristate acetate (pma) stimulated nitroblue tetrazolium (nbt) test showed no reduction (0%) in our patient and partial reduction (58%) in his mother. analysis of the cybb gene showed a deletion of nucleotide g (c.1237delg) exon 10 causing frame shift mutation and early termination of translation (p.val413serfs). this mutation was not previously described. at the moment of diagnosis, his mother was already pregnant, 19 th week of gestation. fetal ultrasound showed that she was carrying a male fetus. the fetal blood sample, obtained by the percutaneous umbilical cord blood sampling, showed male karyotype and the pma stimulated nbt test showed 100% reduction. there were no complication during pregnancy or delivery and a healthy boy was born. the proband patient underwent allogenic hsct, his sister was the identical sibling donor. in the cases where the family-specific mutations are unknown, partial or complete gene deletions can be recognized by multiplex ligase-dependent probe amplification (mlpa) or array comparative genomic hybridization ( features of the patients with ar-hies dock-8 deficient and non-dock-8 deficient group are given in the table 1 . all patients with dock-8 deficiency presented with cutaneous viral infections or early onset and severe atopic dermatitis. many have also food allergy and/or asthma. neurological complications and malignancy were seen in 20% and 27% respectively. sixty seven percent of patients had low t; 83 %, low cd4 levels; 100%, high ige. the latter features were shared between patients with or without dock-8 deficiency, except atopic dermatitis which was mild when present in patients without dock-8 deficiency and ige levels were only mildly high or normal. we identified stk4 and coronin 1a deficiency in two siblings each among the 11 patients who showed overlapping features with ar-hies and do not have dock-8 deficiency. our results showed that patients with dock8 deficiency have early onset and more remarkable eczema, food allergy, asthma, more marked eosinophilia, higher ige, low igm levels and development of malignancy. these features may be helpful in differentiation dock8 patients from patients without dock-8 deficiency. it seems routine lymphocyte subset study results are not helpful for this differentiation. alişan yıldıran 1 , stephan borte 2 murat elli 1 , tunç fışgın 1 1 ondokuz mayıs university, school of medicine, samsun-turkiye 2 immunodeficiency center leipzig (idcl) at klinikum st. georg ggmbh, leipzig-germany hsct might be curative for some pids. our immunology and transplantation center was newly established. we retrospectively reviewed all children with pid who diagnosed and received hsct at ondokuz mayıs university or somewhere between june 2010 and december 2013. twenty-two patients were identified. four of them were referred to us for hsct from other centers. the median age was 6 months (1 month-10 yr) at hsct. patients' diagnoses were scid (n = 11), chs (n = 2), leukocyte adhesion deficiency (n=2), mhc class ii deficiency (n=2), chronic granulomatous syndrome (n = 2), hlh (n = 1), was (n = 1) and omenn's syndrome (n = 1). seven patients received hla-matched related hsct; twelve haploidentical hsct and two matched unrelated hsct. one scid patient died just after her diagnosis. two patients developed bcgosis secondary to reactivation of pretransplant vaccination. one of them died due to hemophagocytic bone marrow aplasia, and the other has recovered. five patients had graft failure; two of them received no conditioning regimens because of general health status and the other because of cmv infection. at a median follow up of 6 months (range 0-64), 15 patients are alive, with overall survival of 68 %. we conclude that; our clinic undertakes an important duty in our region for pid patients. also, different pid's could be seen in our region. nerological complication 20 1 patient (epilepsy, brain infarct) outcome 3 died (one with post -bmt comp., 2 with malignancy all alive department of pediatrics and adolescent medicine 1 department of allergology, rheumatology and clinical immunology, university children's hospital, university medical centre, ljubljana, slovenia. developed.there is no severe life-threatening complications development. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been documented. invasive c.laurentii infection has not been reported in patients with any form of primary immunodeficiency disorder emphasizing the true rarity of disease due to this fungus. two groups have recently reported that dectin-1 deficiency due to the mutation of clec7a or premature termination of the dectin-1 signal transduction molecule card9 may predispose patients to chronic mucocutaneous candidiasis (cmc). we studied the frequency of clec7a mutation in 76 healthy individuals, 10 patients with the hyper-ige syndrome (hies), and 8 patients with autoimmune polyendocrine syndrome type 1 (aps-1), all aged between 2 to 60 years. genomic dna was isolated from peripheral blood. monocytes and monocyte-derived dendritic cells (mddcs) were used to study the phenotypic expression of dectin-1. clec7a gene was sequenced with the big dye terminator cycle sequencing kit. mononuclear cells (mcs) were isolated from heparin-treated venous blood. mdccs were obtained by culturing monocytes isolated by immunomagnetic cell separation assay. receptor expression was assessed by flow cytometry. secretion of il-17a by mcs stimulated with killed c. albicans blastoconidia was assessed by elisa. we report here on healthy individuals with homozygous (one 38year-old man) or heterozygous (32 men and 44 women) tyr238x mutation in the dectin-1 gene but no signs of cmc. dectin-1 levels on monocytes and mddcs were negligible in the homozygous man and the heterozygous individuals displayed intermediate levels of dectin-1, between those of the homozygous man and the wild-type controls. markedly lower levels of il-17a production were observed in the cells of the man with the homozygous mutation than in the control cells. levels of production of this cytokine were intermediate in heterozygotes. the frequency of tyr238x heterozygous individuals was 21% among healthy donors and 10 % in patients with hies and 20 % in patients with aps1. importantly, the 10 year-old hies girl with heterozygous tyr238x mutations has never had mucocutaneous candidiasis. we suggest that dectin-1 receptor-mediated immunity is redundant for host defense against cmc, possibly due to the involvement of multiple lectin receptors in the recognition and uptake of candida. chronic mucocutaneous candidiasis (cmc) is a heterogenous group with recurrent chronic candida infections spesifically involving nails, skin and oropharynx. several immunodeficiencies as dock-8 deficiency, severe combined immune deficiency, autoimmune polyglandular syndrome type 1 (apeced), il-12rβ1 and il-12p40 deficiencies, card 9, stat-3 and stat-1 can cause cmc. we report here a 3 years old boy, born to consaginous parents with onicomycosis, moniliasis, recurrent pneumonia, recurrent herpetic lesions, autoimmune thyroiditis and trombocytopenia. last admission was due to generalized tonic-clonic convulsion and mycotic aneurism on middle cerebral artery was detected. flow cytometry revealed cd4 lymphopenia, immunoglobulin values were in normal range. polymorphism on exon 6 for aire gene (t/c heterozygot g227g) and heterozygot stat1 mutation (c.501 > a; q167h) were detected. various stat1 gof mutations (affecting the coiled-coin domain or the dna-binding domain) have been systematically associated with susceptibility to cmc. autoimmune manifestations associated with stat-1 mutations have been attributed to increased type 1 interferon. the aneurism formation is not elucidated whether it's due to candida infection or vascular damage directly affected by stat-1 mutation. purpose: chronic granulomatous disease (cgd) is a rare genetic disease of phagocytic system. affected patients commonly present with bacterial infections associated with pneumonia, abscesses and lymphadenitis. in this study, we investigated the clinical and laboratory findings of our cgd patients. materials and methods: the demographic data (age at diagnosis, initial presenting symptoms, family history, follow-up period), mutation analysis, therapy options, complications, radiological findings and prognosis were evaluated retrospectively. results: among 9 cgd patients, autosomal recessive form was detected in 4 of them. the age at onset was statistically lower in x' linked cgd patients than ar form (11.2 ± 7.6 mo vs 43.5 ± 21.5 mo; p = 0.001).respiratory tract infections (sinusitis, otitis, pneumonia) and recurrent abscesses were more commonly seen at onset. microbiological culture revealed a. fumigates from lung biopsy in one patient and s.marcescens from blood specimen in other ones. bcgitis was observed in one patient and five patients received anti-tb therapy. non-infectious complications were granulomatous uveitis, recurrent pericardial effusion, skin granuloma, noduler formation in lung and brain area. conclusion: due to high rate of consanguinity, autosomal recessive inheritance was observed highly in our patient cohort. since, patients with cgd are susceptible to tuberculosis and bcg complications; initiation of tuberculosis prophylaxis is advisable in countries where bcg is still administrated at birth. key words: chronic granulomatous disease, consanguinity, bcg. acquired immunodeficiency research center, isfahan university of medical sciences, isfahan, iranbackground: defects of the immune system in primary immunodeficient diseases (pids) predispose individuals to recurrent infections. complex genetic components for susceptibility to mycobacterial disease have been suggested. natural human immunity to the mycobacteria group, including mycobacterium tuberculosis(mtb), bacille calmette-guérin (bcg) or nontuberculous mycobacteria (ntm) relies on the functional il-12/23-ifn-γ integrity of macrophages (monocyte/dendritic cell) connecting to key: cord-257684-4b66lenw authors: salenger, rawn; etchill, eric w.; ad, niv; matthew, thomas; alejo, diane; whitman, glenn; lawton, jennifer s.; lau, christine l.; gammie, charles f.; gammie, james s. title: the surge after the surge: cardiac surgery post-covid-19 date: 2020-05-04 journal: ann thorac surg doi: 10.1016/j.athoracsur.2020.04.018 sha: doc_id: 257684 cord_uid: 4b66lenw abstract background the covid-19 pandemic has dramatically reduced adult cardiac surgery case volumes as institutions and surgeons curtail non-urgent operations. there will be a progressive increase in deferred cases during the pandemic that will require completion within a limited time frame once restrictions ease. we investigated the impact of various levels of increased post-pandemic hospital operating capacity on the time to clear the backlog of deferred cases. methods we collected data from four cardiac surgery programs across two health systems. we recorded case rates at baseline and during the covid-19 pandemic. we created a mathematical model to quantify the cumulative surgical backlog based on the projected pandemic duration. we then used our model to predict the time required to clear the backlog depending on the level of increased operating capacity. results cardiac surgery volumes fell to 54% of baseline after restrictions were implemented. assuming a service restoration date of either june 1 or july 1, we calculated the need to perform 216% or 263% of monthly baseline volume, respectively, to clear the backlog in one month. the actual duration required to clear the backlog is highly dependent on hospital capacity in the post-covid time period, and ranges from one to eight months depending on when services are restored and degree of increased capacity. conclusions cardiac surgical operating capacity during the covid-19 recovery period will have a dramatic impact on the time to clear the deferred cases backlog. inadequate operating capacity may cause substantial delays and increase morbidity and mortality. if only pre-pandemic capacity is available, the backlog will never clear. background. the covid-19 pandemic has dramatically reduced adult cardiac surgery case volumes as institutions and surgeons curtail non-urgent operations. there will be a progressive increase in deferred cases during the pandemic that will require completion within a limited time frame once restrictions ease. we investigated the impact of various levels of increased postpandemic hospital operating capacity on the time to clear the backlog of deferred cases. we collected data from four cardiac surgery programs across two health systems. we recorded case rates at baseline and during the covid-19 pandemic. we created a mathematical model to quantify the cumulative surgical backlog based on the projected pandemic duration. we then used our model to predict the time required to clear the backlog depending on the level of increased operating capacity. results. cardiac surgery volumes fell to 54% of baseline after restrictions were implemented. assuming a service restoration date of either june 1 or july 1, we calculated the need to perform 216% or 263% of monthly baseline volume, respectively, to clear the backlog in one month. the actual duration required to clear the backlog is highly dependent on hospital capacity in the post-covid time period, and ranges from one to eight months depending on when services are restored and degree of increased capacity. cardiac surgical operating capacity during the covid-19 recovery period will have a dramatic impact on the time to clear the deferred cases backlog. inadequate operating capacity may cause substantial delays and increase morbidity and mortality. if only prepandemic capacity is available, the backlog will never clear. key words: cardiac surgery; covid-19; surgical capacity; coronary artery bypass grafting the covid-19 pandemic has resulted in dramatically lower adult cardiac surgery case volumes, as institutions and surgeons are following state and federal guidelines to curtail non-urgent operations. there will be a progressive increase in deferred cases during the pandemic that will require completion within a limited time frame once pandemic restrictions ease, in order to avoid excess morbidity and mortality [1] [2] [3] . by definition, programs will have to achieve higher daily case rates than prior to the pandemic in order to service the excess demand. the postpandemic surge in adult cardiac surgery could overwhelm current hospital resources without proper planning. data were collected from four institutions across two health systems. each system included two large academic centers and two affiliated community cardiac surgery programs. first, we calculated a combined baseline case rate across centers, pre-pandemic, by quantifying all cases done from january 2019 to february 29, 2020. inclusion criteria included any adult cardiac surgery case being performed in the operating room with a cardiac surgeon as primary or co-surgeon. heart and lung transplant, ventricular assist device, and transcatheter aortic and mitral valve replacements were included. primary extracorporeal membrane oxygenation (ecmo) cases and pediatric cases were excluded. next, we calculated the combined rate of cases during the early part of the pandemic. raw data was collected from march 16 th through april 12 th and this was used to extrapolate a new "pandemic" surgical case rate. march 16 th was the day after the state of emergency was declared in the state of maryland. we calculated the number of patients that would have surgery deferred during the pandemic, and the length of time necessary to service the backlog in a timely fashion once restrictions were lifted. mathematical models were used to investigate the impact of the date that restrictions are lifted, and variable levels of hospital operating capacity on the time that would be required to address the backlog. to calculate the current backlog (b) on any given day (t), we developed the equation b = (r 0 -r p ) (t -t 0 ). this is the difference in the daily case rates multiplied by the number of days that difference had existed. t1 was then defined as the day that restrictions were eased and full operating capacity regained. next, the number of operating days necessary to perform all the backlogged cases, defined as backlog time (bt), was calculated. the model was designed to predict the length of time required to clear the backlog based on the amount of extra operating capacity a hospital could achieve. our prediction model defined t 0 as the day the pandemic began, and t 1 as the day restrictions were lifted. each hospital's post-pandemic daily case rate would be defined by an acceleration factor (a) multiplied by the original prepandemic case rate, r 0 . the time required to clear the backlog of cases, bt, was then calculated. bt was defined by the equation bt = (1 -r p /r 0 ) (t1 -t0) / (a-1). the acceleration factor (a) represented how much surgical throughput a program could achieve above baseline. this factor would have to be greater than 1 in order to incorporate some amount of deferred cases into the operating schedule in addition to baseline cases. cases were initially curtailed in early to mid-march 2020 and then more drastically in april as recommendations to postpone all but emergency operations became stronger. baseline total case rate for the four combined programs was 306 adult cardiac surgery cases per month. during the pandemic that rate initially fell to 234, then to 164 cases per month once full restrictions were implemented (54% of normal). during this time the number of deferred cases would be estimated to grow to 356 by june 1 and 498 by july 1. this represents 216% to 263% of normal average monthly volume depending on the duration of the pandemic (figure 1 ). the daily case rates were also calculated. we assumed 20 operative weekdays per month. for the combined health systems, the average daily case rate pre-pandemic, defined as r 0 , was 15.3. the daily case rate during the pandemic, defined as r p , was calculated to be 8.2 (table 1) . we also calculated the baseline and pandemic daily case rates for three broad categories of cases including coronary artery bypass grafting (cabg), open valve surgery, and transcatheter aortic and mitral valve replacements (tavr/tmvr) (figure 1 ). combined cabg/valve cases were counted as cabg because we felt that the patient's coronary disease would often dictate more urgent triage (figure 2 ). isolated valve cases were more likely to be deferred than patients with isolated or concomitant coronary disease. transcatheter cases were treated differently between the health systems. one system's transcatheter case rate fell from 34 per month to 8 cases per month, while the other system increased their transcatheter cases from 21 per month to 32 cases per month. this equated to approximately a 75% decrease in one system's transcatheter cases and a 30% increase within the neighboring system. this differential treatment of transcatheter cases partially mitigated the overall backlog accumulation. cabg cases and valves were triaged similarly by the health systems and these case rates fell by 50% and 71% respectively. next, we used our mathematical model to predict the number of cardiac surgery cases deferred during the pandemic, and the length of time required to operate on the backlog, dependent on the amount of increased operating capacity institutions could achieve. time to clear the backlog, bt, was calculated using our mathematical model (figure 3 ). this was considered for the possibility of restored services on june 1 or july 1 with varied levels of case capacity (figure 4 ). assuming services were restored june 1, the backlog could be cleared between 1-6 months, depending on degree of increased capacity. if services resumed july 1, the cardiac surgery backlog would be estimated to take between 2-8 months to clear according to the increased capacity achieved. progressively increasing the operating capacity had a dramatic effect on reducing the time to clear the backlog. if capacity is not increased above baseline (acceleration factor = 1.0), the backlog will never be cleared. in response to the covid-19 pandemic, adult cardiac surgery programs have dramatically curtailed operating activity, limiting cases to urgent and life-threatening emergencies. this slowdown has resulted in a growing backlog of adult cardiac surgery cases which will require surgical therapy in a finite time frame to improve quality of life and survival. our study analyzed the deliberate deceleration in adult cardiac surgery cases that has occurred at the two busiest cardiac surgical programs in the state of maryland in response to the covid 19 pandemic. we observed a 54% drop in cardiac surgical volume, and subsequently a backlog that would require a monthly operating volume of 216% to 263% of baseline. since this would be challenging to accomplish in one month, we predicted that the amount of time necessary to clear the backlog would range from 1 to 8 months based on varied estimates of post-pandemic increased operational capacity. our data further suggest that deferred cardiac patients were triaged differently during the pandemic and that the composition of the waiting list is directly related to the triaging mechanism. as expected, isolated valve patients were preferentially deferred compared to patients with any coronary artery disease. we were surprised to see the differences in transcatheter case triage between the two systems. one system deferred the majority of transcatheter cases, while the other increased their rate, mainly tavr cases. although our data did not include reasons for triage decisions, this may have been due to differing thresholds for observing patients with severe aortic stenosis and uncertainty regarding best practice when deferring cardiac services. others have attempted to characterize the needs for surgical services after natural disasters by establishing a baseline surgical rate and then examining the incidence of injury during a disaster [4] . a study of the nepal earthquakes in 2015 demonstrated an acute need for surgical therapy that was more than double the annual nepalese operating capacity, highlighting the need to be able to increase surgical capacity at critical times. another study focused on the aftermath of hurricane charley and found a thirty-two percent increase in adverse medical outcomes for patients with chronic medical conditions related to both increased demand and shortfalls in care delivery [5] . similar to our investigation, these studies focused on increased healthcare demand following unpredictable events. our analysis differed because we analyzed the effect of an unexpected viral pandemic that necessitated a nearly 50% decline in surgical volume followed by an anticipated surge in surgical demand. our study is novel in that we prospectively attempted to predict system capacity needs in order to service the anticipated surgical surge. this will require local hospitals and health systems to examine capacity, set priorities, and plan for adequate hospital capacity to service the backlog of cardiovascular patients [6, 7] . the global cardiac surgery response post-covid will be complicated by prior exhaustion of hospital supplies and human resources from the pandemic, and competition for resources and operating room availability with other surgical and medical subspecialties, which will also have backlogs. the recently published joint statement from the american college of surgeons and others provides guidance on when to safely reopen operating room capacity and includes recommendations for testing, personal protective equipment, and case prioritization [8] . we believe that it will be important to maintain an incident command approach during the late phases of the covid epidemic as we transition to re-establishing, and increasing, our prepandemic workflow. planning should begin now, and crucially, prioritize patients with significant risks arising from delayed operations. the risks of deferring needed cardiac treatment have been previously demonstrated. reported mortality rate while waiting for surgical or transcatheter aortic valve replacement can be as high as 3.7% at one month and 11.6% at six months [2] . for patients awaiting coronary surgery, median waiting list mortality rates of 2.6% per month have been reported with mortality risk increasing 11% per month. additionally, 12% of patients experienced a myocardial infarction while on the waiting list [1, 3] . we have attempted to estimate the resources required in planning for the post-pandemic surge in adult cardiac surgical volume. the capacity estimates for a given health system would depend on the percent increase in productivity targeted post pandemic. by utilizing our mathematical model, a health system could predict the time required for servicing their surgical backlog and adjust operating capacity accordingly. actualizing a plan to increase capacity may occur gradually and this will further increase waiting time for deferred patients. increasing capacity will also require proportional adjustments in resources such as operating rooms, icu and telemetry beds, and provider staff. for the majority of hospitals, or time is the most expensive resource. consequently, increasing icu and telemetry capacity to avoid operating room holds and cancellations is likely the most cost-effective approach to achieving increased throughput. the number of additional beds, nurses, and advanced practice providers needed can be calculated based on baseline resources. maximizing efficiency through early telemetry discharge, decreasing intensive care length of stay, level loading elective cases in the or, temporarily altering staffing models, and staggering cases can help increase throughput capacity [9, 10] . another possible solution is to temporarily relocate some non-cardiac surgical cases to other facilities within a health system. in fact, maintaining the incident command center set up by many hospitals and utilizing some of the same workforce and bed solutions employed during the pandemic may be of value in delivering critical cardiac services in a timely fashion. our analysis has several limitations. firstly, we have assumed that historical rates of surgery will accurately predict future rates. this may be incorrect for several reasons. during the pandemic patients may be treated with alternative medical and interventional therapy, obviating the need for surgery. patients treated medically with symptomatic relief may decide not to pursue more aggressive treatment. other patients may be reluctant to incur out of pocket costs during this time of financial difficulty. additionally, there will unfortunately be some degree of attrition of patients from cardiovascular and covid-related mortality. the pent up demand could also accrue more slowly than calculated. cardiac surgeons sit at the bottom of a virtual funnel which begins with patients seeking medical care, referral to a cardiologist, diagnostic testing, and finally evaluation for surgery. each step along this pathway will be delayed during, and likely for some time after, the pandemic. delays would function as a governor on the acceleration of post-pandemic cardiac surgical demand. our study does, however, provide concerning data delineating a large group of patients with surgical level cardiovascular disease whose treatment has been necessarily deferred. our hope is that our modeling can be used to calculate the productivity increase necessary to treat patients in an acceptable time frame. further study could help better define the overall effects of a pandemic on the care of cardiovascular patients. although challenging and costly, early post-pandemic operating capacity will need to be greater than the original daily capacity to satisfy the pent-up surgical demand. not planning for post pandemic volume could result in a second wave of non-covid cardiovascular mortality. mortality on the waiting list for coronary artery bypass grafting: incidence and risk factors mortality while waiting for aortic valve replacement waiting times and prioritisation for coronary artery bypass surgery in new zealand surgical care required for populations affected by climate-related natural disasters: a global estimation medical interventions following natural disasters: missing out on chronic medical needs design of a model to predict surge capacity bottlenecks for burn mass casualties at a large academic medical center surgical subspecialty block utilization and capacity planning: a minimal cost analysis model roadmap for resuming elective surgery after covid-19 pandemic n.d balancing operating theatre and bed capacity in a cardiothoracic centre development and evaluation of a prospective staffing model to improve retention key: cord-257801-rgzmpoxv authors: keeling, matt j; hollingsworth, t. deirdre; read, jonathan m title: the efficacy of contact tracing for the containment of the 2019 novel coronavirus (covid-19). date: 2020-02-17 journal: nan doi: 10.1101/2020.02.14.20023036 sha: doc_id: 257801 cord_uid: rgzmpoxv contact tracing is a central public health response to infectious disease outbreaks, especially in the early stages of an outbreak when specific treatments are limited. importation of novel coronavirus (covid-19) from china and elsewhere into the united kingdom highlights the need to understand the impact of contact tracing as a control measure. using detailed survey information on social encounters coupled to predictive models, we investigate the likely efficacy of the current uk definition of a close contact (within 2 meters for 15 minutes or more) and the distribution of secondary cases that may go untraced. taking recent estimates for covid-19 transmission, we show that less than 1 in 5 cases will generate any subsequent untraced cases, although this comes at a high logistical burden with an average of 36.1 individuals (95th percentiles 0-182) traced per case. changes to the definition of a close contact can reduce this burden, but with increased risk of untraced cases; we estimate that any definition where close contact requires more than 4 hours of contact is likely to lead to uncontrolled spread. kingdom highlights the need to understand the impact of contact tracing as a control measure. using detailed survey information on social encounters coupled to predictive models, we investigate the likely efficacy of the current uk definition of a close contact (within 2 meters for 15 minutes or more) and the distribution of secondary cases that may go untraced. taking recent estimates for covid-19 transmission, we show that less than 1 in 5 cases will generate any subsequent untraced cases, although this comes at a high logistical burden with an average of 36.1 individuals (95 th percentiles 0-182) traced per case. changes to the definition of a close contact can reduce this burden, but with increased risk of untraced cases; we estimate that any definition where close contact requires more than 4 hours of contact is likely to lead to uncontrolled spread. contact tracing is the main public health response to importations of rare or emerging infectious diseases, and was implemented in the uk during the 'containment stage' of the 2009 influenza pandemic (mclean et al 2010) . in more recent years, contact tracing was also a valuable tool following importation of ebola virus disease into the uk in 2014 (crook et al 2017) and the cases of monkeypox in the uk in 2018 (vaughan et al 2018). in general, contact tracing is a highly effective and robust strategy given sufficient resources. the main advantages are that it can identify potentially infected individuals before severe symptoms emerge, and if conducted sufficiently quickly can prevent onward transmission from the secondary cases. contact tracing has proved hugely successful in the treatment of sexually transmitted infections, where the definition of a contact is relatively straightforward, where infection is often asymptomatic and where the time-scales of transmission are slow (hogben et al 2016 , rönn et al 2017 . in contrast, the use of contact tracing for novel invading pathogens has received less quantitative consideration, in part due to greater uncertainties over social contact structure (although see ahmed et al 2018 , hoang et al 2019 . modelling studies have often focused on quantifying the importance of pre-symptomatic and pretracing infectiousness, but are usually based on statistical distributions of contact networks (fraser et al 2004 , kwok et al 2019 . here we leverage detailed social network data from the uk to model both transmission and the act of tracing, and identify the implications of contact tracing for containment of a novel pathogen, using parameters for the novel coronavirus (covid-19) (read et al 2020 , li et al 2020 . we characterised contact patterns in the uk using a postal and online cross-sectional survey, which asked participants to report the number of social encounters with unique individuals during a given day, as well as the duration and typical frequency of those encounters (danon et al 2012 (danon et al , 2013 . in total, 5,802 respondents reported more than 50,000 encounters -one of the biggest studies of its kind to date. the encounter patterns of this study were in good qualitative agreement with other similar studies of social interactions (mossong et al 2008 , isella et al 2010 . in this study, the daily encounter data was first extrapolated to generate a pattern of contacts over a 14 day period (replicating random encounters and increasing the total duration associated regular contacts), to act as the basis for transmission and contact tracing simulations. using this extrapolated data, we can classify interactions into those which satisfy the definition of a close contact for the purpose of contact tracing. from our social encounter data we can also distinguish interactions with people who could be later identified and traced, from those with unidentifiable strangers (schematic figure 1). we assume that all contact of longer than 1 hour or repeated contacts can be identified and traced, whereas shorter meetings with people for the first time are strangers who are unidentifiable. the second element of the simulation is to determine who gets infected from a source case chosen representatively from the survey respondents. this transmission process is stochastic, accounting for both the time spent with each contact and the infectivity on each day (see appendix). taken together these two predictions allow us to bound the efficacy of contact tracing. one of the most notable features of human social contacts is the huge variability in the number and strength of contacts -which is reflected as variation in both the number of secondary cases and the number of individuals that match the contact-tracing definition (figure 2). using preliminary estimates of covid-19 transmission (average latent period 4 days, average effective infectious period 1.61 days, r 0 =3.11 and assuming a simple seir formulation (read et al 2020)) we compute the distribution of epidemiological, social and contact tracing characteristics across the population. extrapolating the data from the social contact survey suggests that the average number of contacts over a 14 day period is 217, although the distribution is significantly over dispersed (with a median of 90 and around 3% of individuals having >1,000 total contacts). of these total encounters, an average of 59 contacts (27%) meet the definition of a close-contact (in contact for >15 minutes, phe 2020) and of these close-contacts we predict an average of 36 (61%) to be individuals known to the infected case that can be traced. therefore, simply considering social contacts, it is clear that there are very many short duration contacts which do not meet the definition of a close contact, and although unlikely to become infected may pose a risk due to their greater abundance. given that the risk of infection increases with duration of contact, the distribution of cases effectively represents a biased sample of all contacts. as expected, given the model assumptions, the expected number of total secondary cases agrees with the assumed r 0 (mean=3.11, median=2, and 95th percentiles 0-10). given that these cases are most likely to be those contacts of the longest duration, we predict that 95% of cases match the definition of a close contact. however, not all of these contacts will be identifiable; assuming that all repeated contacts and contact of longer than 1 hour can be traced, we predict that 93% of all cases meet the definition and can be identified. however, because of the extreme heterogeneity in contacts between individuals and the stochastic nature of transmission, we would still expect 15% of all primary cases to generate at least one secondary case that cannot be identified. aggregating across all individuals and under the optimistic assumption that all the contact tracing can be performed rapidly, we expect contact tracing to reduce the basic reproductive ratio from 3.11 to 0.21 -enabling the outbreak to be contained (figure 2). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . rapid and effective contact tracing can therefore be highly effective in the early control of covid-19, but places substantial demands on the local public-health authorities. each new case requires an average of 36 individuals to be traced, with 8.7% of cases having more than 100 close traceable contacts (figure 2). we therefore consider the implications of changing the definition of a close contact. clearly a more strict definition of a close contact (requiring more contact time) reduces the burden on the health services as fewer contacts need to be traced, but also increases the risk of cases being missed. figure 3 provides a quantitative assessment of changes to the close contact definition. definitions requiring more than four hours of contact, are unlikely to control an outbreak as the expected number of untraced secondly cases is greater than one. this therefore places a strict bound on level of contact tracing required. the added benefit from definitions shorter than 1 hour has relatively little impact on the mean number of untraced cases (figure 3b), but does reduce the probability that some untraced contacts occur. throughout we have used a value of r 0 that represents a population-level average once the local infection has become established. however, the first invasion into any new population or social setting generally has a larger expected number of secondary cases. the first invader enters a completely susceptible population; moreover all their close contacts (eg family members) are susceptible. in contrast, due to clustering of contacts, most secondary cases will be in a landscape with a depleted number of susceptibles -as close contacts such as family members will already have been exposed to the primary case. this susceptible depletion in the local social network may help to explain the change in r t over time reported for covid-19 (yang et al 2020). we therefore consider the impact of different values of the initial reproductive ratio (figure 4), which could capture this social aspect, or could represent heterogeneity between individuals in the amount of virus shed, or could inform about innate differences in behaviour between china and the uk. given the strong biasing of transmission towards long-duration contacts, the impact of varying initial reproductive ratio is less extreme than might be expected; it is only for the highest values of initial reproductive ratio simulated (>9.8) that contact tracing fails to find more than one case such that infection can escape. mathematical models have an important role to play in preparedness for novel infectious diseases, allowing policy makers to plan for potential public health scenarios before they arise. however, in such scenarios reliable data is often limited, so predictions of long term dynamics are generally associated with wide confidence intervals. in contrast, while short term predictions are subject to greater stochasticity, the distribution of possible behaviours can be readily captured. here we have investigated contact tracing of a close-contact pathogen, using 2019 novel coronavirus (covid-19) as the example, and considered the efficacy of contact tracing as a control measure. this work brings together a detailed survey of social encounters together with bespoke mathematical modelling of the transmission and tracing processes. given the huge heterogeneties present in social encounters (both in terms of duration and number) mathematical models are vital to interpret the interplay between a low number of high risk encounters (e.g., household members) and the high number of low-risk less-identifiable encounters (e.g., commuters or retail customers). the uk currently defines a close contact as 15 minutes within 2 meters over two weeks before detection (phe 2020) . under this definition, there are unlikely to be many untraced secondary cases, although the burden of tracing could be large. relaxing the definition of a contact (such that longer contact durations are needed) lessens this burden but at the greater risk of undetected cases ( figure 3) . surprisingly, small changes to the reproductive ratio, within the bounds estimated from early data ( figure 4) or even changes to the distribution of infectivity, are predicted to have a relatively modest impact of the success of contact tracing illustrating the robustness of this control measure. our model has addressed the simple and optimistic question of whether contact tracing is sufficient to identify secondary infections. the public health implications of this tracing are more complex, and depend on the relative timing of events and the treatment of identified contacts. for contact tracing to be an effective public health measure requires secondary cases to be discovered before they become infectious; hence the time from the primary case becoming infectious to the tracing of their contacts needs to be shorter than the incubation period. longer time scales would allow tertiary cases to be infected and would snowball the tracing process. in addition, those contacts that are traced either need to be effectively screened for infection and quarantined or otherwise isolated so that they do not pose a risk to others. therefore, while contact tracing has the potential to control covid-19 (and other close-contact pathogens) the ultimate success relies on the speed and efficacy with which suspect contacts can be contained. with contacts positioned by their total contact duration. here, the definition of a contact is someone with whom the index case encountered for 15 minutes or longer. some contacts will be identifiable (green), while others will be unidentifiable (orange). a definition of contact that is too restrictive and inappropriate for the infection means some encounters may fail to meet the definition yet may be at risk of infection; these excluded contacts could be identifiable (light grey) or unidentifiable (orange). (b) examples of ego-centric networks collected by the survey. the participant (ego) is the blue central triangle; circles represent individual contacts, squares represent groups of contacts (size of group indicated). colours represent social settings of encounters (red=home, cyan=work/school, yellow=travel, pink=other) . larger symbol sizes represent longer contact durations, while a closer proximity to the ego indicates the contact is more frequently encountered. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . figure 3. impact of different assumptions for the definition of a close-contact. (a) the total number of contacts traced (b) the number of secondary contacts that are not traced (c) the probability that at least one secondary case is not traced. for (a) and (b) the crosses mark the mean value, boxes contain the 50th percentiles while bars contact the 95th percentiles, colours correspond to those in figure 1a prob at least one untraced case . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . figure 4. impact of different values for the initial reproduction number of the primary case; changing this does not affect the the number of contacts traced (a) the number of secondary contacts that are not traced (b) the probability that at least one secondary case is not traced. for (a) the crosses mark the mean value, boxes contain the 50th percentiles while bars contact the 95th percentiles, colours correspond to those in figure 1a -distributions are across all respondents to the survey and across stochastic realisations. (based on an seir model with latent period 4, infectious period 1.61, r 0 =3.11). prob at least one untraced case . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10. 1101 effectiveness of workplace social distancing measures in reducing influenza transmission: a systematic review lack of secondary transmission of ebola virus from healthcare worker to 238 contacts social encounter networks: collective properties and disease transmission social encounter networks: characterizing great britain factors that make an infectious disease outbreak controllable a systematic review of social contact surveys to inform transmission models of closecontact infections what's in a crowd? analysis of face-to-face behavioral networks epidemic models of contact tracing: systematic review of transmission studies of severe acute respiratory syndrome and middle east respiratory syndrome. computational and structural biotechnology journal early transmission dynamics in wuhan, china, of novel coronaviurs-infected pneumonia pandemic (h1n1) 2009 influenza in the uk: clinical and epidemiological findings from the first few hundred (ff100) cases. epidemiology & infection social contacts and mixing patterns relevant to the spread of infectious diseases novel coronavirus 2019-ncov: early estimation of epidemiological parameters and epidemic predictions from the contact tracing data, we extrapolate to the estimate the duration of contact ",$ % , between individual i (the respondent) and individual j (the contact) on day d. we then define a close contact of i as all contacts j:where in the uk, we have defined the total contact time t as 15 minutes over a duration d of two weeks before detection and isolation of individual i (phe 2020).the probability of transmission to individual j from individual i is then calculated as:where $ is an estimate of the transmission rate from individual i on day d. key: cord-262787-3a3c8ee1 authors: ray, debashree; salvatore, maxwell; bhattacharyya, rupam; wang, lili; mohammed, shariq; purkayastha, soumik; halder, aritra; rix, alexander; barker, daniel; kleinsasser, michael; zhou, yiwang; song, peter; bose, debraj; banerjee, mousumi; baladandayuthapani, veerabhadran; ghosh, parikshit; mukherjee, bhramar title: predictions, role of interventions and effects of a historic national lockdown in india's response to the covid-19 pandemic: data science call to arms date: 2020-04-18 journal: nan doi: 10.1101/2020.04.15.20067256 sha: doc_id: 262787 cord_uid: 3a3c8ee1 importance: india has taken strong and early public health measures for arresting the spread of the covid-19 epidemic. with only 536 covid-19 cases and 11 fatalities, india a democracy of 1.34 billion people took the historic decision of a 21-day national lockdown on march 25. the lockdown was further extended to may 3, soon after the analysis of this paper was completed. objective: to study the shortand long-term impact of an initial 21-day lockdown on the total number of covid-19 cases in india compared to other less severe non-pharmaceutical interventions using epidemiological forecasting models and bayesian estimation algorithms; to compare effects of hypothetical durations of lockdown from an epidemiological perspective; to study alternative explanations for slower growth rate of the virus outbreak in india, including exploring the association of the number of cases and average monthly temperature; and finally, to outline the pivotal role of reliable and transparent data, reproducible data science methods, tools and products as we reopen the country and prepare for a post lock-down phase of the pandemic. design, setting, and participants: we use the daily data on the number of covid-19 cases, of recovered and of deaths from march 1 until april 7, 2020 from the 2019 novel coronavirus visual dashboard operated by the johns hopkins university center for systems science and engineering (jhu csse). additionally, we use covid-19 incidence counts data from kaggle and the monthly average temperature of major cities across the world from wikipedia. main outcome and measures: the current time-series data on daily proportions of cases and removed (recovered and death combined) from india are analyzed using an extended version of the standard sir (susceptible, infected, and removed) model. the esir model incorporates time-varying transmission rates that help us predict the effect of lockdown compared to other hypothetical interventions on the number of cases at future time points. a markov chain monte carlo implementation of this model provided predicted proportions of the cases at future time points along with credible intervals (ci). results: our predicted cumulative number of covid-19 cases in india on april 30 assuming a 1-week delay in people's adherence to a 21-day lockdown (march 25 april 14) and a gradual, moderate resumption of daily activities after april 14 is 9,181 with upper 95% ci of 72,245. in comparison, the predicted cumulative number of cases under "no intervention" and "social distancing and travel bans without lockdown" are 358 thousand and 46 thousand (upper 95% ci of nearly 2.3 million and 0.3 million) respectively. an effective lockdown can prevent roughly 343 thousand (upper 95% ci 1.8 million) and 2.4 million (upper 95% ci 38.4 million) covid-19 cases nationwide compared to social distancing alone by may 15 and june 15, respectively. when comparing a 21-day lockdown with a hypothetical lockdown of longer duration, we find that 28-, 42-, and 56-day lockdowns can approximately prevent 238 thousand (upper 95% ci 2.3 million), 622 thousand (upper 95% ci 4.3 million), 781 thousand (upper 95% ci 4.6 million) cases by june 15, respectively. we find some suggestive evidence that the covid-19 incidence rates worldwide are negatively associated with temperature in a crude unadjusted analysis with pearson correlation estimates [95% confidence interval] between average monthly temperature and total monthly incidence around the world being -0.185 [-0.548, 0.236] for january, -0.110 [-0.362, 0.157] for february, and -0.173 [-0.314, -0.026] for march. conclusions and relevance: the lockdown, if implemented correctly in the end, has a high chance of reducing the total number of covid-19 cases in the short term, and buy india invaluable time to prepare its healthcare and disease monitoring system. our analysis shows we need to have some measures of suppression in place after the lockdown for the best outcome. we cannot heavily rely on the hypothetical prevention governed by meteorological factors such as temperature based on current evidence. from an epidemiological perspective, a longer lockdown between 42-56 days is preferable. however, the lockdown comes at a tremendous price to social and economic health through a contagion process not dissimilar to that of the coronavirus itself. data can play a defining role as we design post-lockdown testing, reopening and resource allocation strategies. software: our contribution to data science includes an interactive and dynamic app (covind19.org) with shortand long-term projections updated daily that can help inform policy and practice related to covid-19 in india. anyone can visualize the observed data for india and create predictions under hypothetical scenarios with quantification of uncertainties. we make our prediction codes freely available (https://github.com/umich-cphds/cov-ind-19) for reproducible science and for other covid-19 affected countries to use them for their prediction and data visualization work. 622 thousand (upper 95% ci 4.3 million), 781 thousand (upper 95% ci 4.6 million) cases by june 15, respectively. we find some suggestive evidence that the covid-19 incidence rates worldwide are negatively associated with temperature in a crude unadjusted analysis with pearson correlation estimates [95% confidence interval] between average monthly temperature and total monthly incidence around the world being -0.185 [-0.548 the lockdown, if implemented correctly in the end, has a high chance of reducing the total number of covid-19 cases in the short term, and buy india invaluable time to prepare its healthcare and disease monitoring system. our analysis shows we need to have some measures of suppression in place after the lockdown for the best outcome. we cannot heavily rely on the hypothetical prevention governed by meteorological factors such as temperature based on current evidence. from an epidemiological perspective, a longer lockdown between 42-56 days is preferable. however, the lockdown comes at a tremendous price to social and economic health through a contagion process not dissimilar to that of the coronavirus itself. data can play a defining role as we design post-lockdown testing, reopening and resource allocation strategies. software: our contribution to data science includes an interactive and dynamic app (covind19.org) with short-and long-term projections updated daily that can help inform policy and practice related to covid-19 in india. anyone can visualize the observed data for india and create predictions under hypothetical scenarios with quantification of uncertainties. we make our prediction codes freely available (https://github.com/umich-cphds/cov-ind19) for reproducible science and for other covid-19 affected countries to use them for their prediction and data visualization work. four months since the first case of covid-19 in wuhan, china, the sars-cov-2 virus has engulfed the world and has been declared a global pandemic. 1 the number of confirmed cases worldwide stands at a staggering 1,930,780 (as of 9:20 am est april 14, 2020, microsoft bing coronavirus tracker 2 ). of these, 10,815 confirmed cases are from india (figure 1) , the world's largest democracy with a population of 1.34 billion (compare china at 1.39 billion and usa at 325.7 million). 3 india has been vigilant and wise in instituting the right public health interventions at the right time including sealing the borders with travel ban/canceling almost all visas, closing schools and colleges in certain states and diligently following up with community inspection of suspected/exposed cases with respect to adherence of quarantine recommendations ( table 1) . on march 24, india took the historic decision of a 21-day national lockdown starting march 25, when it had reported only 536 covid-19 cases and 11 fatalities. in the subsequent days we have seen a steady growth in the number of new cases and fatalities, with growth rates slower than other affected countries but in 21 days, the curve has not yet "turned the corner" or showed a steady decline in the number of newly diagnosed cases (figure 2 ). while india seems to have done relatively well in controlling the number of confirmed cases compared to other countries in the early phase of the pandemic (figure 2) , there is a critical missing or unknown component in this assessment: "the number of truly affected cases," which depends on the extent of testing, the accuracy of the test results and, in particular, the frequency and scale of testing of asymptomatic cases who may have been exposed. the frequency of testing has been low in india. according to the indian council of medical research (icmr), only 130,792 subjects have been tested as of april 9. 4 when there is no approved vaccine or drug for treating covid-19, entering phase 2 or phase 3 of escalation will have devastating consequences on both the already overstretched healthcare system of india, and india's large at-risk sub-populations (supplementary table 1 ). as seen for other countries like the us or italy, covid-19 enters gradually and then explodes suddenly. we provide a table listing other highly affected countries along with their first reported case, initial interventions, crude fatality rates, and active case counts in supplementary table 2 for reference. in this article, we take a data-driven approach to explore five extremely time-sensitive and important questions that india faces today in light of the covid-19 outbreak and the national lockdown: (a) how many cases can india expect at the end of the lockdown period? (b) when will the curve in india reach its apex and will the number of cases go back up after lockdown is lifted? (c) can summer temperatures thwart the outbreak in india? (d) how can the government and the people of india prepare for this crisis during and after the lockdown? (e) how critical is it to have reliable data, data science methods and tools as we envision a long-term strategy during and after the lockdown? this work is the result of the collective public health conscience of a group of interdisciplinary researchers in different parts of the us and in india. we convened virtually after being quarantined in our homes with alternating waves of fear and inspiration surrounding us. we decided to channel our collective energy to study the defining public health and economic crisis of our time and use our data science expertise to search for answers and solutions that can help covid-19 related policymaking in india. this is our contribution and public service as data scientists. our data science product includes two articles on medium pre 5 and post 6 lockdown announcement, providing critical information for policymakers (reuters, 7 times of india, 8 the guardian, 9 the economic times 10 ) and an interactive app that daily updates forecasts as new case counts are coming in, and publicly available codes for reproducible research. we used the current daily data on number of covid-19 cases, recoveries and deaths in india to predict the number of cases at any given time. 11 we obtained the data (up to april 7) from the 2019 novel coronavirus visual dashboard operated by the johns hopkins university center for systems science and engineering (jhu csse). 12,13 for our temperature analysis, these counts were aggregated to a month-level for each country, that is, we look at the total number of new cases in the months of january, february and march for each country. we obtained the monthly average temperature for major cities in the countries with covid-19 outbreak from wikipedia. 14 we analyzed the data from india with standard epidemiologic tools of modeling disease transmission and estimating the theoretical number of cases at any time. one such epidemiologic model is the susceptible-infected-removed (sir) model, which is guided by a set of differential equations relating the number of susceptible people, the number of infected people (cases) and the number of people who have been removed (either recovered or dead) at any given time. recently, this standard sir model was extended to incorporate time-varying transmission rates or timevarying quarantine protocols and is known as the esir model. 11 when using the esir model with time-varying disease transmission rate, it can depict a series of time-varying changes caused by either external variation like government-initiated macro isolation measures, community-level protective measures and environment changes, or internal variations like mutations and evolutions of the pathogen. the r package for implementing this general model for understanding disease dynamics is publicly available at https://github.com/lilywang1988/esir. to implement the esir model, a bayesian hierarchical framework is assumed where the proportions of infected and the removed people are modeled using a beta-dirichlet state-space model while a latent dirichlet distribution is assumed for the underlying unknown prevalence of the three states. priors for the basic reproductive number r0, disease removal rate (consequently, the transmission rate) and the underlying unobserved prevalence of the susceptible, infected and removed states at the starting time are considered. using the current time series data on the proportions of infected and the removed people, a markov chain monte carlo implementation of this bayesian model provides not only posterior estimation on parameters and prevalence of all the three compartments in the sir model, but also predicted proportions of the infected and the removed people at future time point. the posterior mean estimates of the unobserved prevalence at both observed as well as future time points come along with 95% credible intervals (ci). to get predicted case-counts from the predicted prevalence, we used 1.34 billion as the population of india, thus treating the country as a homogeneous system for the outbreak. 15 we made projections of the cumulative number of cases over a time horizon to assess the shortterm impact of lockdown as well as the long-term impact of lockdown and post-lockdown activities. for the short-term forecast on april 30, we assumed lockdown is implemented until april 14 with either a 1-or a 2-week delay in people's adherence/compliance to lockdown restrictions. we compared these projections with two hypothetical scenarios: (a) no nonpharmaceutical intervention (i.e., a constant disease transmission rate over time since the first case was reported in india), (b) a moderate intervention with social distancing and travel bans only (i.e., a decreased transmission rate compared to no intervention). for the no intervention and the moderate intervention scenarios, we chose the transmission rate and the removal rate such that the means for the prior distribution of the basic reproductive number r0 (the expected number of cases generated by one infected person assuming that the whole population is susceptible) are 2.0 and 1.5 respectively [the change in r0 was created based on what we saw in wuhan 16 ]. the value of 2.0 was estimated based on the early phase data in india. for the current scenario of lockdown, our chosen mean for r0 prior starts with 2.0 during the period of no intervention, drops to 1.5 during the period of moderate intervention, and further drops to 0.8 during the 21-day lockdown period, and moves back up to 1.5 after the lockdown ends as described in figure 3 (assuming a gradual, moderate resumption of daily activities). for the longer-term forecast until june 15, we considered three hypothetical post-lockdown scenarios: (i) people return to normal activities due to the urgent desire for reconnecting after lockdown; (ii) people return to moderate activities as they did during the period with social distancing and travel ban intervention; and (iii) people make a cautious return out of fear for the coronavirus and partake in subdued activities. for these three scenarios, we assume mean for r0 prior moves back up from 0.8 to 2.0, 1.5 and 1.2 respectively three weeks after lockdown ends on april 14. we compared these post-lockdown scenarios with another hypothetical scenario involving perpetual social distancing and travel ban only without any lockdown (we fixed the mean for r0 prior at 1.5 over the entire intervention interval). the changes to r0 values across our simulation scenarios are depicted in figure 3 . to assess the long-term impact of lockdown duration, we considered four scenarios: 21-, 28-, 42-, and 56-day lockdown periods. in all scenarios, we assume mean for r0 prior remains at 0.8 for the duration of the lockdown and returns to 1.5 three weeks after the lockdown period ends (analogous to the "moderate return" scenario). the changes to r0 values across our simulation scenarios are depicted in supplementary figure 4 . there are many hypotheses regarding the slow growth rate of covid-19 cases in many countries, particularly low-and middle-income countries (lmics). some of these hypotheses include the use of bacille calmette guerin (bcg) vaccine, 17 younger population, 18 high daily temperature, 19 use of anti-malarials 20 and host genetics. 21 here, we only explore the temperature hypothesis related to covid-19 incidence. we assessed any correlation between country-wise average monthly temperature and total incidence of covid-19. the monthly average temperature for major cities across the world was used to compute the monthly average temperature for each country experiencing covid-19 outbreak by averaging across the major cities within a country. missing data for average temperature for certain countries was manually appended from www.weatheratlas.com. 22 we computed the pearson correlation coefficient, , between the average monthly temperature and total monthly incidence during each month of january, february and march. we used the fisher's z-transformation to compute z = 0.5 log + ,-. ,/. 0. the standard deviation of , which is known under certain normality assumptions, is used to construct a 95% confidence interval for . the inverse transform of is then used to obtain the 95% confidence interval for the correlation . all calculations were carried out in the rstudio platform. under national lockdown (march 25 -april 14), our predicted cumulative number of covid-19 cases in india on april 30 are 9,181 and 11,626 (upper 95% ci of 72,245 and 84,245) assuming a 1-or 2-week delay (i.e., either a quick or a slow adherence), respectively, in people's adherence to lockdown restrictions and a gradual, moderate resumption of daily activities post-lockdown (figure 4, supplementary figure 1 ). in comparison, the predicted cumulative number of cases under "no intervention" and the "intervention involving social distancing and travel bans without lockdown" are 358 thousand and 46 thousand (upper 95% ci of nearly 2.3 million and 0.3 million) respectively. we are reporting only the upper credible limit here and elsewhere since the lower credible limits are very close to 0 due to the large uncertainty in our predictions arising from many unknowns. we also believe that our point estimates are at best underestimates due to potential under-reporting of case-counts and our model not taking into account the population density, agesex and contact network structure of the whole nation. increase in testing and community transmission may lead to a spike in a single day and that may shift the projection curve significantly upward. regardless of the exact numbers it is clear that, the 21-day lockdown will likely have a strong effect on reducing the predicted number of cases in the short term. we took a close look at what might be coming in the next few months, based on what we have seen in other countries and an epidemiological model that has been gainfully employed to assess the effect of interventions in hubei province. 11 we estimated that 26 (upper 95% ci 135) and 177 (upper 95% ci 2869) cases per 100,000 are avoided by may 15 and june 15, respectively, by instituting a 21-day lockdown with a 1-week delay in people's adherence and a cautious release compared to perpetual social distancing and travel ban (without lockdown) ( figure 5 ). this boils down to preventing roughly 343 thousand (upper 95% ci 1.8 million) and 2.4 million (upper 95% ci 38.4 million) covid-19 cases nationwide by may 15 and june 15, respectively. without some measures of suppression after lockdown is lifted, the impact of lockdown in bringing down the case-counts (the now ubiquitous term, "flattening the curve") can be negated by as early as the first week of june. in fact, in figure 5a , the pre-intervention ("normal") curve first passes the social distancing and travel ban curve on june 8. in particular, if people immediately go back to pre-intervention ("normal") activities post-lockdown, a surge in the predicted case-counts is expected in the long-term beyond what we would have seen if there were only social distancing and travel ban measures without lockdown (7.1 million when post-lockdown activity returns to pre-intervention levels vs. 6.9 million under social distancing and travel ban without a lockdown period on july 31; figure 5 ). we estimated that 195 (upper 95% ci 3494) and 380 (upper 95% ci 6463) cases per 100,000 are avoided by june 15 and july 15 respectively if people are cautious in their activities post-lockdown compared to the scenario where people return to normal preintervention activities. long-term forecasting under slow adherence (2-week delay) can be seen in supplementary figure 2 . . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.15.20067256 doi: medrxiv preprint we took the quick adherence epidemiological models and compared the 21-day lockdown with hypothetical 28-, 42-, and 56-day lockdown scenarios (figure 6 ). when comparing a 21-day lockdown with a hypothetical lockdown of longer duration, we find that 28-, 42-, and 56-day lockdowns can approximately prevent 238 thousand (upper 95% ci 2.3 million), 622 thousand (upper 95% ci 4.3 million), 781 thousand (upper 95% ci 4.6 million) cases by june 15, respectively. a 28-day lockdown does not appear to have a significant impact on cumulative case counts when compared to a 21-day lockdown. however, purely from an epidemiologic perspective, there appears to be some evidence that suggests a 42-or 56-day lockdown would have a more meaningful impact on reducing cumulative covid-19 case counts in india. we note that longer lockdown periods are accompanied by increasing costs to individuals -notably economicand must be considered. our models suggest that some form of post-lockdown suppression (e.g., extension of social distancing measures, limits of gathering size, etc.) is necessary to observe longterm benefits of the lockdown period. lockdown duration study under the slow adherence (2-week delay) scenario can be found in supplementary figure 3 . we did explore some alternative assumptions and conducted thorough sensitivity analysis before settling on the models presented above. in one example, we assumed that there are actually 10 times the number of reported cases to date to reflect potential underreporting of cases due to lack of testing. in another scenario, we assumed these cases occurred in metropolitan areas to reflect a potential intensification of case clustering. in yet a third scenario, we hypothesized that r0 prior starts with 2.5 instead of 2.0 (i.e., a single infected individual would infect 2.5 susceptible individuals, on average, instead of 2). these scenarios did not appreciably change our conclusions in broad qualitative terms, though the exact quantitative projections are quite sensitive to such choices. across these scenarios, the projected total number of infected cases by the entire first phase of the pandemic varied between 2-15% of the population, again showing the significant variability in these numbers. the estimates we present here may appear conservative and are at best underestimates, and, in all cases, our confidence in these projections decreases markedly the farther into the future we try to forecast. it is extremely important to update these models as new data arise. spatial plots for the average monthly temperatures accompanied by total monthly incidence across all countries from january through march indicate a suggestive pattern of increase in community spread across cities and regions specifically along narrow north east-west directions (figure 6 ). countries in these regions consistently exhibit similar weather patterns. however, in the context of india, a gradual rise in the number of cases is observed starting from january through march. . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . the estimates and the 95% confidence interval for the correlation coefficient for january was -0.185 [-0.548, 0.236] with 24 countries having non-zero incidence, for february was -0.110 [-0.362, 0.157] with 56 countries having non-zero incidence, and for march was -0.173 [-0.314, -0.026] with 175 countries having non-zero incidence. although the estimates were negative, the 95% confidence intervals either include zero or the upper limit is close to 0, indicating weak evidence for any claim of negative association between case counts and daily temperature. any such affirmation will require further data and investigation that accounts for many possible sources of confounding. our projections using current daily data on case counts until april 7 in india show that the lockdown, if implemented correctly in the end, has a high chance of reducing the number of covid-19 cases in the short term and buy india invaluable time to prepare its healthcare and disease monitoring system. in the long-term, we need to have some measures of suppression in place after the lockdown is lifted to prevent a massive surge in the number of cases that can quickly overwhelm an already over-stretched indian healthcare system resulting in increased fatalities. specific vulnerable populations will be at higher risk of severity and fatality from covid-19 infection: older persons and persons with pre-existing medical conditions (e.g., high blood pressure, heart disease, lung disease, cancer, diabetes, immunocompromised persons). 24,25 supplementary table 1 provides a description of the approximate number of individuals in these high-risk categories in india. beyond the fragile population characterized by health and economic indicators, we have to remember that healthcare workers and first responders at the front line of this pandemic are amongst the most vulnerable. 16 it is important to note that a massive surge in the number of cases can quickly overwhelm an already over-stretched indian healthcare system. the estimated capacity of hospital beds in india is 70 per 100,000, 26 which is an upper bound on treatment capacity. given an average occupancy rate of 75%, only a quarter of these are available. 27 moreover, critically ill covid-19 patients (about 5-10% of those infected) will require icu beds and ventilator support. india has only 35-58 thousand icu beds with very high occupancy rates and at most 1 ventilator per 2 icu beds. 28 from a purely public health perspective, this analysis shows the impact and necessity of lockdown and subsequent measures of suppression after lockdown is lifted. all the people in india, regardless of their vulnerability to covid-19, need to adhere to the public health guidelines issued by the ministry of health and family welfare in india, and continue to be cautious in their post-lockdown activities to guarantee a long-term benefit of the national lockdown. currently, there are many hypotheses regarding differential covid-19 infection rates and mortality rates across countries. one such hypothesis is that the bcg vaccine -developed a century back for tuberculosis -has a protective effect on the prevalence of covid-19 and related . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . mortality. 29 a recent pre-print found covid-19 attributable mortality in countries with bcg policy is 6 times lower than those without a bcg policy in an ecological analysis, after accounting for country-specific confounders like economic status, percentage elderly (those aged â�¥65 years) in the population, and relative position of each country along the epidemic trajectory. 17 however, the authors caution against over-interpretation of this negative association between bcg use and covid-19 due to limitations of country-level analysis and many sources of unmeasured confounders. another hypothesis is that much like the flu virus, summer temperatures will thwart the covid-19 outbreak. our analysis, based on current data, suggests we cannot rely on the hypothetical prevention (with inconclusive evidence) governed by meteorological factors and need public health actions, regardless of the seasonal weather. the management of this covid-19 crisis requires strong partnership of the government, the scientific community, the health care providers and all citizens of india (and all global citizens). long term surveillance and management of covid-19 crisis is needed with not just public health in mind but also to take care of the economic, social, and psychological trauma that it will leave on the people. reviving the economy will be critical in the coming months. below we recommend some healthcare, social and economic reforms that can counter the negative impact of severe public health interventions, some of which india has already begun to make progress. (a) aggressively increase the number of tests administered daily as there are often asymptomatic cases who are spreading the infection without knowing. it is of utmost importance that india adopt widespread testing to identify and isolate the infected. rt-pcr diagnostic test can provide reliable and faster diagnosis of the sars-cov-2 virus. 30 large scale antibody testing should be launched to assess the true scale of this pandemic. 31 the instrument of isolating nearly everyone with a near universal lockdown not only leads to livelihood losses for millions of families but also starvation for others. as we reopen the country, testing high contact, high density areas and setting up a clever surveillance system is critical. immediately prepare to protect the health care workers and first responders who are at the front line of this pandemic. this involves ensuring a steady supply chain of medical resources (masks, gloves, gowns, ventilators), and protecting our healthcare workers physically and psychologically. full gears (protective suit, medical goggle, cap, face shield, mask and gloves) are absolutely essential when seeing suspected cases. these protection strategies worked in china. 32 (c) reduce all non-essential medical care and expand number of hospital beds, icu beds and ventilators. (d) continue to set up covid-19 testing mobile labs, hospitals and mobile cabins (e.g. by converting stadiums and trains into quarantine and treatment facilities). 33 ensure the healthcare facilities have adequate supply of medications that are currently . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . being recommended. for instance, antiviral drugs "remdesivir and chloroquine are highly effective in the control of 2019-ncov infection in vitro" indicating promise for treating covid-19 patients. 34 recently though, the study 35 finding hydroxycholoroquine as an effective treatment for covid-19 has been retracted for bad study design and not meeting expected scientific standards. 36 (f) use pragmatic real-time data for optimally deploying surveillance, community inspection and health care resources. this is key with limited resources. economic recommendations: (j) provide livelihood assistance over the quarantine period to those who test positive. this will incentivize people to get tested and comply with social isolation protocols. for many people in india, loss of several weeks of earnings can be economically devastating and since symptoms are mild for most infected people, it is unreasonable to expect that all people will tightly follow restrictions unless economically protected. to get a ballpark idea of the fiscal burden involved, assume 1 million detected cases, quarantine of 6 weeks per patient, and inr 10,000 monthly compensation. this adds up to a bill of inr 15 billion, which is roughly 2.5% of the annual healthcare budget of the central government. (k) during periods of social distancing and lockdowns, there is grave livelihood threat to a lot of poor people even if they are uninfected -street hawkers, auto drivers, barbers and shopkeepers, etc. providing a universal basic income (ubi), or some mildly means tested version of it, over the period of disruption is needed for a successful lockdown. (l) to prevent shutdowns in badly affected sectors, the government may provide goods and services tax (gst) credit to firms based on the difference between past and current sales. once the pandemic is over and normal business resumes, expansionary monetary and fiscal policy will be needed to revive macroeconomic health. there are many epidemiological models to predict the course of an infectious disease and even many that are india-specific. 37-39 some use age-structure, contact patterns, spatial information to finesse their prediction. some consider the possibility of a latent number of true cases, only a fraction of which are ascertained and observed. 16 the model we used here is an extension of a . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . standard sir model, called esir model, 11 where we can create hypothetical intervention scenarios in a time dependent manner. the goal of any intervention is to reduce the chance that an infected person meets a susceptible person. we create models for declines/drops in contact probabilities when an intervention is rolled out. thus, there is some intrinsic ad-hocery to our assumptions. any statistical model is wrinkled with such assumptions. similarly, the predictions themselves have large uncertainty (as reflected by the upper credible limits). as we interpret the numbers from any model, let us use caution in not over-interpreting them. a rigorous quantitative treatment often allows us to analyze a problem with clarity and objectivity, but we recommend focusing more on the qualitative takeaway messages from this exercise rather than concentrating on the exact numerical projections or quoting them with certainty. we see tremendous role of data and data science in governing policy as india reopens post lockdown. the release from lockdown will not be in a binary switch on/switch off process but a modulated slow-varying process. we see the following roles and opportunities for pragmatic use of data science in the post-lockdown phase. (a) flexible, athletic, data driven policymaking will need up-to-date numbers and projections at hand, which require granular data, automation and data transparency (b) understand uncertainty in numbers: all models are wrong, some are useful, but note that takeaway messages for intervention forecasting are often the same 40 (c) using technology to create body temperature/expected health status map (e.g., healthweather.us) 41 (d) assess adherence to social distancing using mobile networks, google (e.g., google mobility reports) 42 (e) use survey to identify potential super-spreaders, manage contact network, oversample high risk areas for testing (f) install syndromic surveillance in hospitals, medical claims systems to set up alert; establish expected number of respiratory and flu-like illnesses so departures can set off an alarm. (g) use community health workers in rural areas, community dwellings to identify and isolate cases and conduct cluster testing (h) targeted communication strategies: regarding tests, treatments, contagion level. misinformation and incorrectly analyzed data lead to panic (i) accurate and consistent reporting of case counts and deaths due to covid-19 are extremely critical our statistical modeling and forecasts are not without limitations. we have very few data points and a long time-window to extrapolate for the long-term forecasts. the uncertainty in our predictions is large due to many unknowns arising from model assumptions, population . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . demographics, the number of covid-19 diagnostic tests administered per day, testing criteria, accuracy of the test results, and heterogeneity in implementation of different government-initiated interventions and community-level protective measures across the country. we have neither accounted for age-structure, contact patterns or spatial information to finesse our predictions [37] [38] [39] nor considered the possibility of a latent number of true cases, only a fraction of which are ascertained and observed. 16 increase in frequency and scale of testing, and community transmission of the sars-cov-2 virus may lead to a spike in a single day and that can shift the projection curve significantly upwards. covid-19 hotspots in india are not uniformly spread across the country, and state-level forecasts 43 may be more meaningful for state-level policymaking. we are assuming that the implementation and effects of public health interventions and policies are the same everywhere in india by treating india as a homogeneous unit. future opportunities for improving our model include incorporating contagion network, age-structure, estimating seir model, incorporating test imperfection, and estimating true fatality/death rates. regardless of the caveats in our study, our analyses show the impact and necessity of lockdown and of suppressed activity post-lockdown in india. rather than over-interpreting exact numerical projections, we recommend focusing more on the qualitative takeaway messages. one ideological limitation of considering only the epidemiological perspective of controlling covid-19 transmission in our model is the inability to count excess deaths due to other causes during this period, or the flexibility to factor in reduction in mortality/morbidity due to some other infectious or flu-like illnesses, traffic accidents or health benefits of reduced air pollution levels. a more expansive framework of a cost-benefit analysis is needed as we gather more data and build an integrated landscape of population attributable risks. finally, in our strong commitment to reproducibility and dissemination of our research, we have made the code for our predictions available at github (https://github.com/umich-cphds/cov-ind-19) and created an interactive and dynamic r shiny app (covind19.org) to visualize the observed data and create predictions under hypothetical scenarios with quantification of uncertainties. these forecasts will get updated daily as new data come in. we hope these products will remain our contribution and service as data scientists during this tragic global catastrophe, and the model and methods will be used to analyze data from other countries. our epidemiologic and mathematical calculations make a convincing case for enforcing the 21day national lockdown of the largest democracy in the world, acting early, before the growth of covid-19 infections in india starts to accelerate. we also notice the public health benefit of extending the lockdown by 3-5 weeks in our projections. measures of suppression are needed postlockdown to get long-term benefits from the lockdown. however, these draconian public health . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.15.20067256 doi: medrxiv preprint measures come at a tremendous price to social and economic health that can last months or even years after the restrictions on social mobility are lifted. thus, there is an urgent need for social and economic immunity: not just coverage for testing and treatment for covid-19 for everyone in india, but subsidies and incentives for the common man to survive the consequences of the severe interventions that are needed to stop the coronavirus from creating a massive catastrophe in india. we also illustrate the critical role of data in aiding policy decisions. finally, our message to the public is to proceed with prudence and caution, and not panic or drown in despair. we should draw hope from the success of south korea and china and the initial promising containment in india. we need to support the community around us and help the government of india to manage the crisis with the best strategies, resources and science. the lockdown has given us time to prepare and act, let us make the best use of it. we are still in a state of national and global emergency and it will take a considerable time for humanity to recover from this global pandemic and return to normalcy. in the meantime, we root for public health, for innovation and science, for home testing kits [there is none yet], 44 for fda approved drugs [solidarity trial], 45 and for a vaccine [5 clinical trials ongoing]. 46 in these frightening times, we find inspiration in the power of the common people and the magic of human kindness. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . figure 7 . left: country-wise total monthly incidence of covid-19 in the months of january, february and march. the horizontal lines approximately indicate the equator, the tropic of cancer and the 60n latitude. right: average monthly temperature (in c) during the months of january, february and march. these maps were created by smoothing the counts as well as the average monthly temperature across geographical locations by spatial interpolation. . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.15.20067256 doi: medrxiv preprint supplementary figure 1 . short-term daily growth in cumulative case counts in india assuming a 2-week delay in people's adherence to restrictions. observed data are shown for days up to april 7. predicted future case counts for april 8 until april 30 are based on observed data until april 7 using the esir model. . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . figure 3 . cumulative (a) and incidence (b) graphs for forecasting models assuming a 2-week delay under 21-, 28-, 42-, and 56-day lockdown scenarios using observed data through april 7. . cc-by-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. author/funder, who has granted medrxiv a license 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interventions on covid-19 epidemic in china an interactive web-based dashboard to track covid-19 in real time list of cities by average temperatures evolving epidemiology and impact of non-pharmaceutical interventions on the outbreak of coronavirus disease differentiall covid-19-attributable mortality and bcg vaccine use in countries case-fatality rate and characteristics of patients dying in relation to covid-19 in italy high temperature and high humidity reduce the transmission of covid-19 regulators split on antimalarials for covid-19 covid-19): are you at higher risk for severe illness? the world bank hospital utilizationn statistics: thirty-five year trend analysis, a measure of operational efficiency of a tertiary care teaching institute in south india coronavirus: does india have enough ventilators, hospital beds? the times of india can an old vaccine stop the new coronavirus? the new york times centers for disease control and prevention. cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel new blood tests for antibodies could show true scale of coronavirus pandemic analysis of 25,000 lab-confirmed covid-19 cases in wuhan: epidemiological characteristics and non-pharmaceutical intervention effects india turns trains into isolation wards as covid-19 cases rise remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial hydroxychloroquine-covid-19 study did not meet publishing society's "expected standard prudent public health intervention strategies to control the coronavirus disease 2019 transmission in india: a mathematical model-based approach age-structured impact of social distanncing on the covid-19 epidemic in india covid-19 for india updates coronavirus statistics: what can we trust and what should we ignore? the guardian covid-19 community mobility reports food and drug administration. coronavirus (covid-19) update: fda alerts customers about unauthorized fradulent covid-19 tests world health organization. who director-general's opening remarks at the media briefing on covid-19 -27 covid-19 vaccine tracker. regulatory affairs professionnals society hypertension (women)* 132 â�  based on 2020 est. of 1.38 billion from un department of economic and social affairs * age-standardized defined as within 5-kilometer distance of home or work abbrev.: copd, chronic obstructive pulmonary disease international diabetes federation; nicpr, national institute of cancer prevention and research ⶠdate of 1 st case data obtained from jhu csse time series data on covid-19 â�  microsoft bing covid-19 tracker the authors will like to thank the university of michigan advanced research computing services for enabling daily updates to our models and allocating us abundant computational resources. we will also like to thank professor matthew fox from the boston university school of public health for his valuable comments on our rshiny app, key: cord-272956-0yumc7em authors: gnavi, roberto; demaria, moreno; picariello, roberta; dalmasso, marco; ricceri, fulvio; costa, giuseppe title: therapy with agents acting on the renin-angiotensin system and risk of severe acute respiratory syndrome coronavirus 2 infection date: 2020-05-22 journal: clin infect dis doi: 10.1093/cid/ciaa634 sha: doc_id: 272956 cord_uid: 0yumc7em exposure to agents acting on the renin-angiotensin system was not associated with a risk increase of covid-19 infection in 2 italian matched case-control studies, 1 nested in hypertensive patients and the other in patients with cardiovascular diseases or diabetes. angiotensin-converting enzyme 2 (ace2) is a functional receptor for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that facilitates the entry of the virus into the cells [1] . as agents inhibiting the renin-angiotensin-aldosterone system (raas) could increase the levels of ace2, recent studies have raised concern over the association between these agents and both sars-cov-2 infection and illness severity [2] [3] [4] [5] [6] . consequently, patients treated with ace inhibitors (aceis) or angiotensin ii receptor blockers (arbs), in particular those with diabetes or cardiovascular disease, should be considered at higher risk of developing severe coronavirus disease 2019 (covid-19) infection (cvi), and of experiencing unfavorable outcomes [2, 3] . therefore, it has been suggested that these patients should be carefully monitored or even have their therapy substituted with calcium channel blockers [3, 7] . however, the hypothesis of a link between agents inhibiting the raas and cvi is still under debate [7, 8] , and whether patients should switch to other antihypertensive therapies is controversial. research to clarify the role of raas blockade therapies and cvi is highly needed [7, 9] . in the first half of february 2020, the first cases of covid-19 infection were recorded in italy [10] , that, at present, is one of the leading countries in the world for both the number of diagnosed cases and for case fatality. diabetes, hypertension, and cardiovascular diseases, conditions that are frequently treated with aceis or arbs, are among the most prevalent comorbidities in these patients [11, 12] . as, to the best of our knowledge, a relationship between acei or arb treatments and increased risk of cvi has never been demonstrated [8] , the aim of the present study was to determine whether an association exists between therapies based on agents acting on the raas and cvi in 2 populations at greater risk of being diagnosed with sars-cov-2 infection: hypertensive patients and patients who were affected by a cardio-cerebrovascular disease. we conducted 2 population-based case-control studies nested in 2 cohorts built using administrative data from the piedmont region (4 400 000 inhabitants in northwest italy, with a high rate of cvi). the population is covered by an automated system of databases, which record, among others, all drugs dispensed from all regional pharmacies, all hospital discharges, and a regional register of persons with diabetes. from the beginning of the cvi epidemics a surveillance system was implemented to collect all cases identified by reverse-transcription polymerase chain reaction testing for sars-cov-2. these archives can be linked together by a unique anonymous identifier that is encrypted to protect the patient's privacy. from the regional surveillance system, we obtained a random sample of 1000 confirmed cases of cvi occurring in the first month of the epidemic from 22 february 2020 (beginning of the epidemic) to 23 march 2020, who were linked to the 2 populations, to include only cases within cdd or hy. from the same population sources, we randomly selected 5 controls for each case, matched for year of birth and sex. cvi cases were ineligible for resampling as controls. exposure to aceis or arbs the regional drug database was used to identify cases and controls who had been prescribed aceis or arbs at any time from 1 june to 31 october 2019 (last available month), considering atc codes c09a or c09b for aceis, and c09c or c09d for arbs. the risks of cvi associated with drug dispensation of aceis or arbs, considered both separately and together (to take into account possible switch from one medication to the other), were estimated by fitting conditional logistic regression models, expressed as odds ratios (ors) and corresponding 95% confidence intervals (cis). to take into account the dose of medication prescribed (ie, to explore for a dose-response relationship), the number of boxes prescribed was grouped into 4 classes (0, 1-6, 7-12, and > 12). all calculations were made using sas version 9.4 software (sas institute, cary, north carolina). out of 804 909 hy and 337 059 cdd subjects, we identified 316 and 171 cases, respectively, of cvi, who were matched with 1580 and 855 controls. in the hy population, 58.5% were male and the mean age was 71.4 years, whereas the mean age of the cdd population was 74.5 years and 78.4% were male. among the hy population, 68.0% of cases and 73.0% of controls had at least 1 prescription of agents acting on the raas, while among the cdd population, 54.4% of cases and 55.6% of controls received at least 1 prescription. in both populations, there were no differences between cases and controls by size of the municipality of residence (supplementary table) . in neither of the 2 populations was the prescription associated with the risk of sars-cov-2 infection. in the hy population, ors for aceis, arbs, and the combination of the 2 were, respectively, 0.89 (95% ci, .70-1.15), 0.90 (95% ci, .70-1.17), and 0.78 (95% ci, .60-1.02). in the cdd population, ors for aceis, arbs, and the combination of the 2 were, respectively, 0.92 (95% ci, .64-1.32), 1.03 (95% ci, .70-1.50), and 0.95 (95% ci, .68-1.34). there was no association with the level of exposition, thus excluding a dose-response relationship ( table 1) . as a sensitivity analysis, we considered that some controls could be unknown cases of cvi and thus a differential misclassification could have occurred. we repeated our analysis for the hy population exposed to aceis or arbs (or, 0.78) considering both 10% and 20% misclassification of controls. ors showed a slight change only, to 0.81 and 0.89, respectively. the question whether therapy with agents acting on the raas increases the susceptibility to cvi has raised concern among practitioners and citizens alike [2, 7] , with conflicting opinions [8] but no sound evidence [9] . the key message of our analysis is that no association, regardless of causality, could be determined. our study has several strengths. we used a nested casecontrol design enrolling both cases and controls within 2 unselected populations of subjects at increased risk of developing cvi. the consistency of ors in these 2 independent populations can be viewed as a confirmation of our results. the study was conducted using data retrieved from the regional surveillance system of confirmed cvi cases. all agents acting on the raas were included, and, as they are dispensed and reimbursed only by prescription, we are confident to have included all dispensations. moreover, the potential confounding due to differences in access to diagnosis is probably low, given no differences in the size of municipality. our study also has potential limitations. given the high prevalence of undiagnosed or unknown cases of cvi in the general population, differential misclassification of controls is likely to have occurred. however, even assuming 20% of misclassification, the ors changed slightly, confirming the absence of association. second, the use of a database of dispensed drugs rather than usage data might have overestimated the use aceis and arbs; in addition, not considering the last 4 months closer to the onset of the disease could have slightly affected the prevalence of drug users; however, it is unlikely that these would have affected cases and controls differently. finally, given the small sample size, an association between agents acting on the raas and cvi cannot be ruled out. despite the above limitations, at the time of this writing, our study is the first contribution to explore the question whether agents acting on raas increase the risk of being infected by sars-cov-2, and the first available evidence of safety of this class of drugs. we are aware that further epidemiological research is desirable, ideally considering larger cohort studies to confirm our first findings. however, as studies gathering a large population of confirmed cases require time and may be demanding, smaller but more timely studies can contribute to give answers to urgent public health questions. furthermore, as our study was limited to explore whether raas therapy increases the risk of sars-cov-2 infection, the question whether raas therapy worsens the outcomes of cvi patients remains to be clarified. in conclusion, on the basis of our study, as also mentioned by the major cardiology societies [13, 14] , there is no reason to modify current antihypertensive therapy. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. .893 abbreviations: acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker; cdd, circulatory disease/diabetes; ci, confidence interval; or, odds ratio. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor covid-19 and the cardiovascular system are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. what is the evidence? renin-angiotensin-aldosterone system inhibitors in patients with covid-19 rapid response: re: preventing a covid-19 pandemic: ace inhibitors as a potential risk factor for fatal covid-19 at present there is no evidence to abandon reninangiotensin system blockers is there an association between covid-19 mortality and the renin-angiotensin-system-a call for epidemiologic investigation case-fatality rate and characteristics of patients dying in relation to covid-19 in italy prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers hfsa/acc/aha statement addresses concerns acknowledgments. the authors thank the consorzio per il sistema informativo del piemonte for its essential contribution in prompt data gathering and data management, and dr chiara pasqualini of the regional unit for infective diseases, azienda sanitaria locale alessandria, for her valuable contribution in collecting and accessing data of the regional surveillance system.potential conflicts of interest. key: cord-022597-9b1a8cri authors: nan title: hematopoietic tumors date: 2009-05-15 journal: withrow & macewen's small animal clinical oncology doi: 10.1016/b978-072160558-6.50034-4 sha: doc_id: 22597 cord_uid: 9b1a8cri nan the etiology of canine lymphoma is largely unknown and likely multifactorial; however, current investigations are shedding significant light on the subject. recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, have been and currently are being applied to investigations of chromosomal aberrations in dogs with lymphoma. [10] [11] [12] breen's group has documented gain of canine chromosomes 13 and 31 and loss of chromosome 14 as the most common aberrations in a group of 25 cases analyzed. 11 the recent publication of the canine genome and the commercial availability of canine gene microarrays (e.g., genechip canine genome 2.0 array; affymetrix santa clara, california) certainly will lead to advances in our understanding of the genetic events in lymphoma in the very near future. 13 several genetic predispositions have been reported for a pedigree of bull mastiffs, 14 a group of related otter hounds, a family of rottweilers, and a breeding pair of unrelated scottish terriers. 15 germ line and somatic genetic mutation in the p53 tumor suppressor gene (see chapter 2) and the n-ras gene have been documented in bull mastiffs and in a dog with lymphoma, respectively. [16] [17] [18] in addition, differences in the prevalence of immunophenotypic subtypes of lymphoma among different breeds have been shown to indicate heritable risks. 19,19b epigenetic modifications also have been investigated in dogs with lymphoma; deoxyribonucleic acid (dna) hypomethylation (see chapter 1, section a) was a feature of neoplastic cells in most lymphoma cases and in one third of the leukemia cases investigated and likely is involved in malignant transformation of lymphoid cells. 20 in humans, characteristic chromosomal abnormalities are being described with increasing frequency as more precise banding and other high-resolution techniques are applied. chromosomal aberrations are nonrandom in human lymphoma, and several aberrations serve as markers for various subtypes of lymphoma. in addition, several oncogenes that may play a role in the pathogenesis of lymphoma have been detected based on the identification of cytogenetic abnormalities. [21] [22] [23] chromosomal aberrations also have been reported in canine lymphoma. [10] [11] [12] 24 a study of 61 dogs with lymphoma demonstrated a treatment advantage in dogs with trisomy of chromosome 13 (25% of the dogs studied), as evidenced by an increase in duration of the first remission and overall survival time. 24 as our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various canine tumor types, and some associated with lymphoma have been identified. altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and death-pathway alterations are common in human lymphomas and likely are also involved in the dog. as mentioned earlier, n-ras and p53 aberrations, although rare in dogs, have been implicated in some dogs with lymphoma. [16] [17] [18] [25] [26] [27] telomerase activity (see chapter 14, section d) also has been documented in canine lymphoma tissues. [28] [29] [30] alterations in cellular death-pathways, particularly the bcl-2 family of proapoptotic and antiapoptotic governing molecules, have been implicated in human non-hodgkin's lymphoma 31 and currently are under investigation in canine lymphoma. the hypothesis that a retrovirus may be involved in the pathogenesis of canine lymphoma has not been confirmed. 32 however, viral particles with properties similar to those of retroviruses have been identified in short-term cultures of canine lymphoma tissue. [33] [34] [35] [36] in physician-based oncology, a direct association has been made between helicobacter infections and the development of gastric lymphoma. 37 although this has not been shown definitively in dogs, evidence indicates that helicobacter infection in laboratory beagles results in gastric lymphoid follicle formation, which is considered a precursor of mucosa-associated lymphoid tissue (malt) lymphoma in humans. 38 some evidence has accumulated that implicates phenoxyacetic acid herbicides, particularly 2,4-dichlorophenoxyacetic acid (2,4-d) , in the development of human non-hodgkin's lymphoma. 39, 40 a population case control study of non-hodgkin's lymphoma in kansas farmers reported a twofold to sixfold higher risk in individuals who frequently mixed or applied herbicides (specifically 2,4-d). 41 a published, hospital-based case control study of dogs indicated that owners in households with dogs that developed malignant lymphoma applied 2,4-d herbicides to their lawn or employed commercial lawn care companies to treat their yard more frequently than owners of dogs without lymphoma. 42 the risk of canine lymphoma was reported to rise twofold (odds ratio, 1.3) with four or more yearly applications of 2,4-d. the results of this study since have drawn criticism, and three follow-up investigations have not validated the assertion of increased risk. [43] [44] [45] in another study, dogs exposed to lawn treatment within 7 days of application were more than 50 times more likely to have urine levels of 2,4-d of 50 µg/l or higher. 46 the highest concentration was noted 2 days after application. in a study of 28 dogs with lymphoma, the tumors of 20 dogs with known exposure to 2,4-d were analyzed using polymerase chain reaction (pcr) technology for cellular n-ras oncogene mutations. 47 the ras genes influence cell proliferation and may induce differentiation through signal transduction pathways. mutation in the ras genes results in ras proteins that promote cell growth. one dog in the series showed a mutation of n-ras, indicating that such mutations are uncommon in canine lymphoma, a finding similar to that for humans with lymphoma. 47 in an environmental case control study performed in europe, two variables, residency in industrial areas and use of chemicals (defined as paints or solvents) by owners, modestly increased the risk of lymphoma; however, no link was found between the use of pesticides and risk. 48 a weak association between lymphoma in dogs and exposure to strong magnetic fields was observed in a preliminary epidemiologic study. 49 in this hospital-based, case control study, dogs categorized as having high or very high exposure had an increased risk of lymphoma (odds ratio, 1.8). more thorough studies are necessary to evaluate this association further. impaired immune function has been identified in dogs with lymphoma. 50, 51 immune system alterations in the dog (e.g., immune-mediated thrombocytopenia), independent of age and gender, have been associated with a higher risk of subsequent development of lymphoma compared to the normal population. 52, 53 additional evidence for the role of the immune system in the development of lymphoma comes from observation in human transplantation patients. individuals with immunosuppression have a higher risk of lymphoreticular cancer, 54, 55 and organ transplant patients have a higher incidence of non-hodgkin's lymphoma. 56 a case of lymphoma that developed in a dog after treatment with cyclosporine, although only one case, supports a link to immunosuppressive therapy in the species. 57 the classification of malignant lymphoma in dogs can be distinguished on the basis of anatomic location, histologic criteria, and immunophenotypic characteristics. the most common anatomic forms of lymphoma, in order of decreasing prevalence, are the multicentric, craniomediastinal, gastrointestinal, and cutaneous forms. primary extranodal forms, which can occur in any location outside the lymphatic system, include the eyes, central nervous system (cns), bone, testes, bladder, heart, and nasal cavity. eighty percent of dogs with lymphoma develop the multicentric form, which is distinguished by the presence of superficial lymphadenopathy (figure 31-1) . 58 lymph node enlargement usually is painless, rubbery, and discrete and may be localized initially to the mandibular and prescapular nodes. most animals are asymptomatic at the time of presentation, but approximately 20% to 40% have a history of weight loss, lethargy, anorexia, and febrile episodes. 59, 60 diffuse pulmonary infiltration also may be seen in 27% to 34% of affected dogs, as detected by radiographic changes (figure 31 -2). [61] [62] [63] [64] based on bronchoalveolar lavage, the actual incidence of lung involvement may be higher. 62, 65 hepatosplenomegaly is the most common manifestation of abdominal involvement and usually is associated with an advanced stage of multicentric disease. the alimentary form of lymphoma is much less common, usually accounting for 5% to 7% of all canine lymphomas. 58 this form is reported to be more common in male dogs than female dogs, 8 similar to observations in humans. 66 dogs with infiltrative disease of the intestinal tract show weight loss, anorexia, panhypoproteinemia, and evidence of malabsorption. 67, 68 primary gastrointestinal (gi) lymphoma in dogs usually occurs multifocally and diffusely throughout the submucosa and lamina propria of the small intestine, with frequent superficial ulceration and occasional transmural infiltration of the serosa. lymphocytic-plasmacytic inflammation can be seen adjacent to or distant from the primary tumor. 67 pathologically, some of these neoplasms may resemble plasma cell tumors, and aberrant production of immunoglobulins may occur. histopathologically, distinguishing between gastrointestinal lymphoma and lymphocytic-plasmacytic enteritis (lpe) can be difficult. 67 some have suggested that lpe may be a prelymphomatous change in the gi tract. a syndrome of immunoproliferative intestinal disease characterized by lymphocytic-plasmacytic enteritis has been described in basenjis, which subsequently develop gastrointestinal lymphoma. 69 in addition, plasma cell-rich areas with heterogeneous lymphomatous infiltration may resemble lesions of lpe. only a few reports specifically identify the immunophenotype of the lymphocyte subpopulations in alimentary lymphoma. historically, it was presumed that they most likely originate from b cells; however, recent evidence suggests that most gastrointestinal lymphomas in dogs originate from t cells. 70 the boxer and shar-pei breeds appear to be overrepresented in alimentary lymphoma and are reported to have morphologic features (epitheliotropism) consistent with t-cell disease. 71 lateral thoracic radiograph of a dog with diffuse interstitial infiltration with lymphoma secondary to multicentric lymphoma. the mediastinal form of lymphoma occurs in approximately 5% of cases. 58 this form is characterized by enlargement of the craniomediastinal lymph nodes or the thymus, or both ( figure 31-3) . however, as previously noted, 20% of dogs with multicentric lymphoma have radiographic evidence of craniomediastinal lymphadenopathy. 63 hypercalcemia is reported to occur in 10% to 40% of dogs with lymphoma and is most common with the mediastinal form. 72, 73 in a study of 37 dogs with lymphoma and hypercalcemia, 16 (43%) had mediastinal lymphoma. 74 the mediastinal form in dogs is most commonly associated with a t-cell phenotype. 60, 75 cutaneous lymphoma can be solitary or more generalized and usually is classified as epitheliotropic (mycosis fungoides) or nonepitheliotropic. [76] [77] [78] [79] [80] [81] [82] [83] [84] cutaneous lymphoma may also involve the oral mucosa, 76 and extracutaneous involvement can occur, most often in the lymph nodes, spleen, liver, and bone marrow. canine epitheliotropic cutaneous lymphoma is the most common form of cutaneous lymphoma and usually originates from t cells, 85 similar to its development in humans. in dogs the t cells are cd8+ cells, whereas in humans they are mostly cd4+ cells. 86 a rare form of cutaneous t-cell lymphoma is characterized by generalized skin involvement with evidence of circulating malignant t cells in the peripheral blood. these lymphocytes usually are large (15 to 20 mm in diameter) and have folded, grooved nuclei. in humans this is called sézary syndrome, 87 which also has been reported in dogs and cats. 79, 80, 88, 89 b-cell cutaneous lymphomas usually spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis. hepatosplenic lymphoma is a relatively uncommon, distinct presentation in the dog marked by a lack of peripheral lymphadenopathy in the face of hepatic, splenic, and bone marrow infiltration with malignant lymphocytes, usually of t-cell origin. biologically, this form of lymphoma is extremely aggressive and poorly responsive to therapy. in humans the tumor usually is composed of γδt cells (i.e., t cells that express the γδt-cell receptor), and this immunophenotype has been confirmed in at least one dog in the veterinary literature. [90] [91] [92] intravascular (angiotrophic, angioendotheliomatosis) lymphoma is a distinct form of lymphoma defined as proliferations of neoplastic lymphocytes within the lumen and wall of blood vessels in the absence of a primary extravascular mass or leukemia. it has been reported several times in the veterinary literature, and in most cases it involves the central and peripheral nervous system (including the eye). [93] [94] [95] [96] [97] [98] the b-cell immunophenotype is most common in humans; however, in most reported cases in dogs, the origin is either t cell or null cell (neither b nor t cell), although one case of a b-cell phenotype has been reported. lymphomas arise from a clonal expansion of lymphoid cells with distinctive morphologic and immunophenotypic features. many histologic systems have been used to classify non-hodgkin's lymphoma (nhl) in humans, and some of these have been applied to lymphoma in the dog and other species. [99] [100] [101] [102] [103] [104] [105] [106] [107] the national cancer institute (nci) of working formulation and the updated kiel system have been adapted to canine tumors with some success (tables 31-1 and . the world health organization (who) also publishes a histologic classification scheme, which uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic tumors in domestic animals. 107 this system incorporates both histologic and immunohistologic criteria (b-and t-cell immunophenotype). the clinical relevance of this system is likely to be high; however, it awaits further investigation. more recently, the oncology committee of the american college of veterinary pathologists (acvp) has established a lymphoma subcommittee to formulate a classification system for lymphoma that has clinical relevance. the subcommittee has obtained information from an international group of veterinary oncologists and pathologists, and its report is expected to be completed by 2008. the working formulation (wf) was developed to allow investigators to "translate" among the numerous classification systems so that clinical trials could be compared in humans. most of the larger compilations agree that most canine lymphomas are intermediate or high grade; however, diffuse immunoblastic forms appear to predominate in the united states, whereas the follicular large cell variations predominate in europe. a comparison of european and american classifications is warranted based on this discrepancy. the wf categorizes tumors according to pattern (diffuse or follicular) and cell type (e.g., small cleaved cell, large cell, immunoblastic), but it does not include information about the immunophenotype of the tumor. 103 the wf subtypes are related to the biology of the tumor and patient survival. the updated kiel classification includes the architectural pattern, morphology (centroblastic, centrocytic, or immunoblastic), and immunophenotype (b cell or t cell) of the tumor cells. 102 in both systems, the tumors then can be categorized as low-grade, intermediate-grade, or high-grade malignancies. low-grade lymphomas composed of small cells with a low mitotic rate typically progress slowly and are associated with long survival times but are incurable. high-grade lymphomas with a high mitotic rate progress rapidly but are more likely to respond to chemotherapy and, in humans, are potentially curable. several features of canine lymphomas become apparent when the wf or updated kiel classification is applied. the most striking difference between canine and human lymphomas is the scarcity of follicular lymphomas in the dog. [108] [109] [110] [111] some diffuse lymphomas in the dog initially may be follicular, but these may progress to the more aggressive, diffuse form by the time of diagnostic biopsy. only a small percentage of canine lymphomas (5.3% to 29%) are considered lowgrade tumors. 60, 104, 105, 112 most low-grade small cell lymphomas are t cell in origin. 105 high-grade lymphomas occur frequently if the diffuse large cell lymphomas, classified as intermediategrade in the wf, are considered high-grade, as in the updated kiel classification (in which they are labeled diffuse centroblastic lymphomas). canine lymphoblastic lymphomas are uncommon. 72, 104 most high-grade lymphomas are of b-cell origin. 105 however, a documented difference exists in the prevalence of the various lymphoma immunophenotypes based on breed. 19 for example, cocker spaniels and doberman pinschers are more likely to develop b-cell lymphoma, boxers are more likely to have t-cell lymphoma, and golden retrievers appear to have an equal likelihood of b-and t-cell tumors. to be clinically useful, these classification systems in the end must yield information about response to therapy, maintenance of remission, and survival. some studies suggest that the subtypes in the wf can be correlated with survival, and the kiel system may be useful for predicting relapse. 113, 114 in most studies, high-grade lymphomas show a complete response to chemotherapy significantly more often than low-grade tumors. however, dogs with low-grade tumors may live a long time without aggressive chemotherapy. dogs with t-cell lymphomas have shown a lower rate of complete response to chemotherapy and shorter remission and survival times than dogs with b-cell tumors. 60, 75, 112, 113 furthermore, t-cell lymphomas tend to be associated with hypercalcemia. [115] [116] [117] in the veterinary literature, 60% to 80% of lymphoma cases in dogs are b-cell lymphoma; t-cell lymphomas account for 10% to 38%; mixed b-and t-cell disease accounts for as many as 22%; and null cell tumors (i.e., neither b-cell nor t-cell immunoreactive) represent fewer than 5%.* the development of monoclonal antibodies to detect specific markers on canine lymphocytes has made immunophenotyping of tumors in dogs routinely available in many commercial laboratories. such techniques also can be performed on paraffin-embedded samples and on cytologic specimens obtained by fine-needle aspiration. [118] [119] [120] [121] [122] the rappaport classification system, proposed in 1956 for human nhl, described the architectural pattern (follicular or diffuse) and the cytologic features (well differentiated, poorly differentiated, or histiocytic) of the tumors. 99, 123 the term histiocytic was applied to tumors in which most of the lymphocytes had larger diameters, more vesicular nuclei, and more prominent nucleoli than those of lymphocytic or undifferentiated lymphomas; it also was used because the malignant cells have some morphologic features of benign histiocytes. however, immunophenotyping has failed to document a biologic relationship between these cells and true histiocytes, therefore the term now is largely considered a misnomer. furthermore, this subgroup of histiocytic lymphomas included tumors with different morphologic and immunophenotypic features. the rappaport classification has not been useful in providing prognostic information or in guiding therapy in dogs with lymphoma because of the low number of follicular tumors in dogs, the problematic "histiocytic" subgroup, and the failure to account for different morphologic and immunologic cell types. 100 one criticism of these classification systems is that they fail to include extranodal lymphomas as a separate category. although differences between nodal and extranodal tumors in biologic behavior and prognosis are well recognized, comparative information about the histogenesis of these tumors has been lacking. for example, in humans, small cell lymphomas arising from malt are composed of cells with a different immunophenotype from that of other small cell lymphomas (i.e., malt lymphomas typically are negative for both cd5 and cd10). 124-126 except for cutaneous lymphoid neoplasms, detailed characterization of extranodal lymphomas in dogs has not been done. although cutaneous lymphoma is a heterogeneous group of neoplasms that includes an epitheliotropic form resembling mycosis fungoides and a nonepitheliotropic form, most cutaneous lymphomas have a t-cell phenotype. 85, 127 to summarize, it is important to determine the histologic grade of canine lymphomas as low (small lymphocytic or centrocytic lymphomas) or intermediate to high (diffuse large cell, centroblastic, and immunoblastic lymphomas). furthermore, determining the immunophenotype of the tumor provides useful information. response rates to chemotherapy are better in animals with b-cell tumors and intermediate-to highgrade lymphomas. dogs with low-grade lymphomas can have long survival times without aggressive therapy. the clinical signs associated with canine lymphoma are variable and depend on the extent and location of the tumor. multicentric lymphoma is the most common form (80%), and generalized painless lymphadenopathy (see figure 31 -1) is the most consistent finding. in addition, hepatosplenomegaly and bone marrow involvement are common. most dogs with multicentric lymphoma do not have dramatic signs of systemic illness (who substage a) (box 31-1), however, a large array of nonspecific signs can occur, such as anorexia, weight loss, vomiting, diarrhea, emaciation, ascites, dyspnea, polydipsia, polyuria, and fever (who substage b). 59, 60, [128] [129] [130] dogs with t-cell lymphoma are more likely to have constitutional signs (i.e., substage b). 118 polydipsia and polyuria are particularly evident in dogs with hypercalcemia of malignancy. dogs also may have a history of or clinical signs related to blood dyscrasias secondary to marked tumor infiltration of the bone marrow (myelophthisis) or paraneoplastic anemia, thrombocytopenia, or neutropenia. these signs could include fever, sepsis, anemia, and hemorrhage. dogs with gastrointestinal or alimentary lymphoma usually have nonspecific gi signs, such as vomiting, diarrhea, weight loss, and malabsorption. [66] [67] [68] the mesenteric lymph nodes, spleen, and liver may be involved. the mediastinal form of lymphoma is characterized by enlargement of the craniomediastinal structures or thymus or both (see figure 31 -3, a), and clinical signs are associated with the extent of disease or polydipsia and polyuria from hypercalcemia. these patients commonly have respiratory distress caused by a space-occupying mass and pleural effusion, exercise intolerance, and possibly regurgitation. dogs with mediastinal lymphoma also may have precaval syndrome, characterized by pitting edema of the head, neck, and forelimbs secondary to tumor compression or invasion of the cranial vena cava (figure 31-4) . signs in dogs with extranodal lymphoma depend on the specific organ involved. cutaneous lymphoma usually is generalized or multifocal. [76] [77] [78] [79] [80] [81] [82] [83] [84] tumors occur as nodules, plaques, ulcers, and erythremic or exfoliative dermatitis. epitheliotropic t-cell lymphoma (e.g., mycosis fungoides) has a chronic clinical course with three apparent clinical stages. initially, scaling, alopecia, and pruritus are seen ( figure 31 -5, a). as the disease progresses, the skin becomes more erythematous, thickened, ulcerated, and exudative. the final stage is characterized by proliferative plaques and nodules with progressive ulceration (figure 31-5, b) . oral involvement also may occur, which can appear as multicentric, erythematous, plaquelike lesions or nodules on the gums and lips ( figure 31 -5, c). 76 dogs with primary cns lymphoma may have either multifocal or solitary involvement. 131-133 seizures, paralysis, and paresis may be noted. ocular lymphoma is characterized by infiltration and thickening of the iris, uveitis, hypopyon, hyphema, posterior synechia, and glaucoma ( figure 31-6 ). 134 in one study of 94 cases of canine multicentric lymphoma, 37% had ocular changes consistent with lymphoma, and in a series of 102 cases of uveitis in dogs, the condition occurred secondary to lymphoma in 17% of the cases. 135, 136 anterior uveitis was most often seen in advanced stage disease (stage v). dogs with intravascular lymphoma usually have signs related to cns, peripheral nervous system (pns), or ocular involvement, [93] [94] [95] [96] [97] [98] including paraparesis, ataxia, hyperesthesia, seizures, blindness, lethargy, anorexia, weight loss, diarrhea, polyuria, polydipsia, and intermittent fever. dogs with pure hepatosplenic lymphoma usually have nonspecific signs such as lethargy, inappetence, and weakness. the differential diagnosis of lymphadenopathy depends on the dog's travel history (i.e., relative to infectious disease) and the size, consistency, and location of the affected lymph nodes. other causes of lymphadenopathy include bacterial and viral infections, parasites (toxoplasma and leishmania spp.), rickettsial organisms (salmon poisoning, ehrlichia sp.), and fungal agents (blastomyces and histoplasma spp.). the potential for hypercalcemia to accompany systemic hormone-like substance, parathyroid hormone-related peptide (pthrp), elaborated by neoplastic cells; however, it also can be related to the production of several other humoral factors, including interleukin-1 (il-1), tumor necrosis factor-alpha (tnf-α), transforming growth factor-beta (tgf-β), and vitamin d analogs (e.g., 1,25-dihydroxyvitamin d). 125,141-144 several investigators have reported that hypercalcemia in dogs with lymphoma is most commonly associated with t-cell lymphoma. 60, 113, 117, 118 other paraneoplastic syndromes that may be encountered include monoclonal gammopathies, neuropathies, and cancer cachexia. 145,146 for most animals suspected of having lymphoma, the diagnostic evaluation should include a thorough physical examination; complete blood count (cbc) with a differential cell count, including a platelet count; serum biochemistry profile; and urinalysis. ultimately, obtaining tissue or cytologic specimens for a definitive diagnosis is essential. a thorough physical examination should include palpation of all assessable lymph nodes, including those palpable by rectal examination; in the authors' experience, a significant proportion of dogs have rectal polyps consisting of aggregates of neoplastic lymphocytes. the mucous membranes should be inspected for pallor, icterus, petechiae, and ulceration, because these signs may indicate anemia or thrombocytopenia secondary to myelophthisis or immune-mediated disease or may be evidence of major organ failure or uremia. abdominal palpation may reveal organomegaly, intestinal wall thickening, or mesenteric lymphadenopathy. thoracic auscultation may reveal the presence of a mediastinal mass or pleural effusion or both. an ocular examination that includes funduscopic assessment may reveal abnormalities such as uveitis, retinal hemorrhage, and ocular infiltration in approximately one third to one half of dogs with lymphoma. 135, 136 complete blood count, biochemistry profile, and urinalysis anemia, the most common lymphoma-related hematologic abnormality, 137,138 usually is normochromic and normocytic (nonregenerative), consistent with anemia of chronic disease. however, hemolytic anemia may also occur, and regenerative anemias may reflect concomitant blood loss or hemolysis. in addition, if significant myelophthisis is present, the anemia may be accompanied by thrombocytopenia and leukopenia. in animals with anemia or evidence of bleeding, a reticulocyte count, platelet count, and coagulation studies also may be indicated. thrombocytopenia may be seen in 30% to 50% of cases, but bleeding is seldom a clinical problem. 138 (i.e., stage v disease) from primary lymphoblastic leukemia (discussed later) is important, because the prognoses are entirely different. hypoproteinemia is observed more often in animals with alimentary lymphoma. if the dogs has a high total protein or evidence of an increased globulin fraction on a chemistry profile, serum proteins should be evaluated by serum electrophoresis. monoclonal gammopathies have been reported to occur in approximately 6% of dogs with lymphoma. 145 serum biochemical abnormalities often reflect the anatomic site involved. in addition, approximately 15% of dogs with lymphoma are hypercalcemic (30% to 40% of those with mediastinal involvement and approximately 35% of those with t-cell lymphomas). 74, 118, 147 in cases of hypercalcemia of unknown origin, lymphoma should always be considered high on the differential disease list, and diagnostics directed at this possibility should be undertaken (see chapter 5) . the presence of hypercalcemia also can serve as a marker for response to therapy. elevations in serum urea nitrogen and creatinine can occur secondary to renal infiltration with tumor, hypercalcemic nephrosis, or prerenal azotemia from dehydration. increases in liver-specific enzyme activity or bilirubin concentrations may result from hepatic parenchymal infiltration. serum globulin elevations, usually monoclonal, occur infrequently with b-cell lymphoma. a urinalysis is part of the minimum database used to assess renal function and the urinary tract. for example, isosthenuria and proteinuria in the absence of an active sediment may indicate renal disease, and hematuria may result from a hemostatic abnormality. it is important to remember that isosthenuria in azotemic dogs with hypercalcemia does not necessarily indicate renal disease, because the high calcium levels interfere with tubular concentration capabilities through disruption of antidiuretic hormone (adh) control. abnormalities in serum levels of alpha fetoprotein, alpha-1 glycoprotein, zinc, chromium, iron, and endostatin also have been investigated in dogs with lymphoma. [149] [150] [151] [152] the clinical and biologic significance of these alterations has yet to be elucidated. morphologic examination of the tissue and cells that comprise the tumor is essential to the diagnosis of lymphoma. care should be taken to avoid lymph nodes from reactive areas (e.g., mandibular lymph nodes); the prescapular or popliteal lymph nodes are preferable. also, lymphoid cells are fragile, and in preparing smears of aspirated material, only gentle pressure should be applied in spreading the material on the slides. in most cases, a diagnosis of lymphoma can be made on evaluation of fine-needle aspirates of affected lymph nodes or other tissues. typically, most of the cells are large lymphoid cells (larger than neutrophils), and they may have visible nucleoli and basophilic cytoplasm ( figure 31-7 , a) or fine chromatin with indistinct nucleoli. because tissue architecture is not maintained in cytologic specimens, effacement of the lymph node or capsular disruption cannot be detected. therefore, marked reactive hyperplasia characterized by increased numbers of large lymphoid cells may be difficult to distinguish from lymphoma, and small cell lymphomas may have few cytologic clues that point to their malignancy. also, classification of lymphoma into the subcategories that comprise the low-, intermediate-, and high-grade forms, which has been attempted using the cytologic appearance and immunophenotypic analysis, 153 is performed most accurately on histologic sections. for accurate histopathologic evaluation, an entire lymph node, including the capsule, should be removed, placed in buffered formalin, and submitted to a pathologist. although needle core biopsies may be satisfactory, it is important to avoid crush artifact or inadequate sample size. most pathologists prefer whole node biopsies because they provide the maximum amount of information. effacement of the normal nodal architecture by neoplastic lymphocytes and capsular disruption are characteristic findings ( figure 31 -7, c and d). diagnostic ultrasonography and ultrasound-guided fineneedle aspiration or needle biopsy have been very useful for evaluation of involvement of the liver, spleen, or abdominal lymph nodes. [154] [155] [156] [157] [158] if possible, the diagnosis should be made by sampling peripheral nodes, avoiding percutaneous biopsies of the liver and spleen. however, if there is no peripheral node involvement, it is appropriate to biopsy affected tissues in the abdominal cavity. with alimentary lymphoma, an open surgical wedge biopsy of the intestine must be obtained, ideally without entering the intestinal lumen, and adequate tissue must be obtained; this is important because of the difficulty involved in differentiating lymphoma from lpe. endoscopic biopsies may be inadequate because only a superficial specimen is obtained; however, more aggressive endoscopic biopsy techniques combined with more accurate histopathologic assessments are improving the diagnostic yield of these less invasive techniques. 159 in many dogs with primary gastrointestinal lymphoma, an inflammatory, nonneoplastic infiltrate (e.g., lpe) may be misdiagnosed on biopsy specimens that are too superficial. cytologic examination of cerebrospinal fluid (csf), thoracic fluid, or aspirates of an intracavitary mass is indicated in animals with cns disease, pleural effusion, or an intrathoracic mass, respectively. in one study of dogs with cns involvement, csf analysis was diagnostic in seven of eight dogs. 131 the characteristics of the csf included an elevated nucleated cell count in the seven dogs, and 95% to 100% of the cells were atypical lymphoid cells. the csf protein concentration was higher in five of seven dogs, ranging from 34 to 310 mg/dl (the reference interval was less than 20 mg/dl). for cutaneous lymphoma, punch biopsies (3 to 4 mm) should be taken from the most representative and infiltrative, but not infected, skin lesions. staging procedures for cutaneous lymphoma vary, and the stage has shown no prognostic importance. 160 molecular techniques can be used to establish a diagnosis of lymphoma or to further characterize a tumor after the initial diagnosis has been made. tissues and cells from peripheral blood, lymph nodes, or other sites can be analyzed by histochemical and cytochemical, immunohistochemical and immunocytochemical, flow cytometric, and pcr techniques. for example, the tumor's immunophenotype (b cell or t cell), proliferation rate (e.g., expression of ki-67, proliferating cell nuclear antigen [pcna], and argyrophilic nucleolar organizer regions [agnors]), and clonality (pcr for antigen receptor gene rearrangement [parr]) can be determined. 60, 105, 113, [119] [120] [121] [122] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] the availability of such analyses is increasing, although currently only the immunophenotype consistently predicts the prognosis in dogs. part iv • specific malignancies in the small animal patient immunophenotyping typically is used to determine the type of cells that make up the lymphoma, but sometimes the technique is helpful for making the diagnosis of lymphoma. when a diverse population of lymphocytes is expected in a tissue, the presence of a homogeneous population of the same immunophenotype is supportive of a neoplastic process. the immunophenotype of a lymphocyte is identified by determining the expression of molecules specific for b cells (e.g., cd79a) and t cells (e.g., cd3). although tumor cells sometimes have morphologic characteristics that typify a particular immunophenotype, exceptions occur, and morphology cannot be used as the sole determinant of the type of lymphocyte. for example, in a series of nine high-grade t-cell lymphomas and leukemias in dogs, the cells had a plasmacytoid appearance, typically associated with b-cell lymphoma. 171 similarly, anatomic location does not always predict the immunophenotype. in a series of 44 cases of gastrointestinal lymphoma in dogs, often considered a neoplasm of b cells, the neoplastic cells were identified as t cells (cd3 positivity) in 75% of the cases. 70 for accurate determination of the immunophenotype, antibodies against lymphocyte markers are applied to tissue sections (immunohistochemistry), cytologic specimens (immunocytochemistry), or individual cells in a fluid medium (flow cytometry). flow cytometric evaluation of cells from needle aspirates also is feasible. 172 for t cells, markers include cd3 (pan t), cd4 (helper t), and cd8 (cytotoxic t); for b cells, the markers are cd79a (see figure 31 -7, b) and cd21. 173 interestingly, aberrant expression of cd molecules has been reported in canine lymphoma. in a study of 59 dogs with lymphoma, tumor cells from six dogs were positive for both t-and b-cell cd markers; however, a clonality assay (discussed later) revealed clonality for either the t-cell or the immunoglobulin receptor but not both. this indicates that in some cases of b-and t-cell lymphoma, the malignant cells may coexpress b-and t-cell cd markers. 119 antibodies against these molecules are used to determine the immunophenotype; however, they also have a potential use as a therapeutic modality if tumor cells could be targeted using these antibodies. histologic assessment of markers of multidrug resistance and apoptotic pathways (e.g., p-glycoprotein, p53, and bcl-2 proteins) currently are being evaluated in dogs with lymphoma. however, their significance requires further evaluation. 16, 163, [174] [175] [176] [177] clonality assay occasionally the diagnosis of lymphoma and the differentiation of malignant verses benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses are necessary to help confirm a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically should be derived from expansion of a single malignant clone characterized by a particular dna region unique to that tumor. for example, in a dog with t-cell lymphoma, all the malignant cells should have the same dna sequence for the variable region of the t-cell receptor (tcr) gene; likewise, in a dog with b-cell lymphoma, the tumor cells should have identical dna sequences in the variable region of the immunoglobulin receptor gene. conversely, in reactive lymphocytosis, the cells are polyclonal for their antigen receptors. using this knowledge, investigators have used polymerase chain reaction (pcr) pcr techniques to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in dogs ( figure 31-8) . [167] [168] [169] [170] uniform size of polymerase chain reaction (pcr) products as an indicator of clonality. each panel shows four pcr reactions on a single dna sample. the first lane (left) in each panel is a positive control that indicates that dna is present (any nonrearranged gene would be an appropriate target for this reaction). the middle two lanes represent two different reactions that amplify immunoglobulin cdr3, and the fourth lane shows tcrγ cdr3 amplification. the samples are separated on a polyacrylamide gel. a, lymph node aspirate from a normal dog. b, lymph node aspirate from a dog with histologically confirmed multicentric b-cell lymphoma. c, lymph node aspirate from a dog with histologically confirmed t-cell lymphoma. (from avery pr, avery ac: vet clin pathol 33:196-207, 2004.) in physician-based medicine, such assays of clonality are approximately 70% to 90% sensitive and have a false positive rate of approximately 5%, and recent studies report similar rates in the dog. 169 false negative and false positive results can occur with clonality assays. for example, cells from a dog with lymphoma may be negative for clonality if the clonal segment of dna is not detected with the primers used, if the malignant cells are natural killer (nk) cells (rare), or if the malignant cells are present in too low a frequency to be detected. 169 false positive results occur rarely with some infectious diseases, such as ehrlichiosis and lyme disease. in these cases a diagnosis should be made only after the results of all the diagnostic tests are considered, including histologic and cytologic evaluation, immunophenotyping, and clonality studies, in conjunction with the signalment and physical findings. molecular techniques, in addition to aiding the diagnosis, could also be useful in determining early recurrence, more accurate clinical staging, and so-called molecular remission rates, because they are more sensitive than standard cytologic assessment for the peripheral blood, bone marrow, and lymph nodes. after a diagnosis has been established, the extent of disease should be determined and correlated to the clinical stage of disease. the who staging system routinely used to stage dogs with lymphoma is presented in box 31-1. most dogs (more than 80%) are presented in advanced stages (stage iii or iv). some type of imaging and an assessment of bone marrow involvement may be indicated for staging. the degree to which thorough staging is implemented depends on three factors: whether the result will alter the treatment plan; whether relevant prognostic information will be gleamed; and whether the client needs to know. in addition, when different protocols are compared with respect to efficacy, consistent and similar staging systems should be used to avoid so-called stage migration, which results when one staging methodology is more accurate than another. 178 the effect of stage migration currently is being evaluated in veterinary patients with lymphoma, and until the concept has been thoroughly explored, the impact on the prognosis should be considered when different published outcomes are compared. a bone marrow aspirate or biopsy (from the proximal humerus or iliac crest) is indicated for complete staging and in dogs with anemia, lymphocytosis, peripheral lymphocyte atypia, or other peripheral cytopenia. in one study of 53 dogs with lymphoma, 28% had circulating malignant cells and were considered leukemic, whereas bone marrow examination indicated involvement in 57% of the dogs. 179 the presence of a few prolymphocytes and large lymphocytes with nucleoli in the circulation of dogs with lymphoma may indicate bone marrow involvement. it is important to remember that these cells also can be seen with gi parasitism, immune-mediated hemolytic anemia, and other immune-mediated diseases. recently, circulating tumor cells in dogs with stage iii lymphoma were identified using a clonality assay (parr). 168 the assay is more sensitive than routine microscopy in detecting malignant cells in circulation, but the correlation of these results with staging and prognosis has not been determined. bone marrow evaluation offers prognostically valuable information, but if the client is committed to treatment regardless of the stage of disease, it is not necessary. evaluation of thoracic and abdominal radiographs may be important in determining the extent of internal involvement. approximately 60% to 75% of dogs with multicentric lymphoma have abnormalities on thoracic radiographs; one third have evidence of pulmonary infiltrates, and two thirds have thoracic lymphadenopathy (sternal and tracheobronchial lymph nodes) and widening of the cranial mediastinum (see . [61] [62] [63] [64] craniomediastinal lymphadenopathy is detected in 20% of dogs with lymphoma. 63 abdominal radiographs reveal evidence of sublumbar (iliac) and/or mesenteric lymph node, spleen, or liver involvement in approximately 50% of cases. 63, 64 in the authors' practice, in typical cases of canine multicentric lymphoma, imaging is limited to thoracic radiographs because no prognostic difference exists between dogs with stage iii disease and those with stage iv disease (i.e., liver or spleen involvement); however, the presence of craniomediastinal lymphadenopathy is prognostically significant (see prognosis later in this section). if clinical signs attributable to abdominal disease are present, further imaging of the abdomen is warranted. in addition, as stated previously, abdominal ultrasonography can be important for obtaining ultrasound-guided intraabdominal samples for diagnosis. it is also useful for the diagnosis of gastrointestinal and hepatosplenic lymphoma. [90] [91] [92] 180, 181 advanced imaging modalities, including computed tomography (ct), magnetic resonance imaging (mri), and positron emission/computed tomography (pet/ct), are becoming more commonplace in veterinary practice, and their usefulness is only now being fully determined. [182] [183] [184] [185] [186] the therapeutic approach to a particular patient with lymphoma is determined by the stage and substage of disease, the presence or absence of paraneoplastic disease, the patient's overall physiologic status, the financial and time commitments of the client, and the client's level of comfort with regard to the likelihood of treatment-related side effects. because most canine lymphomas are intermediate-to high-grade tumors, histopathologic characterization has played a less important role in determining the optimal treatment. without treatment, most dogs with lymphoma die of the disease in 4 to 6 weeks. 145 with few exceptions, canine lymphoma is considered a systemic disease and therefore requires systemic therapy to achieve remission and prolonged survival. systemic chemotherapy continues to be the therapy of choice for canine lymphoma. in general, combination chemotherapy protocols are superior in efficacy to single agent protocols. single agent protocols, except for doxorubicin, have a lower response rate that is not as durable as combination chemotherapy. in rare cases in which lymphoma is limited to one site (especially an extranodal site), the animal can be treated with a local modality, such as surgery or radiation therapy, as long as the caregiver and clinician are committed to diligent re-evaluation to document subsequent systemic involvement. many chemotherapy protocols for dogs with lymphoma have been developed over the past 15 to 20 years (table 31-3) . 59, 61, 72, 128, [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] most complex combination protocols are modifications of chop protocols initially designed for human oncologic use. chop represents combinations of cyclophosphamide (c), doxorubicin (represented by the h, for hydroxydaunorubicin), vincristine (o, oncovin) and prednisone (p). conventional chemotherapy induces complete remission (cr) in approximately 60% to 90% of dogs, with median survival times of 6 to 12 months depending on the protocol used. approximately 20% to 25% of dogs live 2 years or longer after initiation of these protocols (figure 31-9) . response rates and the duration of response vary according to the presence or absence of prognostic factors discussed in the prognosis section. the cost to the owner depends on the drug or drugs selected, the size of the animal, the frequency of administration, and the laboratory tests needed to monitor toxicity. dogs that respond to chemotherapy and achieve complete remission usually are free of clinical signs associated with lymphoma and subsequently return to a very good quality of life. treating dogs with lymphoma is gratifying, because a high percentage have a complete response. most dogs tolerate chemotherapy well, and in our experience only a minority of dogs develop significant toxicity. studies assessing clients' perceptions of medical treatment for cancer in general and lymphoma in particular generally report a positive experience; most owners felt that the treatment was worthwhile, that it resulted in improvement in their pet's well-being, and that the animal's quality of life during treatment was good. 202,203 very few clients expressed regret about having the lymphoma treated with a multidrug protocol. with lymphoma, the fundamental goals of chemotherapy are to induce a complete and durable (longer than 6 months) first remission (induction), to reinduce remission when the tumor recrudesces (or the patient relapses) after achievement of a remission (reinduction), and finally to induce remissions when the cancer fails to respond to induction or reinduction using drugs not present in standard protocols (rescue). a previously unanswered question in the treatment of lymphoma was whether long-term maintenance chemotherapy was useful after an initial course of aggressive induction chemotherapy that lasted 6 months or less. long-term maintenance chemotherapy has been shown to be ineffective in humans with hodgkin's disease, nhl, and multiple myeloma. however, the initial induction course of chemotherapy in humans is much more aggressive than that used in veterinary patients. although no randomized studies have been performed to address the therapeutic benefit of long-term maintenance chemotherapy in dogs, comparisons of dogs treated with chop-based protocols in which all treatment was stopped after 6 months of induction therapy 129 were compared with sequentially treated, historical controls that received a nearly identical protocol that included long-term maintenance therapy. 60 the dogs that received the shortened, less expensive, no-maintenance protocol had comparable remission and survival durations and were more likely to achieve second remissions when they relapsed after completion of chemotherapy than their counterparts that received long-term maintenance therapy. other studies, although not prospective randomized trials, suggest that aggressive induction or discontinuous therapy (i.e., induction without maintenance) is as good as or superior to protocols that use an extended maintenance phase. 190, 193, 194, 200 these data, taken together, suggest that maintenance therapy is not necessary and indeed may be inappropriate for dogs with lymphoma that are treated with similar combination chemotherapy protocols. single agent chemotherapy with known activity for dogs with lymphoma agents include vinblastine, actinomycin-d, mitoxantrone, chlorambucil, methotrexate, dtic, 9-aminocamptothecin, ifosfamide, cytosine arabinoside, gemcitabine, lomustine, and dolastatin-10. 206-218 of these, cytosine arabinoside, ifosfamide, dolastatin-10, and gemcitabine appear to have only minimal activity. except for doxorubicin, induction with single agent chemotherapy does not result in durable remission durations compared with standard combination protocols. the efficacy of incorporating these newer drugs with single agent activity into standard combination protocols awaits further investigation. providing precise treatment recommendations for the wide variety of clinical settings of dogs with lymphoma is difficult, especially in light of the plethora of published combination drug protocols (see table 31 -3). because of the large and ever increasing number of protocols available, several factors should be considered and discussed with caregivers on a case-by-case basis in making the choice of protocol. these factors include the cost, time commitment, efficacy, toxicity, and experience of the clinician with the protocols in question. with the increased availability of generic drugs, protocols are becoming affordable to a larger segment of veterinary clients. in general, more complex combination chemotherapy protocols are more expensive, more time-consuming (i.e., requiring repeated office visits and closer monitoring), and more likely to result in toxicity than simpler, single agent protocols. however, as a general rule, more complex combination protocols result in longer remission and survival durations than single agent protocols. the earliest treatment protocol used in veterinary patients was a non-doxorubicin-based combination chemotherapy (i.e., cyclophosphamide, vincristine, and prednisone [cvp]), a relatively simple, easy protocol that is well-tolerated and results in a 60% to 70% cr rate and a median survival time of 6 to 7 months. 187,188 however, it has been clearly established that the standard of care combination protocols used in dogs with lymphoma include doxorubicin, and all are essentially variations on the chop protocols previously discussed and listed in table 31 -3. although many of these chop protocols include l-asparaginase, which is added either at initiation or at varying times throughout the protocol, several studies have confirmed that the addition of l-asparaginase in induction protocols does not result in clinically relevant increases in remission rate, speed of attaining remission, or first-remission duration; therefore its use is best reserved for rescue situations (discussed later). 128,190,219,220 regardless of the veterinary chop-based combination protocol used, they all generally result in an 80% to 90% cr rate with median survival times of 12 months. about 25% of dogs are long-term survivors (longer than 2 years), and some are cured. the chop protocol used by the authors at the time of publication (box 31-3) generally is well tolerated by dogs. this protocol does not have a maintenance therapy arm, and all treatments stop at 19 weeks if the animal is in complete remission. if client factors or other considerations preclude a chop-based protocol, single agent doxorubicin can be offered as an alternative, with the patient receiving five doxorubicin treatments (30 mg/m 2 given intravenously every 3 weeks). the expected complete response rate will range from 50% to 75% with an anticipated median survival time of 6 to 8 months. 128, 188, 189, 197 if financial or other client concerns preclude the use of more aggressive systemic chemotherapy, prednisone therapy alone (2 mg/kg given orally daily) often results in a short-lived remission of approximately 1 to 2 months. in these cases, it is important to inform clients that should they decide to pursue more aggressive therapy later, dogs with previous prednisone therapy are more likely to develop multiple drug resistance (mdr) while receiving single agent prednisone and to have shorter remission and survival durations with subsequent combination protocols. this is especially true after long-term prednisone therapy and in dogs that have experienced a recurrence while receiving prednisone. 195,221 therefore, the earlier a client opts for more aggressive therapy, the more likely it is that a durable response will result. a cbc should be performed before each chemotherapy treatment. a neutrophil count of at least 2000/ml and a platelet count of 50,000/ml should be present before the chemotherapeutic drugs are administered. if the neutrophil count is below 2000/ml, it is best to wait 5 to 7 days and repeat the cbc. if the count has risen above 2000 cells/ml, the drug can be safely administered. a caveat to these restrictions: in dogs presented before initiation of chemotherapy that have low neutrophil and platelet counts because of bone marrow effacement, myelosuppressive chemotherapy is instituted in the face of cytopenias to "open" the bone marrow and allow counts to normalize. with regard to breeds likely to have mdr-1 gene mutations (e.g., collies; see chapter 11) and which therefore are at risk for serious, unexpected chemotherapy toxicity, the authors initiate a chop protocol out of sequence, beginning with non-mdr-1-associated drugs, such as cyclophosphamide. this ensures treatment of the lymphoma while allowing sufficient time for analysis of mdr-1 gene mutations (see chapter 11) before mdr-1-associated drugs are initiated. eventually, most dogs that achieve a remission relapse or experience a recrudescence of lymphoma. this usually represents the emergence of tumor clones that are inherently more resistant to chemotherapy than the original tumor; so-called mdr clones that either were initially drug resistant or became so after exposure to selected chemotherapeutic agents. 175, [222] [223] [224] evidence suggests that in recurrent lymphoma in dogs, tumor cells are more likely to express the mdr-1 gene that encodes the protein transmembrane drug pump often associated with multiple drug resistance. 174, 175, 224 other causes of relapse after chemotherapy include inadequate dosing and frequency of administration of chemotherapy and failure to achieve high concentrations of chemotherapeutic drugs in certain sites, such as the central nervous system. at the first recurrence of lymphoma, reinduction should be attempted first by reintroducing the induction protocol that was successful initially. special attention must be given to the cumulative dose of doxorubicin that will result from reinduction; also, a baseline cardiac assessment, the use of cardioprotectants, alternative drug choices, and client education should all be considered. in general, the likelihood of a response and the length of the reinduction are half those seen in the initial therapy; however, some animals enjoy long-term reinductions, especially if the patient had completed the initial induction regimen and was off chemotherapy when the relapse occurred. a reinduction rate of nearly 90% can be expected in dogs that have completed chop-based protocols and then relapse while off therapy. 129 if reinduction fails or the dog does not respond to the initial induction, use of so-called rescue agents or rescue protocols can be attempted. these are drugs or drug combinations that typically are not found in the standard chop protocol and are withheld for use in cases of drug resistance. the most common rescue protocols most commonly used in dogs include single agent or combination use of actinomycin d, mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), a doxorubicin/dacarbazine combination, lomustine (ccnu), l-asparaginase, and the combination mechlorethamine, vincristine (oncovin) procarbazine, and prednisone (mopp). 176, 206, [209] [210] [211] [225] [226] [227] [228] [229] overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable, and median response times of 1.5 to 2.5 months are typical. a small number of animals will enjoy longer rescue durations. despite the plethora of published chemotherapy protocols for dogs with lymphoma, it appears that veterinary medicine has achieved about as much as it can from the currently available chemotherapeutic drugs in standard settings, because no dramatic improvement has been made in the 12-month median survival "wall" and the 25% 2-year survival rate. advances in remission and survival durations await the development of new methods of delivering or targeting old chemotherapeutic drugs and the development of new generation chemotherapeutics or novel nonchemotherapeutic treatment modalities. mechanisms of avoiding or abrogating mdr, enhancing tumor apoptosis (programmed cell death), and targeting treatments with immunoconjugates (i.e., antibody-directed therapies), as well as novel immunomodulatory therapies, are all active areas of investigation in both human and veterinary medicine. drug resistance can develop in cancer patients after exposure to selected chemotherapeutic agents and often is associated with expression of p-glycoprotein (see chapter 11) . p-glycoprotein acts as a drug efflux pump that actively extrudes drugs from tumor cells, preventing a cytotoxic drug from reaching the cellular site of action. mdr and p-glycoprotein are controlled by the mdr-1 gene. 222,223,230 mdr has been reported in canine lymphoma after treatment with chemotherapy. 174, 224, 231 in one study, expression levels of messenger ribonucleic acid (mrna) that encodes the canine mdr-1 gene was characterized in canine cell lines and lymphomas. 231 although expression of mdr-1 mrna correlated with in vitro drug sensitivity, it did not correlate with in vivo doxorubicin sensitivity in dogs with lymphoma in this study. methods of increasing the time that tumor cells are exposed to chemotherapeutic drugs theoretically should enhance tumor killing. these methods could intralymphatic (il) administration of an autologous killed lymphoma tumor cell vaccine has been done in dogs in which remission was achieved with a combination chemotherapy protocol. in a study that compared 28 dogs that received chemotherapy and then il vaccination with 30 dogs that received chemotherapy alone, the median remission times were 98 and 28 days, respectively (p < 0.024). 238 unfortunately, the survival times for the two groups were not significantly different (305 days for chemotherapy plus il vaccine and 184 days for chemotherapy alone). dogs that responded had significant increases in specific antibody to lymphoma antigens compared to those that did not respond. another immunotherapy approach involved mab-231, a murine-derived anticanine monoclonal antibody (igg2a). this antibody mediates antibody-dependent cellular cytotoxicity (addc) and complement-mediated cellular cytotoxicity (cmcc), 239,240 and it prevented outgrowth of canine lymphoma xenografts in nude mice. 241 in a noncontrolled clinical study, 215 dogs were treated with chemotherapy (l-asparaginase, vincristine, cyclophosphamide, and doxorubicin). 242 after two cycles of chemotherapy, 174 dogs had achieved complete remission and were treated with an intravenous infusion of mab-231 daily for 5 days. the median survival time of the dogs treated with mab-231 was 493 days. the 2-year survival rate was 15.6%. the median number of chemotherapy cycles in the first year was three, and the median number of mab-231 cycles was 1.5. the mab-231 antibody went off the commercial market in the mid-1990s. definitive randomized trials to determine its effectiveness are still lacking. most dogs with lymphoma have the multicentric form and need systemic chemotherapy for effective treatment of the disease. however, surgery has been used to treat solitary lymphoma (early stage i) or solitary extranodal disease. in such cases, careful staging is necessary to rule out multicentric involvement before the local disease is treated. the benefit of surgical removal of the spleen in dogs with massive splenomegaly remains unclear. 243,244 in a published report, 16 dogs with lymphoma underwent splenectomy for a massive spleen and subsequently were treated with chemotherapy. within 6 weeks of splenectomy, five of the 16 dogs died of disseminated intravascular coagulation (dic) and sepsis. the remaining 11 dogs had a complete response rate of 66%, and seven of these, which were followed until their death, had a median survival time of 14 months. splenectomy should be considered only if the lymphoma is in remission in other sites and if the splenic enlargement is caused by lymphoma that is not responsive to chemotherapy. in dogs with lymphoma, splenectomy also can be considered as a treatment for uncontrolled hemolytic anemia and persistent thrombocytopenia. the role of radiotherapy for the management of lymphoma in dogs currently is under investigation. the use of whole body irradiation without bone marrow transplantation has yielded poor results. however, radiation therapy may be indicated in selected cases. 245-248 the indications are: • local stage i or stage ii disease (i.e., nasal lymphoma, cns lymphoma) • palliation of local disease (e.g., mandibular lymphadenopathy, rectal lymphoma, mediastinal lymphoma accompanied by precaval syndrome, localized bone involvement) • whole body irradiation combined with bone marrow or stem cell transplantation • staged half-body irradiation after chemotherapyinduced remission the use of staged half-body irradiation after achieving remission with induction chemotherapy has undergone preliminary investigation as a form of consolidation or maintenance. 247, 248 in these investigations, radiation therapy is delivered either to the cranial or the caudal half of the dog's body in two consecutive 4 gray daily fractions; then, after a rest of 3 or 4 weeks, the other half of the body is irradiated in a similar fashion. although these preliminary investigations were not randomized, their results suggest that radiation therapy, either after completion of chemotherapy or sandwiched between chemotherapy sessions, when dogs are in either complete or partial remission, is safe and should be investigated more extensively to determine whether a significant therapeutic gain could be realized. in general, the veterinary literature offers little information on the treatment of the various extranodal forms of lymphoma in dogs, therefore the ability to predict outcome is limited. the authors recommend that, after extensive staging, local therapies (e.g., surgery, local radiation therapy) should be used in cases in which disease is localized to a solitary site. if multiple extranodal sites are involved or if they are part of a more generalized process, systemic chemotherapy should be given. most dogs with alimentary lymphomas have diffuse involvement of the intestinal tract. involvement of local lymph nodes and the liver is common. chemotherapy in dogs with diffuse disease has been reported to be unrewarding for the most part. 67 however, in the authors' experience, more aggressive, chop-based protocols (which are used extensively for multicentric lymphoma in dogs) have resulted in several cases of durable remission for alimentary lymphoma. solitary alimentary lymphomas are rare in the dog, but if the tumor is localized and can be removed surgically, the results with or without follow-up chemotherapy can be encouraging. most cns lymphoma in dogs results from metastasis of multicentric lymphoma. however, primary central nervous system lymphoma (pcnsl) has been reported. 131, 132 if tumors are localized, local radiation therapy should be considered. few studies have reported on the use of chemotherapy. in one study, cytosine arabinoside (ara-c) at a dosage of 20 mg/m 2 was given intrathecally by bolus injection after withdrawal of an equal volume of csf. 131 the dose was diluted in 2 to 4 ml of lactated ringer's solution and was injected twice weekly for a total of six treatments. this treatment was combined with systemic chemotherapy and cns irradiation. overall, the response rates were low and of short duration (several weeks to months). the treatment of cutaneous lymphoma depends on the extent of disease. solitary lesions may be treated with surgical excision or radiation therapy. fractionated radiation therapy (to a total dose of 30 to 45 gy) has been associated with long-term control. 80 diffuse non-t-cell lymphoma is best managed with combination chemotherapy, although the response rate is less than in multicentric lymphoma. in one study, investigators reported that combination chemotherapy with cyclophosphamide, vincristine (oncovin), cytosine arabinoside, and prednisone (coap) induced longterm remission in some cases. 249 five of six dogs with diffuse non-t-cell cutaneous lymphoma attained a complete or partial remission, with a median remission duration of longer than 250 days and a median survival of longer than 399 days. 249 retinoids, such as isotretinoin (accutane) and etretinate (tegison), have been used successfully in canine and human t-cell cutaneous lymphoma. 82, 250 in one study, 12 dogs with cutaneous lymphoma were treated with isotretinoin (3 to 4 mg/kg given orally daily), and two were treated with etretinate (1.25 to 1.45 mg/kg given orally daily, continuously). eleven of these 14 dogs had t-cell cutaneous lymphoma, and six of the 14 dogs achieved remission. in another study, four dogs with t-cell lymphoma were treated successfully with isotretinoin for 13, 11, 10, and 5 months. 82 in the authors' experience, retinoid treatment must be given for at least 2 months to note a response. polyethylene glycol (peg)-l-asparaginase (30 mg/kg given intramuscularly weekly) induces responses in dogs with cutaneous t-cell lymphomas, although remissions are not durable, and no cures have been noted. 251 prednisone may also be necessary to control pruritus. in the authors' experience, pegylated-liposomal doxorubicin (doxil) has produced remissions in approximately 40% of cases. although most of these were short-lived responses, remissions of 1 year or longer occasionally have occurred. a preliminary abstract reported activity for oral ccnu with cutaneous lymphoma in dogs. 252 all seven dogs (five with mycosis fungoides [mf] and two with nonepitheliotropic disease) that were treated with ccnu (50 mg/m 2 given orally every 3 weeks) achieved a complete response, and two of those responses were relatively durable (7 and 15 months). a larger cohort needs to be treated before definitive response rates and durations can be given. topical chemotherapy is another strategy for treating cutaneous t-cell lymphoma, although it is rarely used in veterinary medicine because of "patient compliance" problems. mechlorethamine (mustargen) can be applied topically as an aqueous solution or an ointment base. the aqueous solution is prepared by combining 10 mg of mechlorethamine with 50 ml of tap water. the ointment is prepared by mixing 90 mg of mechlorethamine with 10 ml of absolute alcohol and further combining enough xipamide (aquaphor) to prepare 900 g of ointment. hair must be removed before application. gloves must be used when the drug is applied, because mechlorethamine is carcinogenic and can cause contact hypersensitivity in humans. the response to therapy varies, and the treatment often is only palliative. 253 the prognosis for canine lymphoma varies and depends on a number of factors, such as the location of disease, the extent of disease (the clinical stage), the presence or absence of clinical signs (the substage), the histologic grade, the immunophenotype (t cell or b cell), exposure to previous chemotherapy or corticosteroids and subsequent development of mdr (see chapter 11), altered cell death processes (apoptosis), the proliferation rate of the tumor, the presence of concurrent medical problems or paraneoplastic conditions (e.g., hypercalcemia, weight loss, and liver insufficiency), and possibly gender.* although canine lymphoma is rarely curable (fewer than 10% of cases), complete responses and a good quality of life during extended remissions and survival are typical. factors that have been shown to influence survival are shown in table 31 -5. the two factors most consistently identified as being prognostically important in dogs with lymphoma are the immunophenotype and who substage (see figure 31 -9). many reports have confirmed that dogs with cd3-immunoreactive tumors (i.e., t-cell derivation) are associated with significantly shorter remission and survival durations. † this holds true primarily for dogs with multicentric lymphoma, because the immunophenotype of solitary or extranodal forms of lymphoma has not been thoroughly investigated with respect to the prognosis. in addition, it has been shown that dogs with b-cell lymphomas that express lower than normal levels of b5 antigen (expressed in 95% of nonneoplastic lymphocytes) also have shorter remission and survival durations. 75 dogs with who substage b disease (i.e., clinically ill) also do poorly compared to dogs with substage adisease (see figure 31 -9). ‡ dogs with stage i or stage ii disease have a better prognosis than dogs with more advanced disease (stage iii, iv, or v). 187, 188 in some studies, an elevated serum calcium (over 11.9 mg/dl) has been shown to be a negative prognostic factor 72, 257 ; however, this rarely holds true with multivariate analysis because hypercalcemia is associated with the t-cell phenotype. the histologic grade (subtype) has been found to influence the prognosis in some studies; however, our ability to predict outcome based on subtype is still quite limited. dogs with lymphoma classified as intermediate-or high-grade (large cell, centroblastic, and immunoblastic) tend to respond to chemotherapy but can relapse early. dogs with low-grade lymphomas (small lymphocytic or centrocytic) have a lower response rate to chemotherapy, yet have a survival advantage over dogs with intermediate-or high-grade lymphomas ( figure 31 -10) in that the course of disease may be more indolent. 113 using the working formulation, dogs with low-grade lymphomas have a survival advantage compared to dogs with intermediate-or high-grade tumors. 78 recently, proliferative assays (e.g., analysis of bromodeoxyuridine [brdu] uptake, ki-67 antibody reactivity, and agnor indices) to measure the proliferative activity of tumor cells have been shown to provide significant prognostic information in dogs treated with combination chemotherapy. 60, 163, 254, [258] [259] [260] however, the results of different studies are contradictory. in two trials, dogs that had tumors with short doubling times, high agnor frequencies, or high ki-67 immunoreactivity had a better prognosis than dogs with long doubling times or low agnor frequencies. 60, 163 in other trials, the low-proliferating tumor groups were associated with a better prognosis. 254, 259 in one trial, the proportion of tumor cells undergoing apoptosis was modestly predictive of remission duration. 163 the anatomic site of disease also has considerable prognostic importance. primary diffuse cutaneous, diffuse gastrointestinal, hepatosplenic, and primary cns lymphomas tend to be associated with a poor prognosis. cutaneous lymphoma tends to progress slowly, and in our experience the responses to systemic chemotherapy are less durable. localized lymphomas in the skin can be managed with radiation therapy or surgery or both, and these tumors have a better prognosis. in some dogs with lymphoma, significant involvement in the bone marrow may be present (i.e., tumor cells comprise more than 50% of all nucleated cells) and circulating malignant lymphocytes may be present in the peripheral blood. these dogs tend to have an overall poor prognosis. in some cases it is difficult to determine whether the disease arises from the bone marrow (e.g., acute lymphoblastic leukemia [all]) or is a diffuse lymphoma with extensive involvement in the marrow. immunophenotyping is helpful in these cases, because the tumor cells in all typically are cd34 positive. gender has been shown to influence the prognosis in some studies. 59, 191 neutered females tend to have a better prognosis. males may have a higher incidence of the t-cell phenotype, which may account for the poorer prognosis. 60 other reported potential biomarkers of the prognosis include circulating levels of glutathione-s-transferase, thymidine kinase, and vascular endothelial growth factor (vegf). 261-263 one report suggests that a history of chronic inflammatory disease of several types predicts a likelihood of early relapse. 264 these putative prognostic indicators require confirmation in larger trials. lymphoid leukemia is the proliferation of neoplastic lymphocytes, which usually originate in the bone marrow but occasionally may originate in the spleen. the neoplastic cells may or may not be circulating in the peripheral blood. lymphocytic leukemia is more common than nonlymphocytic leukemia and other myeloproliferative disorders (mpds). the true incidence is not known. in a series of 30 cases of all, german shepherds accounted for 27% of the cases, and the male to female (m:f) ratio was 3:2. 265 in this study, the median age was 5.5 years (range, 1 to 12 years), and eight dogs were younger than 4 years of age. recently, all was reported in a 12-week-old greyhound. 266 well-differentiated or chronic lymphocytic leukemia (cll) is seen less frequently than all but more commonly than mpd. the median age is 10 to 12 years. the m:f ratio has been reported as 1.8:1 (22 dogs) and approximately 2.3:1 in 15 dogs with granular lymphocytic (gl) t-cell cll. 267 in a large study of 73 dogs with cll, no gender predilection was found in dogs with b-cell or non-gl cll; female dogs were overrepresented among dogs with gl cll, (m:f ratio, 1:1.7). 268,269 as with lymphoma, the etiology of lymphoid leukemia is unknown. retroviruses have been implicated in diverse animal species such as cats, cattle, fish, snakes, birds, rodents, and nonhuman primates. a retroviral cause in dogs has not been proved. however, a retrovirus with morphology typical of lentiviruses has been isolated from mononuclear cells obtained from the peripheral blood of a dog with all. 270 in humans, acute leukemia has been associated with exposure to radiation, benzene, phenylbutazone, and antineoplastic agents. [271] [272] [273] [274] [275] [276] the alkylating agents can cause chromosomal damage and are clearly carcinogenic. 276 human t-lymphotropic virus type 1 (htlv-1) is a proven cause of leukemia in a large cohort of human patients from the southern islands of japan. 277,278 the etiology of cll is less clear, but genetic factors likely are important. extrapolation of predisposing factors across species is not warranted, and the etiologic factors for dogs may be quite different from those for humans, given the difference in the predominant immunophenotype of the neoplastic cells (discussed later). in all, the blast cells infiltrate the bone marrow, and the result is variable degrees of anemia, thrombocytopenia, and neutropenia. infiltration of the spleen and liver is common, and extramedullary sites (e.g., the nervous system, bone, and gi tract) also may be involved. some animals may have lymph node involvement and develop generalized lymphadenopathy. 265 the lymphocytes of cll are virtually indistinguishable morphologically from normal small lymphocytes, and they have a low proliferation rate. the accumulation of lymphocytes likely results from their prolonged life span. in b-cell cll, the marrow is infiltrated with mature lymphocytes, and the extent of infiltration is less than that seen with all or mpd. the neoplastic cells in gl t-cell cll originate in the spleen, and bone marrow involvement may or may not occur. 173 dogs with cll tend to have a mild anemia, and granulocytes and platelets are only mildly reduced. splenomegaly is common, and lymph nodes can be slightly to moderately enlarged. 269 despite the well-differentiated appearance of the lymphocytes in cll, these cells may function abnormally. part iv • specific malignancies in the small animal patient paraneoplastic syndromes include monoclonal gammopathies, immune-mediated hemolytic anemia, pure red cell aplasia and, rarely, hypercalcemia. 173, 279 hypercalcemia was reported in a giant schnauzer with b-cell cll, which is highly unusual given that hypercalcemia is associated with t-cell lymphoproliferative disorders. in one study of 22 dogs with cll, 68% had monoclonal gammopathies. 269 the immunophenotypes were not reported, but a monoclonal gammopathy is associated with production of immunoglobulins by the leukemic cells (b cells). the immunoglobulin is usually igm or iga. the term macrogammaglobulinemia is used to describe igm gammopathy (see chapter 31, section d). dogs with cll and an igm monoclonal gammopathy are said to have waldenström's macroglobulinemia. dogs can also develop hyperviscosity syndrome (see chapter 31, section d). reports of the immunophenotype of neoplastic cells in canine leukemias are increasing in frequency. one consistent finding in acute leukemias (either all or acute myeloid leukemia) is the presence of cd34 on the blast cells. this marker is useful for distinguishing all (cd34-positive) from cll and lymphoma with bone marrow infiltration (cd34-negative). in one study of 12 cases of cll, eight dogs were associated with a cd8-positive t-cell phenotype. 75 in a larger study of 73 cases, only 26% of the cases had a b-cell immunophenotype, whereas 73% were identified as t-cell cll (cd3-positive). in 54% of these t-cell leukemias, the cells had the morphology of gls, and most of them were cd8-positive. these findings are difficult to reconcile with the high frequency of monoclonal gammopathies reported in the earlier study. it also should be noted that this differs from human clls, which typically are lymphoproliferative diseases of b cells. dogs with all usually have a history of anorexia, weight loss, polyuria, polydipsia, and lethargy. splenomegaly is typical, and other physical abnormalities may include hemorrhages, lymphadenopathy, and hepatomegaly. anemia, thrombocytopenia, and an elevated white blood cell (wbc) count are common. the anemia may be severe and usually is characterized as normocytic and normochromic (nonregenerative). wbc counts usually are increased despite neutropenia because of an increased number of circulating lymphoblasts (more than 14,000 cells/ml). however, some dogs may be leukopenic. 265 neoplastic lymphoblasts may infiltrate the bone marrow extensively, resulting in depression of normal hematopoietic elements. dogs with cll often are asymptomatic, although some owners report lethargy and a decreased appetite. mild lymphadenopathy and splenomegaly may be present. most dogs tend to be mildly anemic (packed cell volume [pcv] less than 35%) and thrombocytopenic (110,000 to 190,000 platelets/ml). the wbc count usually exceeds 30,000 cells/ml but can vary from normal to more than 100,000 cells/ml, owing to an increase in circulating mature lymphocytes. 269 lymphocytosis is persistent, and granulocytes usually are present in normal numbers. in some dogs the disease is identified incidentally when the patient undergoes evaluation for an unrelated problem. the clinician must consider the signalment, history, physical findings, cell morphology, and immunophenotype to diagnose the lymphoid leukemias accurately. a knowledge of the profile of lymphocyte subsets in the peripheral blood of normal dogs is helpful for determining whether a particular subset has expanded. approximately 80% of circulating lymphocytes in dogs are t cells, and about 15% are b cells. nk cells and double-negative (cd4−cd8−) t cells account for the remaining fraction. in the t-cell fraction, helper t cells (cd4+) outnumber cytotoxic t cells (cd8+). 173 lymphocytic leukemia should be a consideration if atypical lymphocytes are in circulation, if the immunophenotype of the lymphocytes in circulation is homogenous, or if a phenotype typically present in low numbers has increased. differential diagnoses for lymphocytosis include certain infectious diseases (e.g., chronic ehrlichiosis), postvaccinal responses in young dogs, il-2 administration, and transient physiologic or epinephrine-induced lymphocytosis. reactive and neoplastic lymphocytoses sometimes are difficult to differentiate. in these cases, pcr assays are used to determine the clonality of the t cell or immunoglobulin receptor genes in a population of lymphocytes (see earlier discussion). 167, 169, 268 infiltration of the bone marrow by neoplastic lymphoid cells is the hallmark of all and is seen in most cases of cll; careful examination of peripheral blood and bone marrow by experienced cytopathologists is essential for establishing a diagnosis of lymphocytic leukemia. if adequate diagnostic bone marrow cannot be obtained by aspiration, a bone marrow core biopsy should be performed. in all, lymphoblasts predominate in the bone marrow and also are present in peripheral blood. infiltration of bone marrow by lymphoblasts is accompanied by a decrease in the granulocytic, erythroid, and megakaryocytic cell lines. perhaps the distinguishing feature of lymphoblasts is the nuclear chromatin pattern, which is more condensed than the chromatin in myeloblasts. 280 lymphoblasts are larger than neutrophils, have a high nucleus-to-cytoplasm ratio, and blue cytoplasm, which in some cases is intensely basophilic ( figure 31-11, a) . nucleoli, although present, are less prominent in lymphoblasts than in myeloblasts. nevertheless, these cells cannot be easily distinguished from blast cells of other hematopoietic lineages, and lineage-specific markers on the cells must be identified by immunocytochemistry or flow cytometry. 278 the most frequently used markers are cd3 for t cells and cd79a for b cells. as mentioned earlier, cd34 is found on blast cells of both lymphoid and myeloid lineages, and positivity for cd34 helps to confirm leukemia of immature cells (acute leukemia) and to distinguish all from stage v lymphoma. immature and differentiating lymphocytes may stain strongly for alkaline phosphatase activity, which suggests that this cytochemical staining procedure is not specific for myeloid leukemia. 278, 280, 281 in b-cell and non-gl t-cell cll, the lymphocytes are small mature cells ( figure 31-11 , b) that appear in excessive numbers in bone marrow (30% or more of all nucleated cells) early in the disease. 269 infiltration becomes more extensive as the disease slowly progresses, and eventually the neoplastic cells replace normal marrow. the lymphoid lineage of the cells in cll typically is easy to identify, and immunophenotyping is done to determine the lymphocyte subset. in dogs, most cases of cll are t-cell (cd3+) proliferations. in gl cll, the neoplastic cells originate in the spleen. 173 a separate clinical staging system has not been developed for lymphocytic leukemia. currently, all dogs with leukemia are classified as stage v based on the who staging system for lymphoma (see box 31-1). although a specific clinical staging system for cll has been used in humans, it has not been evaluated in the dog. 282 similar to other infiltrative bone marrow malignancies, all causes morbidity by suppressing bone marrow function. neutropenia, thrombocytopenia, and anemia may be severe. therapy must be aggressive; to restore hematopoiesis, a 1.5 to 2 logarithmic reduction in leukemic cell numbers (to less than 100 million cells) must be achieved. patients need supportive therapy, such as fresh whole blood, broad-spectrum antibiotics, fluid therapy, and nutritional support. patients must be monitored carefully for bleeding and thrombosis, which may signal the development of dic. all requires aggressive chemotherapy. consistently efficacious protocols for all have not been developed in veterinary medicine. chop-based protocols, similar to those used for lymphoma (see , tend to be used as treatment protocols for dogs with all. in one report on the use of vincristine and prednisone in dogs with all, 40% of the dogs responded to vincristine and prednisone, 20% with a complete remission and 20% with a partial remission. 265 with the addition of doxorubicin and l-asparaginase, it is anticipated that response rates will increase over those previously reported using vincristine and prednisone alone; however, the relative rarity of all limits the ability to identify effective protocols. a b because of the indolent nature of cll in many animals, the question of whether all dogs with the disease should be treated is controversial. 283,284 most oncologists recommend observation over active therapy when the discovery of cll is incidental, when no physical or clinical signs are present, and when no significant hematologic abnormalities are identified. therapy should be instituted if the animal is anemic or thrombocytopenic, is showing evidence of significant lymphadenopathy or hepatosplenomegaly, or has an excessively high wbc count (over 60,000 lymphocytes/ml) (box 31-4). the most effective drug evaluated thus far is chlorambucil. 269 chlorambucil is administered at a dosage of 0.2 mg/kg or 6 mg/m 2 given orally once daily for 7 to 14 days. the dosage then can be reduced to 0.1 mg/kg or 3 mg/m 2 given orally once daily. for long-term maintenance, a dosage of 2 mg/m 2 given orally every other day can be used. the dosage is adjusted according to the clinical response and bone marrow tolerance. chlorambucil should be administered without food to increase the rate of absorption. 285 corticosteroids are lymphocytolytic and lead to cell death by apoptosis. studies in humans have shown that the antitumor activity of chlorambucil combined with prednisone is better than that of chlorambucil alone. 286 when the bone marrow is heavily infiltrated with cll cells, and neutropenia, thrombocytopenia, and anemia occur, use of a more aggressive alkylating agent (usually cyclophosphamide [250 mg/m 2 given once]) when initiating oral chlorambucil and prednisone therapy can be considered to increase the speed of initial remission; however, this modification of the protocol has not been scrutinized in clinical trials. if chlorambucil or cyclophosphamide fails, the choice of treatment is combination chemotherapy similar to that presented in box 31-3. the treatment of cll is primarily palliative, and complete remissions are rare. because of the indolent nature of this disease, however, survival times have been in the range of 1 to 3 years with a good quality of life. 269, 284 in humans, splenectomy has been shown to increase survival significantly in individuals with aggressive forms of cll; however, splenectomy in dogs with cll has not been evaluated. 287 the phenotypic expression of cll usually is stable over months to years. however, the disease may evolve into an acute phase, and some dogs develop a rapidly progressive, pleomorphic (immunoblast) lymphoma. 269 in humans, this is called richter's syndrome. 288 the prognosis for response to treatment is poor for this form. in general, the prognosis for all in the dog is very poor. in a study of 21 dogs treated with vincristine and prednisone, the dogs achieving complete or partial remission (29%) had a median survival time of 120 days, and few dogs survived longer than 8 months with that protocol. 265 in one case report, a dog with all was treated with an infusion of a large volume of fresh canine plasma and whole blood, and a complete remission was maintained for 19 months without additional therapy. this is a very unusual response, which indicates that normal blood contains some antileukemic factor or factors. 289 as stated before, cll is a slowly progressive disease, and some animals do not require therapy. one dog was observed for almost 2 years without treatment. 283 for dogs that are treated, normalization of wbc counts can be expected in 70% of cases. in one report of 17 dogs treated with vincristine, chlorambucil, and prednisone, the median survival time was approximately 12 months, with an expected 30% survival at 2 years. 269 in other studies, dogs treated intermittently with chlorambucil and prednisone have had remissions of 10 to 30 months. 284 david m. vail the lymphomas (malignant lymphoma and lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues, such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. lymphoma is one of the most common neoplasms seen in the cat. the feline leukemia virus (felv) was the most common cause of hematopoietic tumors in the cat during the so-called felv era of the 1960s through the 1980s, when 60% to 70% of lymphoma cases were associated with felv antigenemia. [1] [2] [3] [4] [5] [6] [7] several studies have documented the potential molecular means by which felv can result in lymphoid neoplasia. 8, 9 however, over the past 20 years in north america, a profound change has occurred in the viral status, presentation, signalment, and frequency of anatomic sites in cats with lymphoma, as documented by two large studies of feline lymphoma involving more than 700 cats. 10, 11 the change in the epidemiology and characteristics of lymphoma in cats appears to coincide with the widespread integration of clinically relevant felv diagnostic assays (indirect immunofluorescence assay) and the affected animal elimination regimens of the late 1970s; it was further enhanced by the appearance of commercially available felv vaccines in the late 1980s. the decline in felv-associated lymphoma was mirrored by a decline in the overall prevalence per year of felv positivity in cats tested, as characterized by reports from the tufts veterinary diagnostic laboratory from 1989 to 1997 10,12 and by louwerens' group, 11 which reported a decline in felv association in more than 500 cases of lymphoma in cats brought to the university of california-davis veterinary teaching hospital. in these reports, felv antigenicity represented only 14% to 25% of the cats with lymphoma. the incidence figures for feline lymphoma generated before the use of felv vaccination and testing became widespread suggest that lymphoma accounted for 50% to 90% of all hematopoietic tumors in the cat 13, 14 ; because hematopoietic tumors (lymphoid and myeloid) represent approximately one third of all feline tumors, the estimated incidence of lymphoid neoplasia was 200 per 100,000 cats at risk. 15 in one series of 400 cats with hematopoietic tumors, 61% had lymphoma and 39% had leukemias and mpds (21% of the mpds were categorized as undifferentiated leukemias, most likely myeloid in origin). 16 an important fact that louwerens' study revealed is that despite a sharp drop in felv-associated lymphoma, the overall prevalence of lymphoma in cats is increasing. 11 this appears to be due to an increase in the number of affected cats and the relative frequency of the abdominal (particularly the intestinal) anatomic form of lymphoma in the species (figure 31-12) . as might be expected, along with a shift away from felv antigen-associated tumors came a shift away from the traditional signalment and relative frequency of anatomic sites. 10, 11 this observation is supported outside north america by the similar signalment and anatomic frequency data obtained in australia, where felv infection is quite rare. [17] [18] [19] [20] the median age of approximately 11 years now reported in north america for lymphoma in cats is considerably higher than the median age of 4 to 6 years reported during the felv era. 1,3-6,10,11,21 the median age of cats within various anatomic tumor groupings has not changed, and anatomic forms traditionally associated with felv, such as the mediastinal form, still occur in younger, felv-antigenemic cats. similarly, the alimentary form occurs most often in older, felv-negative cats. 10, 11, [18] [19] [20] table 31 -6 presents an overview of the characteristics of the various anatomic sites of lymphoma in cats. younger cats with lymphoma tend to be felv antigenemic and are more likely to have mediastinal or multicentric lymphoma. most cats with spinal lymphoma (85% to 90%) also are felv antigenemic. 22, 23 older cats usually are felv negative and develop alimentary lymphoma. 10, 17, 19, 20, [24] [25] [26] [27] in a large compilation of australian cases, male cats and the siamese/oriental breeds were overrepresented. 17 similar breed findings have been observed in north america, 11 but similar gender findings have not emerged. 28, 29 the siamese/oriental breeds appear to have a predisposition for the mediastinal form that affects a younger population (median age, 2 years). 11 evidence also indicates that feline immunodeficiency virus (fiv) infection can increase the incidence of lymphoma in cats. [30] [31] [32] [33] [34] [35] [36] [37] in contrast to felv, which plays a direct role in tumorigenesis, fiv appears to have an indirect role, likely secondary to the immunosuppressive affects of the virus. 35 shelton and colleagues 30 determined that fiv infection alone in cats was associated with a fivefold increased risk for the development of lymphomas. coinfection with felv further potentiates the development of lymphoproliferative disorders. experimentally, cats infected with fiv have developed lymphoma in the kidney, alimentary tract, and liver and in multicentric sites. fiv-associated lymphoma is more likely to have a b-cell immunophenotype whereas t-cell type is predominantly associated with felv. 32, 33, 35, 36, [38] [39] [40] [41] some have suggested that fiv infection may be associated more often with alimentary lymphoma of b-cell origin, 40, 41 which may be related to chronic dysregulation of the immune system or activation of oncogenic pathways. however, in another large compilation of cases, fiv antigenemia only rarely was associated with alimentary lymphoma. 10, [24] [25] [26] [27] genetic and molecular factors as discussed in chapter 31, section a, recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, are being used in investigations of chromosomal aberrations in veterinary species with lymphoma. the previously mentioned predisposition of the oriental cat breeds to the development of lymphoma suggests a genetic predisposition and indicates heritable risks. 11, 17 as our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various feline tumor types, and some associated with lymphoma have been identified. altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and deathpathway alterations are common in human lymphomas and likely are also involved in the cat. n-ras aberrations, although rare in cats, have been implicated. 42 telomerase activity (see chapter 14, section d) also has been documented in feline lymphoma tissues. 43, 44 other factors implicated in feline lymphoma include alterations in cellular proliferation and in cell cycle and death (apoptosis) pathways, particularly the cyclindependent kinase cell cycle regulators and the bcl-2 family of proapoptotic and antiapoptotic governing molecules, which have been implicated in human nhl. 45, 46 environmental factors evidence that exposure to environmental tobacco smoke (ets) is a risk factor for lymphoma in humans has prompted investigations in cats. in one report, cats with any exposure to ets had a relative risk of developing lymphoma of 2.4; the relative risk for cats with 5 or more years of exposure was 3.2. 47 immune system alterations in the cat, such as those brought on by fiv infection, have been implicated in the development of lymphoma. [30] [31] [32] [33] [35] [36] [37] in a report on 95 feline renal transplant recipients, nearly 10% developed de novo malignant lymphoma, which bolsters support for a link to immunosuppression in the species. these findings are similar to those seen in immunosuppressed human organ transplant patients. 48 although definitive proof is lacking, a growing body of indirect evidence suggests that lymphoma may be associated with a state of chronic inflammation, such as in intestinal and nasal lymphoma. in particular, an association has been suggested between intestinal lymphoma and inflammatory bowel disease 11,49-51 ; however, others have not found support for this concept. 52 additional support for this association is provided by a recent report that suggests that cats with vaccine site-associated sarcoma, a syndrome directly linked to inflammation, are also at risk for the development of lymphoma. 53 although no direct evidence exists, a link between diet and the development of intestinal lymphoma in cats has been suggested. 11 this association is supported by the relative and absolute increase in the intestinal form of lymphoma over the past 20 years and by the fact that several dietary modifications in cat food have occurred in a similar time frame in response to conditions such as urinary tract disease. further investigation is warranted to prove or disprove such assertions. lymphoma can be classified on the basis of anatomic location and histologic criteria. several systems of anatomic classification exist for lymphoma in the cat. some categorize the disease into mediastinal, alimentary, multicentric, nodal, leukemic, and individual extranodal forms. others have combined various nodal and extranodal forms into categories of atypical, unclassified, and mixed, while still others have combined intestinal, splenic, hepatic, and mesenteric nodal forms into one category (intra-abdominal). for the purposes of this discussion, the "mixed" form is defined as cases involving multiple sites in which no primary site categorization was possible. some discrepancies in the discussion of frequency inevitably will result from the variations in classification used in the literature. the relative frequency of anatomic forms and the immunophenotype may vary with geographic distribution and may be related to genetic and felv strain differences, as well as to the prevalence of felv vaccine use (see table 31 -6). in most larger studies, most cases of lymphoma in cats (approximately 70% to 75%) are of the b-cell immunophenotype; however, the mediastinal, leukemic, and purely hepatic forms are more likely to be of t-cell derivation. 10, 18 cats with t cell-rich b-cell lymphoma (hodgkin's-like lymphoma) and nk-like t-cell lymphoma (non-t, non-b-cell) have been described. 54, 55 as with dogs, most lymphomas in cats have an intermediate-grade or a high-grade histology according to the working formulation (wf) criteria. 18, 56 alimentary/intestinal lymphoma alimentary/intestinal lymphoma can manifest as a purely intestinal infiltration or as a combination of intestinal, mesenteric lymph nodes, and liver involvement. 11 some reports limit the alimentary form to gi involvement, with or without extension to the liver. approximately two thirds to three fourths of reported cases are of the b-cell immunophenotype. most patients are older cats, and very few cases appear to be associated with felv antigenemia. 10, 11, 18, [24] [25] [26] [57] [58] [59] however, some discordance on these points is seen in the literature. in one smaller report, most alimentary cases sampled were found to be of the t-cell immunophenotype. 24 the population size was relatively small in comparison and may represent a geographic variation. the epitheliotropic form of intestinal lymphoma also has been reported to be more commonly t-cell in origin. 49, 57 a canadian group investigated formalin-fixed archival lymphoma tissue in cats and found a nearly even distribution between the b-cell and t-cell immunophenotype; they also found that approximately two thirds of the samples were felv positive by pcr techniques. 2,60-62 some felv-negative tumors may be derived from transformation of multipotent lymphoid or monocyte precursors 61 or fiv-transformed b lymphocytes. 32, 33, 38, 39 the most common site of involvement in the alimentary tract is the small intestine (50% to 80% of cases), followed by the stomach (approximately 25%), ileocecocolic junction, and colon. 25, 26, 62 the tumor can be solitary or diffuse throughout the intestines (figure 31-13) , muscle layers, and intestinal submucosa, resulting in annular thickening that leads to partial or complete intestinal obstruction. in a series of colonic neoplasias part iv • specific malignancies in the small animal patient necropsy specimen from a cat with intestinal lymphoma. note the generalized thickening of the small intestine and the associated mesenteric lymph node involvement (arrows). in cats, lymphoma was the second most common malignancy (41%), exceeded only by adenocarcinoma. 27 an abstract report has described chronic lymphocytic-plasmacytic enteritis in cats that progressed to overt lymphoma after 6 to 18 months of conservative therapy. 63 the mediastinal form can involve the thymus and the mediastinal and sternal lymph nodes. pleural effusion is common. in two large compilations, 63% of cats with thymic disease and 17% of cats with pleural effusion were documented as having lymphoma. 64, 65 occasionally the tumor extends from the thoracic inlet and can be palpated in the ventral neck region. hypercalcemia is common with mediastinal lymphoma in dogs but is rare in cats with lymphoma. [66] [67] [68] pthrp has been detected through immunoradiometric assays in cats with hypercalcemia of malignancy, including one cat with lymphoma. 69 most cats with mediastinal lymphoma are young and felv positive, and their tumors are of the t-cell immunophenotype.* involvement of peripheral lymph nodes alone is very unusual in cats with lymphoma, representing approximately 4% to 10% of cases. 10, 11 in contrast, approximately one fourth of all other anatomic forms of lymphoma have some degree of lymph node involvement. 11 one third of cats with nodal lymphoma are of the t-cell immunophenotype and are felv antigenemic. 10, 18 as lymphoma progresses, bone marrow infiltration with malignant cells and hepatosplenomegaly may develop. recently an uncommon and distinct form of nodal lymphoma in cats, referred to as hodgkin's-like lymphoma, has been reported. this form typically involves solitary or regional nodes of the head and neck and histologically resembles hodgkin's lymphoma in humans. 17, 54, 70 these tumors generally manifest as an enlargement of a single mandibular or cervical node and immunophenotypically are classified as t cell-rich b-cell lymphoma by immunohistochemical analysis. 54, 70 none have been associated with either felv or fiv. several reports of nonneoplastic peripheral lymphadenopathy in cats have been published. [71] [72] [73] [74] [75] this condition resembles lymphoma clinically, and it has histologic features that also may resemble those of lymphoma. [71] [72] [73] [74] [75] affected lymph nodes may be two or three times normal size. in one report, the syndrome was called distinctive peripheral lymph node hyperplasia (dplh) of young cats. 72 these cats tend to be young (2 to 4 years old), and many have had episodes of fever or previous viral infections, or they may have hypergammaglobulinemia (polyclonal gammopathy); most are felv negative. 71 histopathologically, the nodal architecture is severely distorted, showing loss of subcapsular and medullary sinuses. the cell population shows an admixture of histiocytes, lymphocytes, plasma cells, and immunoblasts and occasionally effaced lymphoid follicles. the lymph nodes regress spontaneously in most of these cats. the histologic changes noted in these lymph nodes resemble the histologic features of acquired immunodeficiency syndrome (aids)-related lymphadenopathy in humans. 72 in another report, benign lymphadenopathy in cats was associated with argyrophilic intracellular bacteria. 75 the most common extranodal sites for lymphoma are the kidneys, nasal cavity, eyes, retrobulbar space, cns, and skin. renal lymphoma can be primary or associated with alimentary lymphoma. based on several studies, the median age for cats with renal lymphoma is approximately 7.5 years. one fourth of cases are felv antigenemic, and most are of the b-cell immunophenotype. 10, 11, 18, 76 the frequency of renal lymphoma is reported to be approximately 5% of all lymphomas. extension to the cns is a common sequela to renal lymphoma and occurs in 40% to 50% of treated cats. 76 nasal/paranasal lymphoma usually is a localized disease; however, systemic extension occasionally is seen. 10, 77, 78 neoplasia accounts for most nonviral nasal/paranasal disease in cats, and lymphoma has been reported to represent nearly one third to one half of these cases. [79] [80] [81] it occurs primarily in older, felvnegative cats (median age, 9 to 12 years). at least three fourths of cases are b cell in origin, and most are intermediate grade or high grade histologically. 10,79-81 a subset of epitheliotropic t-cell nasal lymphomas has been reported. 81 cats that are concurrently felv positive are more likely to have concurrent systemic disease. in the older literature, cns lymphoma was most often seen extradurally in the spinal canal of felv-positive cats (85% to 90%). 20, 21, 82 however, in more recent reviews, both spinal cord and intracranial lymphoma occurs mostly in older, felv-negative cats, and spinal cord sites are more commonly intradural than extradural. 83, 84 after meningioma, lymphoma is the second most common tumor involving the cns in cats. 84 feline cns lymphoma may be primary or may occur secondary to multicentric involvement (especially of the renal system or bone marrow). [83] [84] [85] [86] bone involvement is rarely seen radiographically. 87 multiple cord regions and the brain are involved in nearly 50% of cats with spinal lymphoma, and more than 80% of these patients have other organ (e.g., renal) and bone marrow involvement. 20, 21, 83 only one third of cases of intracranial lymphoma are primary and confined to the cns, and all cats tested have been fiv negative. 84 cutaneous lymphoma generally is primary but can be seen secondary to multicentric involvement. it commonly is seen in older cats (median age, 10 to 12 years) and gender sex or breed predominance has not been found. [88] [89] [90] [91] [92] although patients usually are felv negative, one case report using pcr techniques found evidence of felv provirus in tumor dna. 92 cutaneous lymphoma can be solitary or generalized. two forms of cutaneous lymphoma have been distinguished histologically and immunohistochemically. [88] [89] [90] [91] [92] in most species, the epitheliotropic form, sometimes referred to as mycosis fungoides, is composed of t lymphocytes, whereas the nonepitheliotropic form usually is composed of b lymphocytes. in contrast, one report of nonepitheliotropic cutaneous lymphoma in cats found that five of six cases were of t-cell derivation. 93 neoplastic t lymphocytes are large and have abundant cytoplasm and convoluted nuclei (mycosis cells). they usually form intraepidermal nests of five to 10 cells, which are separated from surrounding keratinocytes by a clear space (pautrier's microabscesses). the b-cell lymphomas show lymphocytes deep in the epidermis, with sparing of the papillary dermis and epidermis. a recent report described 23 cases of cutaneous lymphocytosis, an uncommon disease histologically resembling well-differentiated lymphoma. 94 solitary lesions were most common, and all were composed primarily of t-cells, although two thirds had some b-cell aggregates. cutaneous lymphocytosis was characterized as a slowly progressive disorder, but internal organ infiltration developed in a few cases. a cat with cutaneous t-cell lymphoma and circulating atypical lymphocytes has been reported. 91 the circulating cells were lymphocytes with large, hyperchromatic, grooved nuclei. in humans, cutaneous t-cell lymphoma with circulating malignant cells is called sézary syndrome, 95 which also has been reported in dogs. [96] [97] [98] [99] a number of histopathologic grading systems have been used to classify human nhl, including the wf (discussed earlier in the chapter). most recently, the wf was used to classify more than 600 cases of feline lymphoma. 56 low-grade lymphoma was found in 11% of the cases, intermediate-grade disease in 35%, and high-grade lymphoma in 54%. about 1.1% were plasmacytomas. more than one third of the tumors were the immunoblastic type. lymphoblastic lymphoma, a subtype of the high-grade tumor, accounted for less than 3%. similarly, in a large group of cases compiled in australia (n = 118), 90% of the cases were mediumto high-grade disease as classified by the wf. 18 also as discussed earlier in the chapter, who has published a histologic classification scheme that uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic tumors of domestic animals. 100 this system incorporates both histologic criteria and immunohistologic criteria (b-and t-cell immunophenotype). the clinical relevance of this system is likely to be high; however, it awaits further investigation and evaluation. in a series of 28 cases of alimentary lymphoma, 89% were the high-grade lymphoblastic type. 25 however, in another report, many of the intestinal lymphomas had several characteristics of malt, as described in humans; that is, a tendency to remain localized in the lamina propria, a relatively indolent clinical behavior, and an excess of low-grade tumors. 56 the age of affected animals varied considerably with various subtypes, but in general, low-grade tumors tended to develop in older cats (over 10 years of age), and high-grade tumors tended to develop in younger cats (under 6 years of age). a less commonly reported, distinct form of alimentary lymphoma has been described and classified as large granular lymphoma. [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] these lesions are granulated, round cell tumors that also have been called either globule leukocyte tumors or large granular lymphocyte lymphoma, although they probably are variations of the same disease. large granular lymphocytes are characterized by abundant cytoplasm with prominent azurophilic granules. this population of cells includes nk cells and cytotoxic t cells. several reports have identified their immunophenotype as cd3, cd57-like, perforin positive, and cd20 negative; these cells also have been described as having a t-cell receptor gene rearrangement. these tumors commonly originate in the small intestine, especially the jejunum or mesenteric lymph nodes; however, most cases show widespread metastasis to the lung, myocardium, salivary gland, and spinal cord. leukemia also has been reported with this disease. affected cats generally are felv negative. large granular lymphocytes must be differentiated from several other granular cell types that may be found in the small intestine, including enterochromaffin cells, mast cells, and eosinophils. the clinical signs associated with feline lymphoma vary and depend on the location and extent of disease. the alimentary form is most commonly associated with an abdominal mass that originates in the gi tract. the condition often is associated with enlarged mesenteric lymph nodes or other organ involvement. in 50% to 85% of cases, a palpable abdominal mass or thickened bowel loops are present. 24, 25, 27, 62, 111 clinical signs may consist of weight loss, anorexia, diarrhea, and occasional vomiting. in approximately half of cases, the only historical finding is anorexia and weight loss. 25 other reported presentations include abdominal distention, splenomegaly, persistent thrombocytopenia, and pica. hematochezia and tenesmus may be present if the lymphoma involves the colon. 27 polyuria and polydipsia have been reported in approximately 10% of cases. 62 in a small percentage of cases, the patient may have signs consistent with an acute abdomen as a result of intestinal perforation and concurrent peritonitis. 24 clinical signs of the mediastinal form of lymphoma include dyspnea, tachypnea, and a noncompressible anterior mediastinum with dull heart and lung sounds. 20, [111] [112] [113] [114] in rare cases, horner's syndrome may be present as a result of involvement of the sympathetic nerve as it ascends around the first rib, and edema of the head may be caused by pressure on the cranial vena cava. 111 pleural effusion is common; the pleural fluid is characterized by serohemorrhagic to chylous effusion, and in most cases neoplastic cells (lymphoblasts) are identified. 65, 114 cats with the nodal form of lymphoma have variable clinical signs, depending on the location and extent of disease; however, these cats often are depressed and lethargic. peripheral lymphadenopathy as the only physical finding is a very uncommon presentation for cats. as stated earlier, an uncommon and distinct form of nodal lymphoma in cats is hodgkin's-like lymphoma. this form typically involves a single mandibular or cervical node, and the cat usually does not have any overt clinical signs. 54, 70 in cats, unlike in dogs, peripheral lymphadenopathy without organomegaly is more often hyperplastic or reactive and does not represent lymphoma. 73 the extranodal sites include the kidneys, skin, eyes, nasal area, and cns. renal lymphoma is most consistently bilateral, even in cats that appear to have unilateral disease. 76 in general, the kidneys are uniformly enlarged; however, they may also feel lumpy and irregular on palpation. more than 50% cats have signs consistent with renal insufficiency. 76 cats with cns lymphoma most often have signs associated with thoracolumbar involvement. 22, 23 the most common sites are between the second thoracic and fourth lumbar vertebrae. signs include gradual or sudden onset of weakness, upper motor neuron paralysis to the bladder, tail flaccidity, hyperpathia in the region of the lesion, and progressing ataxia. the neurologic dysfunction may be insidious or may progress rapidly. 23 cats with cervical spinal cord or nerve root involvement generally show peracute tetraparesis and diminished sensation in the thoracic limbs. those with cervical root involvement may show root lesions (root signature), such as lameness and hyperesthesia upon shoulder extension. common presenting signs for intracranial lymphoma, in decreasing order of frequency, include anorexia, ataxia, lethargy, altered consciousness, and aggression. 84 cats with nasal lymphoma usually have signs localized to the nasal passage. in a large compilation of cases, the presenting complaints, in decreasing order of frequency, were unilateral nasal discharge (bilateral is less common), facial deformity, dyspnea, and epistaxis. 79-81 stertor, anorexia, epiphora, exophthalmia, sneezing, and regional lymphadenopathy also can occur. cutaneous lymphoma may be solitary or diffuse and manifest with alopecia, erythema, and crusted papules. minimal peripheral lymphadenopathy may also be present. in most cats the signs have a prolonged duration (i.e., several months). [88] [89] [90] [91] [92] 94 nonspecific signs all cats with lymphoma, regardless of the site, may have secondary bone marrow infiltration that leads to anemia and a leukemic blood profile. anemia is a common condition in cats with lymphoma, and at least 50% have moderate to severe nonregenerative anemia. signs related to paraneoplastic hypercalcemia (pu/pd) can occur in cats, but the syndrome is much less common than in the dog. in one survey of hypercalcemia in cats, approximately 10% were diagnosed with lymphoma of various anatomic types. 115 one case of hypereosinophilic paraneoplastic syndrome and one case of symmetric cutaneous necrosis have been reported in cats with lymphoma. 116,117 a number of disease conditions can be confused with lymphoma in cats (table 31-7) . for most cats suspected of having lymphoma or leukemia, the diagnostic evaluation should include a cbc with differential cell count, platelet count, serum chemistry profile, and a test for felv and fiv. bone marrow aspiration or biopsy may be indicated to evaluate for possible involvement and complete staging of the extent of disease. bone marrow evaluation is particularly indicated if anemia, cellular atypia, and leukopenia are present. histopathologic evaluation of lymph nodes or involved organ tissue (procured by means of surgical incision, endoscopy, or needle core biopsy) is essential for a definitive diagnosis. in the cat, lymph node fine-needle aspiration alone is not sufficient in most cases because of the difficulty involved in distinguishing lymphoma from benign hyperplastic lymph node syndromes unique to the species; these include idiopathic peripheral lymphadenopathy, plexiform vascularization of lymph nodes, and peripheral lymph node hyperplasia of young cats. [71] [72] [73] [74] [75] whole lymph node excision is preferred in these cases, because determination of the orientation, invasiveness, and architectural abnormalities may be necessary for diagnosis. additional site-specific cytologic or histologic assessments may be warranted when extranodal sites are suspected. histologic and cytologic samples also can be analyzed by various histochemical and immunohistochemical techniques to determine the immunophenotype (b cell or t cell), tumor proliferation rate (e.g., ki-67, pcna, agnors), and telomerase activity (see chapter 14, section d), and histologic subtype (low, intermediate, or high grade). 10, 18, 26, 34, 43, 44, 46 the availability of such analysis is increasing, but currently none has proved to be predictive of outcome in cats. serum chemistry profiles can help establish the overall health and clinical staging of cats, but the results are not specific for a diagnosis. elevated liver enzymes may indicate hepatic infiltration with lymphoma, and elevated blood urea nitrogen (bun) and creatinine levels may indicate renal lymphoma. for cats with alimentary lymphoma, hypoproteinemia and anemia are reported to occur in up to 23% and 76% of cases, respectively. 25, 118 hypercalcemia is rare in cats but has been reported in patients with lymphoma at various anatomic sites. 25,66-68 elevated globulin levels may indicate the presence of a monoclonal gammopathy with or without serum hyperviscosity (this is a rarely reported paraneoplastic syndrome in cats with lymphoma). 119,120 hypoglycemia was reported in approximately one third of cats with lymphoma in an australian study. 118 serum alpha 1-acid glycoprotein concentrations have been reported to be elevated in cats with lymphoma, but no clinical relevance has been associated with this finding. 121 most cats with alimentary lymphoma develop a palpable abdominal mass or thickened intestinal loops. approximately one third of patients have a mass that can be visualized on abdominal radiographs, and about 90% have ultrasound abnormalities. 24, 25 the ultrasound abnormalities may include mesenteric lymphadenopathy in 33% to 50% of cases, an intestinal mass or thickening in approximately 40% of cases, and possibly splenomegaly (approximately 33% of cats with ultrasonographic changes in the spleen have lymphoma 122 ), hepatomegaly, or effusion. 24, 25 in one compilation of 28 cases, the lesions appeared ultrasonographically to be localized in 70% of cases and diffuse in 30%; however, at surgery only 33% were resectable. 25 for alimentary lymphoma, especially if primary gi lymphoma is suspected, caution must be exercised when endoscopically obtained tissue is used because of the difficulty in differentiating lymphoplasmacytic gastroenteritis from primary, diffuse, intestinal lymphoma. 123 however, diagnosis of this disease by means of endoscopically derived biopsies is improving with more advanced techniques and instrumentation. a wedge biopsy through serosa and muscularis, avoiding the mucosa, may be necessary to establish a diagnosis in cases in which endoscopic samples are equivocal. as an alternative, because nearly half of alimentary lymphomas have secondary mesenteric lymph node involvement, ultrasound-guided biopsy specimens or fine-needle aspirates may be adequate for a diagnosis. for cats with mediastinal lymphoma, diagnostic suspicion may begin with a noncompressible cranial thorax on physical examination and conformation of a mediastinal mass or pleural effusion on a thoracic radiograph (figure 31-14) . fine-needle aspiration of any suspected mass or cytologic evaluation of pleural fluid may be sufficient to establish a diagnosis. in most cats the finding of a monotonous population of lymphoblasts establishes a diagnosis. 65, 124, 125 however, definitive diagnosis of lymphoma in cats with a mediastinal mass and concurrent chylothorax can be challenging. 114 the ct appearance of these lesions has been 127 in chylous effusions, the pleural fluid triglyceride concentration is greater than in the serum; however, anorectic cats have lower triglyceride levels in the pleural fluid. a major differential diagnosis for mediastinal lymphoma is thymoma. the cytologic features of thymoma were recently described, and although these were found to be distinct from lymphoma in many cases, the diagnosis was challenging because of a preponderance of small lymphocytes in thymoma. 128 mast cells can also be seen in up to 50% of aspirate samples from thymomas. diagnostics for most extranodal forms of lymphoma are site specific. in cats suspected of having spinal lymphoma, survey radiographs of the spine rarely reveal osseous lesions. myelograms, ct, or mri is indicated, and in approximately 75% of cases, an extradural or intradural mass is detected. 22, 23, 83, 84 most lesions occur at a thoracolumbar or lumbosacral location. 83 fluoroscopic-guided, fine-needle aspiration of epidural lesions may yield enough tissue for a cytologic diagnosis. in most of the cats evaluated in one study, csf analysis revealed a clear, colorless fluid with a mixed pleocytosis (mean, 140 cells/µl; range, 0 to 1625 cells/ul) and an elevated protein content (mean, 140.7 mg/dl; range, 12 to 405 mg/dl). 23 malignant lymphocytes were identified in six of 17 cats evaluated with csf analysis. in a report of intracranial neoplasia, one of two cats with lymphoma had lymphoblasts in the csf fluid. 84 bone marrow and renal involvement are common in cats suspected of having cns lymphoma, and cytologic assessment of these organs generally is easier than in spinal sites. if nasal lymphoma is suspected, a biopsy specimen can be obtained either by intranasal procurement or by flushing one hemicavity with a bulb syringe and saline while occluding the contralateral cavity and collecting samples flushed out the nasopharynx (figure 31-15 ). thorough staging to make sure that the disease is confined to the nasal passages is recommended, because this presentation can be treated locally with radiotherapy if systemic involvement is ruled out. a ct scan is indicated to determine local involvement and to plan radiotherapy if it is to be pursued. for cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative sites, although overtly infected skin lesions should be avoided. complete staging to rule out systemic disease is also recommended for cats with cutaneous lymphoma, because local therapies can be applied in cases of solitary disease. in the case of renal lymphoma, physical examination findings of massive and often bilateral renomegaly raise the index of suspicion. the radiographic appearance is a smooth to irregular renomegaly ( figure 31-16, a) that is most consistent with either renal lymphoma or polycystic kidney disease. the disease usually is diffuse throughout the renal cortex ( figure 31-16, b) , and transabdominal needle aspiration or core biopsy is diagnostic in most cases. a who staging system exists for the cat that is similar to the one used in the dog (see box 31-1). however, because of the high incidence of visceral involvement in the feline species, another staging system is used more often (box 31-5). 21 because lymphoma in cats is more varied with respect to location and anatomic types, staging systems generally are less helpful for predicting response. occasionally the diagnosis of lymphoma and the differentiation of malignant and benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses may be helpful for confirming a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically is derived from expansion of a single malignant clone. this was discussed earlier in the chapter for lymphoma in the dog (see section a and figure 31-8) , and the same assays can be applied for advanced diagnosis of lymphoma in cats. 85 investigators have used pcr techniques to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in cats, and these techniques appear to be a useful adjunctive diagnostic tool, although they are undergoing a more thorough assessment. molecular techniques may also prove useful for assessing early recurrence, for more accurate clinical staging, and for providing so-called molecular remission rates, because they are more sensitive than the standard cytologic assessment of peripheral blood, bone marrow, or lymph nodes. our knowledge base for treating cats with lymphoma is less well established and less predictable than that for dogs, primarily because of the greater variation in histologic type and anatomic locations observed in felines. the chemotherapeutic agents most often used to treat feline lymphoma are similar to those used for dogs and humans with lymphoma (see section a); they include doxorubicin, vincristine, cyclophosphamide, methotrexate, l-asparaginase, ccnu, and prednisone.* in general, cats tolerate chemotherapy quite well. selected published protocols for the treatment of feline lymphoma are detailed in table 31 -8. most current combination protocols in north america are modifications of chop protocols initially designed for human oncologic use. in europe, cop (i.e., without the addition of doxorubicin) is used more often in cats with lymphoma, and one compilation reported results similar to those for chop. 133 some studies with relatively few case entries have reported limited activity for doxorubicin as a single agent in cats with lymphoma 19, 132 ; however, larger studies using combination protocols have more consistently reported that the addition of doxorubicin is necessary to attain more durable responses. 10, 129 in european and australian studies that reported less favorable doxorubicin responses, the patient populations consisted of a higher proportion of the mediastinal form in siamese cats, a population less frequently included in north american reports. 19, 133 the protocol the authors use in their practice is presented in table 31 -9. this protocol has been used in many cats with various forms of lymphoma and is well tolerated. currently, our canine lymphoma protocols (see section a) discontinue chemotherapy by week 25, and we have sufficient data exist to show that in dogs, this short, maintenance-free protocol is as good as if not superior to longer maintenance protocols. because long-term maintenance protocols have not proved superior to maintenance-free protocols in dogs and humans with lymphoma, the same likely holds true in the cat, although no data exist to document this. until such time as evidence to the contrary exists, we recommend discontinuation of chemotherapy at week 25 in cats that have attained complete remission. although doxorubicin appears to be the most effective agent for treating cats with lymphoma in north america, the species generally is less tolerant of doxorubicin than are dogs, and significant toxicity results when it is used at the dog dosage of 30 mg/m 2 given intravenously every 3 weeks. however, at lower dosages (e.g., 25 mg/m 2 or 1 mg/kg, given intravenously) doxorubicin can be used without significant toxicity. 10, 129 the major toxicities noted with doxorubicin are anorexia, myelosuppression, renal toxicity and, if perivascular leakage occurs, severe tissue damage. clinical doxorubicin-induced cardiac toxicity has not been documented in cats, but no information indicates that cats are resistant to myocardial damage. renal toxicity has been produced experimentally in rats and box 31-5 • single tumor (extranodal) with regional lymph node involvement • two or more nodal areas on the same side of the diaphragm • two single (extranodal) tumors with or without regional lymph node involvement on the same side of the diaphragm • resectable primary gastrointestinal tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes only rabbits and has been reported in cats. [136] [137] [138] in our experience, the incidence is significant enough that renal function in cats should be monitored closely (i.e., serial creatinine and urine specific gravity analysis) before each therapy session. a stealth liposomal form of doxorubicin has been shown to increase the likelihood of renal toxicity and therefore is limited in its use in this species. 138 a small number of cats with lymphoma have been treated with single agent ccnu at dosages ranging from 30 to 60 mg/m 2 given orally every 3 to 6 weeks. 130,131 although activity was noted, only partial responses were reported. little is known about the treatment of solitary or regional nodes of the head and neck that closely resemble hodgkin's lymphoma histologically. 1, 54 one study described the clinical outcome in four cats with this condition; these researchers found that the course of disease was prolonged, but after surgical excision of the single affected node, only one cat showed recurrence 6 months after surgery. 54 a second surgery was performed in this cat, and a second recurrence was noted, again 6 months after excision. cats with granular cell lymphoma or globule leukocyte tumors tend to respond poorly to chemotherapy, although durable responses have been reported. 102 currently, too few cases have been treated with aggressive chemotherapy to allow an assessment of response to therapy for this disease. a distinct form of intestinal lymphoma in cats that is composed of small, mature lymphocytes has been referred to as lymphocytic lymphoma. this syndrome has been reported to respond well to oral prednisone (10 mg/cat/day) and chlorambucil (15 mg/m 2 given once daily for 4 consecutive days out of every 3 weeks). a complete response rate of 69% was reported, with median remission and survival durations of 16 and 17 months, respectively. 62 radiation therapy has been used effectively to treat localized lymphoma, such as epidural, mediastinal, and nasal lymphoma. total doses of 10 to 15 gy usually result in a complete remission. 77 in one study, 10 cats with localized lymphoma were treated with radiation, alone or with chemotherapy, at doses of 8 to 40 gy. 78 eight of the 10 cats achieved complete remission, with a median remission duration of just over 2 years. radiation therapy also has been used effectively to treat nasal lymphoma. 78 complete response is the norm (80% to 100%), and median remission durations exceeding 1.5 years can be expected in felv-negative cats with disease confined to the nasal and paranasal cavities. for nasal lymphoma, radiotherapy appears to be superior to chemotherapy, for which reported median remission durations are 151 to 380 days. 10 very little has been published about the treatment of cutaneous lymphoma or mycosis fungoides in cats; however, a report of a complete response to ccnu exists. 135 cats with a solitary mass should be treated with surgical excision, although clinical staging is necessary to rule out possible internal involvement. combination chemotherapy can be considered if multiple sites are involved. if the disease is localized to a small region, radiation therapy usually is also effective. mycosis fungoides may be treated effectively in dogs with retinoids, such as isotretinoin (accutane) at a dosage of 3 to 4 mg/kg given orally daily or etretinate (tegison) at a dosage of 1.25 mg/kg given orally daily; however, no providing precise treatment recommendations for the wide variety of clinical settings of cats with lymphoma is difficult. our current recommendation is to treat cats with lymphoma using the chop-based protocols (see table 31 -9). doxorubicin alone (25 mg/m 2 given iv every 3 weeks for five total treatments) or palliative prednisone therapy is offered if the client declines more aggressive therapy. nutritional support is especially important, particularly for cats with alimentary lymphoma. a feeding tube may need to be placed in cats undergoing chemotherapy for alimentary lymphoma, particularly if anorexia is a factor (see chapter 16, section b.) ultimately, most cats with lymphoma that are successfully treated with chemotherapy have a relapse of the disease. this often represents a recrudescence of the tumor in a more drug-resistant form. at the first recurrence, reinduction first should be attempted by using the induction protocol that was successful initially. in general, the likelihood of a response and the length of the response are half those for the initial therapy; however, a subset of animals enjoy long-term reinduction. if reinduction fails or if the cat does not respond to the initial induction, so-called rescue agents or rescue protocols can be attempted. these are drugs or drug combinations that typically are not found in the standard chop protocol and are withheld for use in drugresistant cases. a number of rescue protocols have been reported in the veterinary literature and were reviewed previously. 139,140 the most common rescue protocols advocated for cats with resistant relapse include single agent use or combination use of mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), ccnu, and mopp. in general, overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable; median responses of 1.5 to 2 months are the norm. a small subset of animals enjoy longer rescue durations. in general, cats do not enjoy response rates as high or remission and survival durations as long as dogs with lymphoma. complete response rates range from 50% to 70% after combination chemotherapy, and overall median remission and survival durations are approximately 4 and 6 months, respectively. 1,5,6,10,129 however, a significant proportion of cats (25% to 30%) that achieve a complete response with combination chemotherapy enjoy more durable overall remission and survival times (i.e., 1 year or longer). the response rate and the length of response vary according to the presence or absence of several prognostic factors. the wide variation in frequency and the great heterogeneity of anatomic forms of lymphoma in cats make specific prognostications more difficult than in dogs with lymphoma. most studies have lumped anatomic groups together to produce remission and survival data, and individual group numbers generally have been too small to apply statistical analysis with meaningful power. that being said, the factors that appear to be most strongly associated with a more positive prognosis in cats are a complete response to therapy (figure 31-17) , which unfortunately cannot be determined before treatment; negative felv status ( figure 31-18) ; early clinical stage (figure 31-19) ; anatomic location; and addition of doxorubicin to the treatment protocol. 5,10,24,129 unlike in the dog, cd3 immunoreactivity has not been established as a negative prognostic factor in the cat. 10 early reports may contradict more recent studies partly because of the decline in felv-associated lymphoma, and patients reported in the early literature may not equate to more recent populations studied. in general, felv-negative cats that achieve a complete response on chop-based protocols have a high likelihood of long-term survival, proportion alive with approximately 30% alive at 1.5 years after diagnosis. for cats with alimentary lymphoma, median survival times of 7 to 10 months are expected with chemotherapy that includes doxorubicin. 10, 26, 129 in one study of 28 cats with alimentary lymphoma in which the treatment protocol did not include doxorubicin, the median survival time was only 50 days. 25 mediastinal lymphoma in felv-positive young cats is associated with the poorest prognosis, and survivals times of approximately 2 to 3 months are expected with chemotherapy. 6, 10, 19 in contrast, older, felv-negative siamese cats with mediastinal lymphoma appear to experience remission rates approaching 90%, and responses tend to be quite durable. overall, cats with nasal lymphoma have the best prognosis, because local radiotherapy (or chemotherapy if radiotherapy is not available) generally results in excellent control, with median survival times approaching 1.5 years. 10,78 however, cats with nasal lymphoma and concurrent felv infection have much shorter survival durations. renal lymphoma is associated with a shorter survival time, with published medians ranging from 3 to 6 months. 1,6,10,76 in a study by mooney and colleagues, 76 28 cats with primary renal lymphoma were treated with combination chemotherapy. a complete response (cr) was noted in 17 cats (61%), and nine (32%) had a partial response. the median survival times were 4 months for the cats that showed a complete response and 1 month for those that showed a partial response. the duration of response to chemotherapy did not correlate with the degree of renal insufficiency except in cats with a bun higher than 150 mg/dl. extension to the cns occurred in 40% of these treated cats. the investigators revised their chemotherapy protocol to include cytosine arabinoside, which can penetrate the blood barrier, theoretically to prevent or reduce cns metastasis; however, definitive improvement with this modification has not been documented. few studies have reported on the results of treatment of cats with spinal lymphoma. in one study of four cats treated with chemotherapy (l-asparaginase, vincristine, and prednisone) combined with spinal radiation (n = 3) or surgical cytoreduction (n = 1), most of the cats were euthanized by 5 months, although one cat survived 13 months. 23 in another study of nine cats treated with chemotherapy (vincristine, cyclophosphamide, and prednisone), three cats achieved a complete response with a duration of 14 weeks and three achieved a partial response with a duration of 6 weeks. 22 one cat treated with dorsal decompression laminectomy and chemotherapy survived 13 months. although the numbers are small overall, the prognosis for spinal lymphoma is poor. section c of this chapter presents a complete discussion of leukemias and mpds, including a general discussion of hematopoiesis, etiologies, lineage classification, and descriptions. the classification of leukemias in cats is difficult because of the similarity of clinical and pathologic features and the transition, overlap, or mixture of cell types involved. 110, [141] [142] [143] [144] [145] [146] [147] [148] leukemia is a neoplastic proliferation of hematopoietic cells that originate in the bone marrow. cell lineage includes myeloid cells, neutrophils, basophils, eosinophils, monocytes, lymphoid cells, megakaryocytes, and erythrocytes. box 31-6 presents a classification scheme for the leukemias identified in the cat. leukemias also must be classified according to their degree of differentiation. 110 well-differentiated leukemia is referred to as chronic leukemia, and poorly differentiated leukemia usually is called acute leukemia; this distinction is very important in the therapeutic management and prognosis of leukemias. cats with acute leukemia usually show signs of severe anemia (pale mucous membranes), splenomegaly, and febrile episodes. cats with chronic leukemia may have a longer duration of signs and mild anemia with or without splenomegaly. in cats suspected of having leukemia, bone marrow aspiration or biopsy usually is diagnostic. the preferred sites for bone marrow aspiration are the proximal humerus and the iliac crest. cats with acute leukemia are likely to have malignant cellular infiltrates in organs other than the bone marrow. 144 the bone marrow aspirate must contain more than 30% abnormal blast cells to support a diagnosis of an acute leukemia. in cats suspected of having cll, infiltration of the bone marrow with more than 20% mature lymphocytes helps confirm the diagnosis. all cats with leukemia should be tested for felv and fiv. determining the lineage of some leukemias can be challenging; most can be distinguished from one another by histologic appearance, histochemical stains, immunohistochemical analysis of cell surface antigens, and flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 141, 143, 149, 150 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. the french-american-british (fab) classification system (box 31-7) is considered useful in cats with myelodysplastic syndromes, and almost all these patients are felv antigenemic. 151,152 lymphoid leukemia is the most common leukemia in cats. approximately 25% of cats with lymphoma also have a leukemic blood profile. 153 all is the most common lymphoid leukemia. 154 all is characterized by the presence of poorly differentiated lymphoblasts and prolymphocytes in the blood and bone marrow. 155 most cats with all have normal to low wbc counts. a few cats have leucocytosis with circulating blasts. a moderate to marked anemia is common. bone marrow aspiration usually reveals extensive infiltration with lymphoblasts. approximately 60% to 80% of cats with all are felv positive, and most malignant cells have a t-cell phenotype. 156 cll, which is rarely reported in cats, is characterized by the presence of well-differentiated, small, mature lymphocytes in the peripheral blood and bone marrow. 155,156 although most of these cells are of the t-cell lineage (particularly t helper), b-cell cll has been reported. 150, 157, 158 most cats with cll have an elevated wbc count (i.e., over 50,000/µl), and most are felv negative. the total leukocyte count varies and may range from leukopenia to marked leukocytosis. chronic granulocytic leukemia (gl) must be distinguished from leukemoid reactions associated with infections. acute gl is characterized by a large percentage of myeloblasts and/or progranulocytes in the blood and bone marrow. myeloblasts can be difficult to distinguish from lymphoblasts, but the former have a finer chromatin pattern, a smaller nucleus-to-cytoplasm ratio, more prominent or multiple nucleoli, and sometimes cytoplasmic granules. it is not uncommon in cats with gl to have no recognizable neoplastic cells in the peripheral blood. the bone marrow is hypercellular as a result of granulocytic leukemia cells. 141,145,146,159 myelomonocytic leukemia (mml) results from malignant transformation of both neutrophils and monocytes. this form of leukemia is one of the most common forms reported. monocytic leukemia (ml) is a rarely reported leukemia. it generally is considered an acute leukemia regardless of the morphologic appearance of the cells. eosinophilic leukemia (el) is rarely diagnosed in cats and is considered a variant of chronic granulocytic or myeloid leukemia. 148,160 felv has induced el experimentally. 160 mature eosinophils outnumber immature stages, and anemia is uncommon in cats associated with el. cats usually have an eosinophil count of 15,000 cells/µl or higher with or without immature cells in the peripheral blood. the bone marrow shows hyperplasia of eosinophilic precursors, and the myeloid to erythroid ratio (m:e) is significantly increased. organ infiltration, such as in lymph nodes, the spleen, and the liver, can be seen. 161 in establishing a diagnosis of el, it is important to rule out eosinophilic enteritis, parasitism, eosinophilic granuloma complex, and allergic disorders. diagnosing el can be very difficult because of the hypereosinophilic syndrome (hes) seen with other disease conditions in cats. [161] [162] [163] hes is characterized by a marked increase in the eosinophil count, bone marrow hyperplasia of eosinophilic precursors, and multiple organ infiltration by mature eosinophils. most cats have signs related to gi involvement. basophilic leukemia (bl) is considered a variant of chronic granulocytic leukemia. only one confirmed case of basophilic leukemia has been reported in the cat. it is important to differentiate bl from systemic mastocytosis with mast cell leukemia. mast cells have numerous cytoplasmic granules and round nuclei. basophils have segmented nuclei and cytoplasmic granules that superimpose on the nucleus, giving it a moth-eaten appearance. erythremic myelosis [165] [166] [167] [168] erythremic myelosis (em) is an mpd characterized by excessive proliferation of erythroid elements, which results in an increase in nucleated erythrocytes (rubriblasts to metarubricytes). severe anemia is common, and the peripheral blood shows numerous nucleated erythrocytes, moderate to marked anisocytosis, and an increased erythrocyte mean cell volume. the bone marrow contains a preponderance of normal-appearing erythrocyte precursors. some cats undergo blast transformation to myeloblastic, granulocytic, or a poorly differentiated leukemia (previously called reticuloendotheliosis). the transition from erythremic myelosis to erythroleukemia to acute granulocytic leukemia is well recognized in humans. 169 110, 143, 146 erythroleukemia, or acute erythremic myelosis, can develop from blast transformation of erythremic myelosis. primitive erythroid precursors in the blood and bone marrow predominant. primitive cells resembling myeloblasts often are present in low numbers. [170] [171] [172] [173] primary erythrocytosis is rarely reported in cats, and the diagnosis is based on an elevated pcv (65% to 80%) with low to normal serum erythropoietin activity. most clinical signs are associated with an increased red blood cell (rbc) volume, which increases blood volume and viscosity, causing impaired blood flow, stasis, and tissue hypoxia. neurologic signs such as seizures, blindness, and mental depression are common. 172 the oral mucous membranes may appear brick red. it is important to differentiate this condition from secondary polycythemia caused by renal tumors, chronic hypoxia, and right-to-left cardiac shunts. [174] [175] [176] megakaryocytic leukemia is characterized by abnormal megakaryocytic hyperplasia in the bone marrow. the megakaryocytes are morphologically abnormal, and some are small (dwarf megakaryocytes) and have few or no nuclear lobulations. thrombocytopenia or thrombocytosis may be present. in humans this form of leukemia often is associated with extensive marrow fibrosis and an increase in reticulum or collagen. 169 primary thrombocythemia is a very rare, chronic mpd characterized by proliferation of megakaryocytes and elevated platelet counts exceeding 1 million. giant platelets and platelets with abnormal morphology may be seen in the peripheral blood. one case has been reported in the cat. [178] [179] [180] [181] [182] malignant histiocytosis, a rare condition in cats, is characterized by systemic proliferation of malignant macrophages (histiocytes) and their precursors. a distinguishing characteristic of this disease is erythrophagocytosis. hepatosplenomegaly with progressive anemia (sometimes coombs positive) and thrombocytopenia are characteristic. the erythrophagocytosis may be confused with a possible immune-mediated anemia. bone marrow biopsy, rather than aspiration, and splenic biopsy may be necessary to establish a diagnosis. special stains using acid phosphatase and nonspecific esterase with fluoride inhibition (naphthol butyrate substrate) may be necessary to indicate macrophage origin. metaplasia [183] [184] [185] [186] myelofibrosis and myeloid hyperplasia are characterized by abnormal growth and differentiation of erythroid, myeloid, and megakaryocytic cell types with varying proliferation of fibroblasts in the marrow. anemia, leukopenia, or thrombocytopenia or varying combinations are common. myelofibrosis has been diagnosed in felv-positive cats and is directly associated with the virus rather than a consequence of myeloproliferative disorders. myeloid metaplasia may terminate in acute leukemia and therefore may be considered a preleukemic event. the use of chemotherapy to treat all has been disappointing. a 27% complete response rate has been reported with a cop regimen. 1, 10, 138, 187 in 15 cats treated with cop for all, four achieved a cr and 6 cats had a partial response. 1 the median remission was 7 months (range, 1 to 24 months). one report described a short-term remission in a cat with lymphoid leukemia that was treated with a low dose of cytosine arabinoside (10 mg/m 2 given subcutaneously twice daily). 188 cll can be treated with chlorambucil (0.2 mg/kg given orally or 2 mg/cat given every other day) and prednisone at a dose of 1 mg/kg given orally daily. cats with cll have a better prognosis and survive 1 to 2 years when treated with chlorambucil. as in humans and dogs, treatment can be withheld if the patient has no significant clinical signs and no profound cytopenias. in one study, a cat with cll remained stable without chemotherapy for over a year. 158 the prognoses for acute nonlymphoblastic leukemias generally are very poor, although some exceptions exist. a treatment regimen consisting of a combination of cytosine arabinoside and cyclophosphamide and multiple blood transfusions was effective at inducing a response for 3 months in a cat with acute megakaryocytic leukemia. 176 hydroxyurea (hydrea) can be used to treat chronic myeloid leukemia and primary erythrocytosis. cats with primary erythrocytosis that go untreated are reported to survive 6 to 20+ weeks. 170, 172 phlebotomy alone every 2 to 3 months was used to treat one cat, which survived longer than 20 months. 171 hydroxyurea treatment for primary erythrocytosis was used in eight cats, and all survived longer than 1 year. 172 hydroxyurea is available in 500 mg capsules, and the dosage is 25 to 50 mg/kg given orally daily. some cats have been given 500 mg every 5 to 7 days, but methemoglobulinemia and hemolytic anemia with heinz bodies has been seen. 172 a better recommendation is to have the drug formulated into 125 mg capsules, which is a more appropriate dosage. however, care must be used in making these capsules because hydroxyurea is potentially carcinogenic. a recommended treatment schedule for hydroxyurea is 125 mg daily to every other day, depending on the type of leukemia under treatment. the drug is tolerated very well at this dosage. a case of acute ml in a cat undergoing treatment with cytarabine, doxorubicin, vincristine, and prednisolone has been reported; a partial remission was noted for approximately 2 months, and the cat survived for 3 months after diagnosis. 147 a retrospective study subcommittee to re-examine criteria for a classification system and to spearhead large multi-institutional studies to validate the criteria. mpds are uncommon or rare in the dog; they occur 10 times less frequently than lymphoproliferative disorders. 3 accurate information about the incidence and other epidemiologic information await consistent use of a uniform classification system (see later discussion). no known age, breed, or gender predisposition exists, although large breed dogs have been overrepresented in some retrospective studies. [4] [5] [6] [7] [8] [9] [10] [11] [12] in dogs, the etiology of spontaneously occurring leukemia is unknown. genetic and environmental factors (including exposure to radiation, drugs, or toxic chemicals) probably play a role. in humans, acquired chromosomal derangements lead to clonal overgrowth with arrested development. 13 chromosomal abnormalities have been reported in dogs with acute myeloid leukemia (aml), chronic myelogenous leukemia (cml), and lymphoid leukemia. 14, 15 however, because karyotyping is difficult to perform in dogs, owing to the large number and morphologic similarity of their chromosomes and their resistance to banding, definition of the genetic factors in canine mpds awaits application of molecular techniques and use of the canine genome map. 14, 16, 17 certain forms of leukemia in dogs have been produced experimentally by irradiation. [18] [19] [20] in contrast to mpds in cats, no causative viral agent has been demonstrated in dogs, although retrovirus-like budding particles were observed in the neoplastic cells of a dog with granulocytic leukemia. 21 a review of normal hematopoiesis can aid the clinician's understanding of the various manifestations of mpds. hematopoiesis is the process of proliferation, differentiation, and maturation of stem cells into terminally differentiated blood cells (a simplified schematic is presented in figure 31 -20) . pluripotent stem cells differentiate into either lymphopoietic or hematopoietic multipotent stem cells. 22 under the influence of specific regulatory and microenvironmental factors, multipotent stem cells in bone marrow differentiate into progenitor cells committed to a specific hematopoietic cell line, such as erythroid, granulocyticmonocytic, or megakaryocytic cells. maturation results in the production of terminally differentiated blood cells-erythrocytes, granulocytes, monocytes, and platelets-that are delivered to the circulation. in some cases, as in the maturation of reticulocytes to erythrocytes, final development may occur in the spleen. the proliferation and differentiation of hematopoietic cells are controlled by a group of regulatory growth factors. 22, 23 of these, erythropoietin is the best characterized; it regulates erythroid proliferation and differentiation and is produced in the kidneys, where changes in oxygen tension are detected. the myeloid compartment depends on a group of factors, collectively referred to as colony-stimulating factors (csfs). these factors act at the level of the committed progenitor cells but also influence the functional capabilities of mature cells. some of these factors have a broad spectrum of activity; others are more restricted in their target cells and actions. csfs are produced in vitro by a multitude of cell types, including monocytes, macrophages, lymphocytes, and endothelial cells, and these cells likely play a role in the production and regulation of these factors in vivo. the gene for thrombopoietin also has been cloned, and this hormone alone apparently can induce differentiation of megakaryocytes and platelet production. 24 recombinant forms of many of these hormones are becoming increasingly available. the clonal disorders of bone marrow include myeloaplasia (usually referred to as aplastic anemia), myelodysplasia, and myeloproliferation. a preleukemic syndrome, characterized by peripheral pancytopenia and bone marrow hyperplasia with maturation arrest, is more correctly called myelodysplasia because the syndrome does not always progress to overt leukemia. this syndrome has been described in cats, usually in association with felv infection, but it has only rarely been recognized in dogs. [25] [26] [27] [28] mpds may be manifested by abnormalities in any or all of the different cell lines, because hematopoietic cells share a common stem cell. in addition, transformation from one mpd to another may occur. 29 mpds are classified in several ways. the terms acute and chronic refer to the degree of cellular differentiation of the leukemic cells, but these terms also correlate with the biologic behavior of the neoplasm. 30 disorders resulting from uncontrolled proliferation of cells incapable of maturation lead to the accumulation of poorly differentiated, or blast, cells. these disorders have been called acute mpds or acute nonlymphocytic leukemias, but they now are included under the umbrella term acute myeloid leukemia. disorders resulting from unregulated proliferation of cells that exhibit progressive, albeit incomplete and defective, maturation lead to the accumulation of differentiated cells. these disorders are called chronic mpds. they include polycythemia vera, cml and its variants, essential thrombocythemia, and possibly primary myelofibrosis. mpds are further classified by the lineage of the dominant cell type or types, as defined by romanowsky stains, special cytochemical stains, ultrastructural features, and immunologic cell markers, and they recently have been classified into subtypes (see later discussion). acute leukemias have a more sudden onset and are more aggressive. in both acute and chronic disorders, however, abnormalities in proliferation, maturation, and functional characteristics can occur in any hematopoietic cell line. 1 in addition, normal hematopoiesis is adversely affected. animals with leukemia usually have decreased numbers of circulating normal cells. the pathogenesis of the cytopenias is complex and may result in part from production of inhibitory factors. eventually, neoplastic cells displace normal hematopoietic cells, a process called myelophthisis. anemia and thrombocytopenia are particularly common. neutropenia and thrombocytopenia result in infection and hemorrhage, which may be more deleterious to the animal than the primary disease process. despite the disseminated nature of the disease at the time of diagnosis, parenchymal organ dysfunction usually occurs only in very advanced cases of mpd. aml is rare and is characterized by aberrant proliferation of a clone of cells without maturation. this results in the accumulation of immature blast cells in the bone marrow and peripheral blood (figure 31-21) . the wbc count varies, ranging from leukopenia to counts greater than 150,000/ml. the spleen, liver, and lymph nodes commonly are involved, and other tissues may be infiltrated as well, including the tonsils, kidneys, heart, and cns. no characteristic age has been noted, and even very young dogs may be affected. 31 the clinical course of these disorders tends to be rapid. production of normal peripheral blood cells usually is diminished or absent, and anemia, neutropenia, and thrombocytopenia are common. infection and hemorrhage are common sequelae. occasionally, malignant blasts are present in the bone marrow but not the peripheral blood; this is called aleukemic leukemia. subleukemic leukemia suggests a normal or decreased wbc with some neoplastic cells in circulation. in 1985 the animal leukemia study group was formed under the auspices of the american society for veterinary clinical pathology to develop specific morphologic and cytochemical criteria for classifying acute nonlymphocytic leukemias in dogs and cats. recognition of specific subtypes of leukemia is necessary for the accumulation of accurate, useful information about prognosis and response to treatment and for comparison of studies from different sites. in 1991 this group proposed a classification system following adaptation of the french-american-british (fab) system and criteria established by the national cancer institute workshop. 2 the group members examined blood and bone marrow from 49 dogs and cats with mpds. romanowsky-stained specimens were examined first to identify blast cells and their percentages. lineage specificity then was determined using cytochemical markers. the percentage of blasts and the information about lineage specificity were used in combination to classify disorders as acute undifferentiated leukemia (aul), acute myeloid leukemia (aml, subtypes m1 to m5 and m7), and erythroleukemia with or without erythroid predominance (subtypes m6 and m6er) (see box 31-7). except for acute promyelocytic leukemia (subtype m3), all these subtypes have been described in dogs. however, because this modified fab system has only recently been adopted, the names given to these disorders in the literature vary considerably. in addition, in the absence of special cytochemical staining, immunophenotyping, and/or electron microscopy, the specific subtype of leukemia often has been uncertain, making retrospective analysis of epidemiologic information, prognosis, and response to therapy confusing at best. although defining specific subtypes may seem to be an academic exercise owing to the uniformly poor prognosis of acute leukemias, this information is critical to improving the management of these diseases. because of the low incidence of mpds, national and international cooperative efforts are required to accumulate information on the pathogenesis of specific subtypes and their response to different treatment modalities; use of a uniform classification system is an essential first step. different forms of aml are shown in figure 31 -21, a-e. the most frequently reported forms of aml in the dog are acute myeloblastic leukemia (m1 and m2) and acute myelomonocytic leukemia (m4). [3] [4] [5] [6] [7] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [32] [33] [34] [35] [36] [37] [38] [39] megakaryoblastic leukemia (m7) also is well recognized in dogs 8, [40] [41] [42] [43] [44] [45] [46] and may be associated with platelet dysfunction. 44 monocytic leukemias likely have included those with and without monocytic differentiation (m5a and m5b), 9, 47 but in some cases the diagnosis may have been chronic myelomonocytic or chronic monocytic leukemia (see later discussion). there are few reports in dogs of spontaneously occurring erythroleukemia (m6) in which the leukemic cells comprise myeloblasts, monoblasts, and erythroid elements. 48, 49 auls have uncertain lineages, because they are negative for all cytochemical markers. these leukemias should be distinguished from lymphoid leukemias by flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 50 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. chronic mpds are characterized by excessive production of differentiated bone marrow cells, which results in the accumulation of erythrocytes (polycythemia vera), granulocytes and/or monocytes (chronic myelogenous leukemia and its variants), or platelets (essential thrombocythemia). primary myelofibrosis as a clonal disorder of marrow stromal cells, characterized by proliferation of fibroblasts with accumulation of collagen in bone marrow, is not recognized in animals. myelofibrosis is considered a response to injury and may occur secondary to mpds. polycythemia vera (pv) is a clonal disorder of stem cells, although whether the defect is in the pluripotent stem cell or the hematopoietic multipotent stem cell is still unclear. in humans, progenitor cells have an increased sensitivity to insulin-like growth factor 1 (igf-1), which stimulates hematopoiesis. 51 whether this hypersensitivity is the primary defect or occurs secondary to another gene mutation is unknown. in any case, the result is overproduction of rbcs. the disease is rare and must be distinguished from more common causes of polycythemia, including relative and secondary absolute polycythemia (see later discussion). in pv, neoplastic proliferation of the erythroid series with terminal differentiation to rbcs occurs. the disease has been reported mostly in middle-aged dogs, and no breed or gender predilection has been noted. [52] [53] [54] [55] [56] [57] [58] [59] [60] pv is characterized by an increased rbc mass, evidenced by an elevated pcv, rbc count, and hemoglobin concentration. the pcv typically is in the range of 65% to 85%. the bone marrow is hyperplastic, although the m:e tends to be normal. in contrast to the disease in humans, other cell lines do not appear to be involved, and transformation to other mpds has not been reported. the disease in dogs may be more appropriately called primary erythrocytosis. cml is a neoplastic proliferation of the neutrophil series, although concurrent eosinophilic and basophilic differentiation can occur. cml can occur in dogs of any age. 31, [61] [62] [63] [64] [65] neutrophils and neutrophilic precursors accumulate in the bone marrow and peripheral blood and invade other organs. the peripheral wbc count usually, but not always, is higher than 100,000/ml. both immature and mature neutrophils are present (see figure 31 -21, f). mature forms usually are more numerous, but sometimes an "uneven" left shift is present. signs of dysplasia may be evident, including hypersegmentation, ringed nuclei, and giant forms. eosinophils and basophils may also be increased. the bone marrow is characterized by granulocytic hyperplasia, and morphologic abnormalities may not be present. erythroid and megakaryocytic lines may be affected, resulting in anemia, thrombocytopenia or, less commonly, thrombocytosis. cml must be distinguished from severe neutrophilic leukocytosis and "leukemoid reactions" caused by inflammation or immune-mediated diseases. leukemoid reactions also can occur as a paraneoplastic syndrome. in humans with cml, characteristic cytogenetic abnormalities are present in all bone marrow cells, signifying a lesion at the level of an early multipotent stem cell. typically these individuals have a chromosomal translocation, resulting in the philadelphia chromosome. 66 no consistent cytogenetic abnormalities have been demonstrated in spontaneously occurring cml in dogs. variants of cml are chronic myelomonocytic leukemia (cmml) and chronic monocytic leukemia (cmol). these diagnoses are made based on the percentage of monocytes in the leukemic cell population. besides accumulating in the bone marrow and peripheral blood, leukemic cells invade the red pulp of the spleen, the periportal and sinusoidal areas of the liver, and sometimes the lymph nodes. other organs (e.g., the kidneys, heart, and lungs) are less commonly affected. in addition, extramedullary hematopoiesis may be present in the liver and spleen. death usually results from complications of infection or hemorrhage secondary to neutrophil dysfunction and thrombocytopenia. in some cases, cml may terminate in "blast crisis," in which a transformation occurs from a predominance of well-differentiated granulocytes to excessive numbers of poorly differentiated blast cells in the peripheral blood and bone marrow; this phenomenon is well documented in the dog. 61, 62, 64 basophilic leukemia, although rare, has been reported in dogs and is characterized by an elevated wbc count with a high proportion of basophils in the peripheral blood and bone marrow. [67] [68] [69] hepatosplenomegaly, lymphadenopathy, and thrombocytosis may be present, and these dogs have all been anemic. basophilic leukemia should be distinguished from mast cell leukemia (mastocytosis). whether dogs develop eosinophilic leukemia remains in question. reported cases have had high blood eosinophil counts and eosinophilic infiltrates in organs. 70, 71 one dog responded well to treatment with corticosteroids. the distinction between neoplastic proliferation of eosinophils and idiopathic hypereosinophilic syndrome remains elusive. disorders associated with eosinophilia (e.g., parasitism, skin diseases, and diseases of the respiratory and gi tracts) should be considered first in an animal with eosinophilia. one distinguishing feature should be clonality, with reactive eosinophilia comprising polyclonal cells and the neoplastic condition arising from a single clone. as clonality assays become more available, this discrepancy may be resolved. in humans, essential thrombocythemia, or primary thrombocytosis, is characterized by platelet counts that are persistently higher than 600,000/ml. no blast cells are in circulation, and marked megakaryocytic hyperplasia of the bone marrow without myelofibrosis is present. thrombosis and bleeding are the most common sequelae, and most patients have splenomegaly. other mpds, especially pv, should be ruled out, and the patient should have no primary disorders associated with reactive thrombocytosis, 72 such as inflammation, hemolytic anemia, iron deficiency anemia, malignancies, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, or abscence of a spleen. in addition, certain drugs, such as vincristine, can induce thrombocytosis. essential thrombocythemia has been recognized in dogs. 29, [73] [74] [75] [76] in one dog, the platelet count exceeded 4 million/ml, and bizarre giant forms with abnormal granulation were present. the bone marrow contained increased numbers of megakaryocytes and megakaryoblasts, but circulating blast cells were not seen. other findings included splenomegaly, gi bleeding, and increased numbers of circulating basophils. causes of secondary or reactive thrombocytosis were ruled out. 73 basophilia also was reported in a more recent case. 75 in another dog, primary thrombocytosis was diagnosed and then progressed to cml. 29 in some cases reported in the literature as essential thrombocythemia, the dogs had microcytic hypochromic anemias. because iron deficiency anemia is associated with reactive or secondary thrombocytosis, care must be taken to rule out this disorder. however, spurious microcytosis may be reported if a dog has many giant platelets that are counted by an analyzer as small rbcs. 76 microscopic review of the blood film may be helpful in these cases. primary myelofibrosis with clonal proliferation of marrow fibroblasts has not been reported in dogs. 77 in humans, myelofibrosis is characterized by collagen deposition in the bone marrow and increased numbers of megakaryocytes, many of which have morphologic abnormalities. in fact, breakdown of intramedullary megakaryocytes and subsequent release of factors that promote fibroblast proliferation or inhibit collagen breakdown may be the underlying pathogenesis of the fibrosis. 78 focal osteosclerosis sometimes is present. anemia, thrombocytopenia, splenomegaly, and myeloid metaplasia (production of hematopoietic cells outside the bone marrow) are consistent features. the extramedullary hematopoiesis is ineffective at maintaining or restoring normal peripheral blood counts. in dogs, myelofibrosis occurs secondary to mpds, radiation damage, and congenital hemolytic anemias. [79] [80] [81] [82] in some cases the inciting cause is unknown (idiopathic myelofibrosis). concurrent marrow necrosis may occur in cases of ehrlichiosis, septicemia, or drug toxicity (estrogens, cephalosporins), and some speculate that fibroblasts proliferate in response to release of inflammatory mediators associated with the necrosis. 77 myeloid metaplasia has been reported to occur in the liver, spleen, and lungs. 82 extramedullary hematopoiesis is ineffective at preventing or correcting the pancytopenia that eventually develops. dysfunction of the hematopoietic system can be manifested by a variety of abnormalities that comprise myelodysplastic syndromes (mds). in dogs, in which the syndrome is rare, cytopenias usually are seen in two or three lines in the peripheral blood (anemia, neutropenia, and/or thrombocytopenia). other blood abnormalities can include macrocytic erythrocytes and metarubricytosis. the bone marrow typically is normocellular or hypercellular, and dysplastic changes are evident in several cell lines. if blast cells are present, they account for fewer than 30% of all nucleated cells, 2 although this threshold may be altered to fewer than 20%. 16 myelodysplasia sometimes is called preleukemia because in some cases it may progress to an acute leukemia. [25] [26] [27] in humans and in cats, mds are clonal disorders and are considered neoplastic. dogs with mpds have similar presentations regardless of the specific disease entity, although animals with acute mpds have a more acute onset of illness and a more rapid clinical course. a history of lethargy, inappetence, and weight loss is common. clinical signs include emaciation, persistent fever, pallor, petechiation, hepatosplenomegaly and, less commonly, lymphadenopathy and enlarged tonsils. shifting leg lameness, ocular lesions, and recurrent infections also are seen. vomiting, diarrhea, dyspnea, and neurologic signs are variable features. serum chemistries may be within reference intervals but can change if significant organ infiltration occurs. animals with msd may be lethargic and anorectic and may have pallor, fever, and hepatosplenomegaly. in pv, dogs often have erythema of mucous membranes because of the increased rbc mass. some dogs are polydipsic. in addition, neurologic signs may be present, such as disorientation, ataxia, or seizures, and these are thought to be the result of hyperviscosity or hypervolemia. 56 hepatosplenomegaly usually is absent. peripheral blood abnormalities are consistently found. in addition to the presence of neoplastic cells, other abnormalities may be present, including a decrease in the numbers of normal cells of any or all hematopoietic cell lines. occasional nucleated rbcs are present in the blood of about half of dogs with acute nonlymphocytic leukemia. 2 nonregenerative anemia and thrombocytopenia are present in most cases. the anemia usually is normocytic and normochromic, although macrocytic anemia sometimes is present. pathogenic mechanisms include the effects of inhibitory factors, leading to ineffective hematopoiesis, myelophthisis, immune-mediated anemia secondary to neoplasia, and hemorrhage secondary to thrombocytopenia, platelet dysfunction, or dic. anemia is most severe in acute mpds, although both anemia and thrombocytopenia may be milder in animals with the m5 subtype (acute monocytic leukemia). in myelofibrosis, the anemia is characterized by anisocytosis and poikilocytosis. in addition, pancytopenia and leukoerythroblastosis, in which immature erythroid and myeloid cells are in circulation may be present. these phenomena probably result from the replacement of marrow by fibrous tissue, with resultant shearing of red cells and escape of immature cells normally confined to bone marrow. in pv, the pcv is elevated, usually in the range of 65% to 85%. the bone marrow is hyperplastic, and the m:e usually is in the normal range. the neoplastic cells often are defective functionally. platelet dysfunction has been reported in a dog with acute megakaryoblastic leukemia (m7), 44 and in cml, the neutrophils have decreased phagocytic capacity and other abnormalities. one exception to this was a report of cml in a dog in which the neutrophils had enhanced phagocytic capacity and superoxide production. 83 the authors hypothesized that increased synthesis of granulocyte-macrophage colony-stimulating factor (gm-csf) resulted from a lactoferrin deficiency in the neoplastic neutrophils and mediated the enhanced function of these cells. in all cases of mpd, the diagnosis depends on the examination of peripheral blood and bone marrow. acute mpds are diagnosed on the basis of finding blast cells with clearly visible nucleoli in blood and bone marrow. most dogs with acute leukemia have circulating blasts. these cells may be present in low numbers in peripheral blood, and the smear should be searched carefully, especially at the feathered edge. even if blasts are not detected in circulation, indications of bone marrow disease, such as nonregenerative anemia or thrombocytopenia, usually are present. occasionally neoplastic cells can be found in csf in animals with invasion of the cns. smears of aspirates from tissues such as the lymph nodes, spleen, or liver may contain blasts but usually contribute little to the diagnostic workup. examination of blasts stained with standard romanowsky stains may give clues to the lineage of the cells (see figure 31 -21, a to c and e). in aml, in addition to myeloblasts, some progranulocytes with their characteristic azurophilic granules may be present. in myelomonocytic leukemia, the nuclei of the blasts usually are pleomorphic and have round to lobulated forms. in some cells the cytoplasm may contain large azurophilic granules or vacuoles. blasts in megakaryocytic leukemia may contain vacuoles and may have cytoplasmic blebs. in addition, bizarre macroplatelets may be present. although these distinguishing morphologic features may suggest a definitive diagnosis, cytochemical stains or immunophenotyping usually is required to define the lineage of the blasts. several investigators have reported modification of diagnoses after cytochemical staining. 84, 85 it is especially important to distinguish aml from lymphocytic leukemia to provide prognostic information to the owner. the animal leukemia group recommends the following diagnostic criteria (figure 31-22 ). 2 using well-prepared romanowsky-stained blood and bone marrow films, a minimum of 200 cells are counted to determine the leukocyte differential in blood and the percentage of blast cells in bone marrow or blood or both. in bone marrow, blast cells are calculated both as a percentage of all nucleated cells (anc) and nonerythroid cells (nec) and are further characterized using cytochemical markers. [84] [85] [86] neutrophil differentiation is identified by positive staining of blasts for peroxidase, sudan black b, and chloracetate esterase. nonspecific esterases (alpha-naphthyl acetate esterase or alphanaphthyl butyrate esterase), especially if inhibited by sodium fluoride, mark monocytes. canine monocytes may also contain a few peroxidase-positive granules. acetylcholinesterase is a marker for megakaryocytes in dogs and cats. in addition, positive immunostaining for von willebrand's factor (factor viii-related antigen) and platelet glycoproteins on the surface of blasts identifies them as megakaryocyte precursors. 8, [42] [43] [44] [45] [46] alkaline phosphatase (ap) only rarely marks normal cells in dogs and cats but is present in blast cells in acute myeloblastic and myelomonocytic leukemias. however, because of reports of ap activity in lymphoid leukemias in dogs, its specificity as a marker for myeloid cells is not certain. omega-exonuclease is a specific marker for basophils, which are also positive for chloracetate esterase activity. 69 blood and bone marrow differential counts and cytochemical staining should be performed and interpreted by experienced veterinary cytopathologists. if erythroid cells comprise less than 50% of anc and the blast cells account for more than 30%, a diagnosis of aml or aul is made. if erythroid cells are more than 50% of anc and the blast cells are more than 30%, a diagnosis of erythroleukemia (m6) is made. if rubriblasts comprise a significant proportion of the blast cells, a diagnosis of m6er, or erythroleukemia with erythroid predominance, can be made. (it should be noted that in the human aml classification system, the blast threshold has been lowered to 20%.) in some cases, electron microscopy is required to identify the lineage of the blast cells. for example, megakaryocyte precursors are positive for platelet peroxidase activity and contain demarcation membranes and alpha granules. 42, 46 both these features are detected at the ultrastructural level. immunophenotyping, used to identify cell lineages in human patients, awaits development of appropriate markers for animal species (see later discussion). hematopoietic cells from humans with leukemia often have abnormal chromosome patterns. cytogenetic abnormalities have been found in leukemic cells from a small number of dogs. 14,15 it is not clear whether chromosomal aberrations are primary (causative) or secondary (caused by the leukemia). if consistent karyotypic patterns can be identified and correlated with leukemic subtypes, cytogenetic analysis eventually may yield important diagnostic and prognostic information and become a valuable tool for evaluating remission and predicting relapse. although morphologic and cytochemical analyses have formed the mainstay of cell identification, newer technologies now are used routinely to classify leukemias by using monoclonal antibodies to detect antigens associated with certain cell types. cells can be immunophenotyped using flow cytometric analysis or immunocytochemistry. both lymphoid and nonlymphoid acute leukemias are positive for cd34. many lymphocyte markers, including cd3, cd4, cd8, cd18, bone marrow erythroid cells cd21, cd45, cd79, and igg, are available for dogs and can be used to rule out lymphoblastic leukemia in dogs with acute leukemias. 50, 87 other markers include myeloperoxidase (mpo) and cd11b for myeloid cells and cd41 for megakaryoblasts. some overlap in the expression of these cell antigens occurs. for example, canine (but not human) granulocytes express cd4. it is best to use a panel of antibodies (similar to using a battery of cytochemical stains), because antigens often are expressed on multiple lineages, and lineage infidelity can occur. these tests have become more valuable with the availability of canine reagents. currently, the myeloid neoplasm subcommittee of the acvp oncology committee recommends that the following immunophenotyping panel be done on bone marrow and/or blood smears to characterize animal leukemias: for b lymphocytes, cd79a; for t lymphocytes, cd3; for myeloid cells, mpo and cd11b; for megakaryoblasts, cd41; for dendritic cells, cd1c; and for acute leukemias, cd34. 16 owing to the degree of differentiation of cells in chronic mpds, these disorders must be distinguished from nonneoplastic causes of increases in these cell types. to allow a diagnosis of pv, tests first must establish that the polycythemia is absolute rather than relative. in relative polycythemias, plasma volume is decreased from hemoconcentration, dehydration, or hypovolemia, and the absolute red cell mass is not increased. splenic contraction also can result in relative polycythemia. absolute polycythemia, in which the rbc mass is increased, usually occurs secondary to tissue hypoxia, causing appropriate increased production of erythropoietin. in rare cases, erythropoietin may be produced inappropriately by a tumor (e.g., renal cell carcinoma) or in renal disease (pyelonephritis) or localized renal hypoxia. [88] [89] [90] these causes of polycythemia should be eliminated by appropriate laboratory work, thoracic radiographs, arterial blood gas analysis, and renal ultrasonography. in humans with pv, plasma erythropoietin (epo) levels are low. epo levels in dogs with pv tend to be low or low-normal, whereas in animals with secondary absolute polycythemia, the levels are high. 91, 92 samples for determination of epo concentrations should be taken before therapeutic phlebotomy to treat hyperviscosity and, owing to fluctuations in epo levels, should be repeated if results are incongruous with other information. cml in dogs has no pathognomonic features, and other common causes for marked leukocytosis with a left shift (leukemoid reaction) and granulocytic hyperplasia of bone marrow must be eliminated. these include infections, especially pyogenic ones; immunemediated diseases; and other malignant neoplasms. in cml, maturation sometimes appears disorderly, and variation in the size and shape of neutrophils may occur at the same level of maturation. in addition, neoplastic leukocytes may disintegrate more rapidly and appear vacuolated. 31 because of the invasive nature of cml, biopsy of the liver or spleen may also help distinguish true leukemia from a leukemoid reaction, assuming the animal can tolerate the procedure. if characteristic cytogenetic abnormalities can be found in dogs with cml, this analysis may be helpful. basophilic leukemia is diagnosed by finding excessive numbers of basophils in circulation and in bone marrow. basophilic leukemia must be differentiated from mastocytosis based on the morphology of the cell type present. basophils have a segmented nucleus and variably sized granules, whereas mast cells have a round to oval nucleus that may be partly or totally obscured by small, round, metachromatic-staining granules. this distinction usually is easy to make; however, in basophilic leukemia, changes in the morphology of the nucleus and granules make the distinction less clear. 68 essential thrombocythemia has been diagnosed based on finding persistent and excessive thrombocytosis (more than 600,000/ml) without circulating blast cells and in the absence of another mpd (e.g., polycythemia vera), myelofibrosis, or disorders known to cause secondary thrombocytosis. 72 these include iron deficiency anemia, chronic inflammatory diseases, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and absence of a spleen. thrombocytosis is transient in these disorders or abates with resolution of the primary disease. in essential thrombocythemia, platelet morphology may be abnormal, with bizarre giant forms and abnormal granulation. 73 in the bone marrow, megakaryocytic hyperplasia is a consistent feature, and dysplastic changes may be evident in megakaryocytes. 76 spurious hyperkalemia may be present in serum samples from dogs with thrombocytosis from any cause because of the release of potassium from platelets during clot formation. 93 measurement of the potassium in plasma is recommended in these cases and usually demonstrates a potassium concentration within reference interval. platelet aggregability has been variably reported as impaired 73 or enhanced. 76 in the one dog in which it was measured, the plasma thrombopoietin (tpo) concentration was normal. 75 whether tpo plays a role in essential thrombocythemia or is suppressed by the high platelet mass is unclear. elucidation of the pathogenesis of this disorder should be aided by the recent cloning of the genes for thrombopoietin and its receptor, the proto-oncogene mpl. 94 in mds, abnormalities in two or three cell lines usually are manifested in the peripheral blood as neutropenia with or without a left shift, nonregenerative anemia, or thrombocytopenia. other changes include macrocytosis and metarubricytosis. the bone marrow typically is normocellular or hypercellular with an increased m:e, and blasts cells, although increased, account for fewer than 30% of the nucleated cells. this blast threshold may be changed to fewer than 20%, and in a report of 13 dogs with primary or secondary mds, in all but one dog the blast cell percentage was less than 20%. 95 dysplastic changes can be detected in any cell line. dyserythropoiesis is characterized by asynchronous maturation of erythroid cells, typified by large hemoglobinized cells with immature nuclei (megaloblastic change). if the erythroid component is dominant, the mds is called mds-er (see box 31-7). 2, 28 in dysgranulopoiesis, giant neutrophil precursors and abnormalities in nuclear segmentation and cytoplasmic granulation can be seen. finally, dysthrombopoiesis is characterized by giant platelets and micromegakaryocytes. myelofibrosis should be suspected in animals with nonregenerative anemia or pancytopenia; abnormalities in erythrocyte morphology, especially shape; and leukoerythroblastosis. bone marrow aspiration usually is unsuccessful, resulting in a "dry tap"; this necessitates a bone marrow biopsy taken with a jamshidi needle. 96 the specimen is processed for routine histopathologic examination and, if necessary, special stains for fibrous tissue can be used. because myelofibrosis occurs secondary to other diseases of bone marrow (e.g., mpd), chronic hemolytic anemia, or bone marrow necrosis, the clinician should look for a primary disease process. because of the poor response of acute nonlymphocytic leukemias, treatment has been unrewarding to date. however, little information is available on the response of specific subtypes of leukemia to uniform chemotherapeutic protocols, partly because of the rarity of these disease processes and the paucity of cases in the literature. the clinician is advised to contact a veterinary oncologist for advice on new protocols and appropriate management of these cases. the therapeutic goal is to eradicate leukemic cells and re-establish normal hematopoiesis. currently, this is best accomplished by cytoreductive chemotherapy, and the agents most commonly used include a combination of cytosine arabinoside and an anthracycline (e.g., doxorubicin or cyclophosphamide), vincristine, and prednisone.* in humans, the introduction of cytosine arabinoside has been the single most important development in the therapy of acute nonlymphocytic leukemia. 99 in dogs, a regimen of 100 to 200 mg/m 2 of cytosine arabinoside, given by slow infusion (over 12 to 24 hours) daily for 3 days and repeated weekly, has been used. doxorubicin (30 mg/m 2 given intravenously every 2 to 3 weeks) can be administered at intervals alternating with cytosine arabinoside. if remission is achieved, as evidenced by normalization of the hemogram, the coap protocol (cyclophosphamide, vincristine, cytosine arabinoside, and prednisone), as described for canine lymphoma, could be used as maintenance therapy. 7, 97 another protocol that has been used to treat acute myeloblastic leukemia is presented in table 31-10. regardless of the chemotherapy protocol used, significant bone marrow suppression will develop, and intensive supportive care will be necessary. transfusions of whole blood or platelet-rich plasma may be required to treat anemia and thrombocytopenia, and infection should be managed with aggressive antibiotic therapy. because of the generally poor response, the major thrust of therapy may be to provide palliative supportive care. for pv, therapy is directed at reducing the red cell mass. the pcv should be reduced to 50% to 60% or by one sixth of its starting value. phlebotomies should be performed as needed, with administration of appropriate colloid and crystalloid solutions to replace lost electrolytes; 20 ml of whole blood per kilogram of body weight can be removed at regular intervals. 54 in humans, phlebotomy continues to be the therapeutic approach used most frequently. radiophosphorus ( 32 p) has been shown to provide long-term control but can be used only in specialized centers. 100 the chemotherapeutic drug of choice is hydroxyurea, an inhibitor of dna synthesis. this drug should be administered at an initial dosage of 30 mg/kg for 10 days; the dosage then is reduced to 15 mg/kg given orally daily. 56 the major goal of treatment is to maintain the pcv as close to normal as possible. cml is best managed with chemotherapy to control the proliferation of the abnormal cell line and improve the quality of life. hydroxyurea is the most effective agent for treating cml during the chronic phase. 61, 101 the initial dosage is 20 to 25 mg/kg given twice daily. treatment with hydroxyurea should continue until the leukocyte count falls to 15,000 to 20,000 cells/ml. 61, 65, 67 the dosage of hydroxyurea then can be reduced by 50% on a daily basis or to 50 mg/kg given biweeky or triweekly. in humans, the alkylating agent busulfan can be used as an alternative. 102 an effective dosage has not been established in the dog, but following human protocols, 0.1 mg/kg/day given orally is administered until the leukocyte count is reduced to 15,000 to 20,000 cells/ml. despite response to chemotherapy and control for many months, most dogs with cml eventually enter a terminal phase of disease. in one study of seven dogs with cml, four underwent terminal phase blast crisis. 61 in humans, blast crisis may be lymphoid or myeloid. 103 in dogs, the lineage of blast cells often has not been determined. these dogs have a poor prognosis, and the best treatment to consider, if any, would be that listed in table 31 -10. few cases have been reported, but one dog was treated successfully with a combination chemotherapy protocol that included vincristine, cytosine arabinoside, cyclophosphamide, and prednisone. 74 treatment is controversial in humans because of the lack of evidence that asymptomatic patients benefit from chemotherapy. patients with thrombosis or bleeding are given cytoreductive therapy. hydroxyurea is the drug of choice for initially controlling the thrombocytosis. 72 no standard therapeutic regimen exists for mds. often, humans receive no treatment if the cytopenias do not cause clinical signs. transfusions are given when necessary, and patients with fever are evaluated aggressively to detect infections. growth factors, such as erythropoietin, gm-csf, granulocyte colony-stimulating factor (g-csf), and il-3, are sometimes used in patients who require frequent transfusions to increase their blood cell counts and enhance neutrophil function. 104, 105 in one case report, human epo (100 u/kg given subcutaneously every 48 hours) was administered to a dog with msd because of profound anemia. the rationale for the erythropoietin was to promote terminal differentiation of dysplastic erythrocytes. the pcv increased from 12% to 34% by day 19 of epo treatment. this dog remained in remission for longer than 30 months. 28 other factors that induce differentiation of hematopoietic cells include retinoic acid analogs, 106 1,25 dihydroxyvitamin d 3 , 107 interferon-alpha, and conventional chemotherapeutic agents, such as 6-thioguanine and cytosine arabinoside. 108 the propensity of these factors to enhance progression to leukemia is not known in many cases, but the potential risk exists. in general, the prognosis for animals with chronic mpds is better than for dogs with acute mpds, for which the prognosis is grave. the prognosis for pv and chronic myelogenous leukemia is guarded, but significant remissions have been achieved with certain therapeutic regimens and careful monitoring. animals commonly survive a year or longer. 61, 67 the development of blast crisis portends a grave prognosis. the pathophysiology and therapy of nonlymphocytic leukemia in humans is being studied intensively. the mpds have been demonstrated to be clonal, with abnormalities evident in all hematopoietic cell lines. leukemogenesis likely is caused by mutation or amplification of proto-oncogenes in a two-step process that initially involves a single cell and is followed by additional chromosomal alterations that may involve oncogenes. 1, 13 these alterations are manifested as chromosomal abnormalities. environmental factors known to cause leukemia are exposure to high-dose radiation, benzene (chronic exposure), and alkylating agents. 109 new classification systems have incorporated genetic mutations, more accurately reflect prognoses, and facilitate use of consistent categorization among institutions. 110 therapeutic modalities under investigation or development include combination chemotherapy, immunotherapy, cytokine therapy, drug-resistance modulators, proapoptotic agents, antiangiogenic factors, signal transduction-active agents, and bone marrow transplantation. the prognosis for chronic mpds is better than for acute mpds. for acute nonlymphocytic leukemias, the prognosis is better for children than adults; only 10% of adults who receive chemotherapy maintain remissions for longer than 5 years. 109 the spontaneous canine diseases probably occur too infrequently to serve as useful models. mpds have been induced experimentally in the dog by irradiation and transplantation in an attempt to create models for study. many similarities between human and canine mpds exist, and veterinary medicine may benefit from any therapeutic advances made in the human field. plasma cell neoplasms arise when a cell of the b-lymphocyte plasma cell lineage proliferates to form a malignant population of similar cells. this population is believed in most instances to be monoclonal (i.e., derived from a single cell), because the cells typically produce homogenous immunoglobulin, although some examples of biclonal and polyclonal plasma cell neoplasms exist. a wide variety of clinical syndromes are represented by plasma cell neoplasms, including multiple myeloma, igm (waldenstrom's) macroglobulinemia, and solitary plasmacytoma (including solitary osseous plasmacytoma and extramedullary plasmacytoma). based on incidence and severity, multiple myeloma is the most clinically important plasma cell neoplasm. although multiple myeloma (mm) represents fewer than 1% of all malignant tumors in animals, it accounts for approximately 8% of all hematopoietic tumors and 3.6% of all primary and secondary tumors affecting bones in dogs. 1, 2 early studies indicated a male predisposition, 3 but subsequent reports do not suggest a gender predilection for the dog. 1, 4 older dogs are affected most often (average age, 8 to 9 years). 1, 3, 4 in one large case series, german shepherds were overrepresented based on the hospital population. 1 the true incidence of mm in the cat is unknown, although it is a much rarer diagnosis in that species than in the dog. mm accounted for only one of 395 and four of 3248 tumors in two large compilations of feline malignancies and 0.9% of all malignancies and 1.9% of hematologic malignancies in another report. [5] [6] [7] mm occurs in aged cats (median age, 12 to 14 years), most often in domestic shorthairs, and no gender predilection has been consistently reported, although a male preponderance may exist. 4,7,8 mm has not been associated with coronavirus, felv or fiv infection. the etiology of mm for the most part is unknown. genetic predispositions, molecular aberrations, viral infections, chronic immune stimulation, and exposure to carcinogen stimulation all have been suggested as contributing factors. 4, [8] [9] [10] [11] [12] [13] [14] support for a familial association in cats comes from cases reported among siblings. 8 evidence exists that molecular mechanisms of cellular control, including overexpression of cell cycle control components such as cyclin d1(see chapter 2) and receptor tyrosine kinase dysregulation, may be involved in canine myeloma and plasma cell tumors. 13, 14 in rodent models, chronic immune stimulation and exposure to implanted silicone gel has been associated with development of mm, 9, 10 as have chronic infections and prolonged hyposensitization therapy in humans. 11 viral aleutian disease of mink results in monoclonal gammopathies in a small percentage of cases. 12 working in the agricultural industry, petroleum products, and irradiation are known risk factors for the development of mm in humans. [15] [16] [17] progression of solitary plasma cell tumors to mm has been reported in both dogs and cats, and a single case of a b-cell lymphoma that progressed to mm has been reported in the dog. 18, 19 pathology and natural behavior mm is a systemic proliferation of malignant plasma cells or their precursors, which arise as a clone of a single cell that usually involves multiple bone marrow sites. malignant plasma cells can have a varied appearance on histologic sections and cytologic preparations. the degree of differentiation ranges from cells that resemble normal plasma cells in late stages of differentiation ( figure 31 -23) to very large, anaplastic round cells with a high mitotic index, representing early stages of differentiation. 3, 4, 7, 20 in cats with mm, most plasma cells (83% in one report) are immature and have marked atypia, including increased size, multiple nuclei, clefted nuclei, anisocytosis, anisokaryosis, variable nucleus-to-cytoplasm ratios, decreased chromatin density, and variable nucleoli. nearly one fourth of the cells have "flame cell" morphology, characterized by peripheral eosinophilic cytoplasmic processes. 7 malignant plasma cells typically produce an overabundance of a single type or component of immunoglobulin, referred to as the m component ( figure 31-24) . the m component may be represented by any class of the entire immunoglobulin or by only a portion of the molecule, such as the light chain (bence jones protein) or the heavy chain (heavy chain disease). in the dog, the m component usually is represented by either igg or iga immunoglobulin types in nearly equal incidence, whereas the ratio of igg to iga in cats is approximately 5:1.* if the m component is the igm type, the term macroglobulinemia (also waldenstrom's macroglobulinemia) often is applied. several cases of biclonal gammopathy in dogs and cats have been reported. 7, 8, [26] [27] [28] [29] [30] two cases of nonsecretory mm have been reported in dogs. 31 rarely, cryoglobulinemia has been reported in dogs with mm and igm macroglobulinemia, and it has been reported in a cat with igg myeloma. 4, [32] [33] [34] cryoglobulins are paraproteins that are insoluble at temperatures below 37°c. they require blood collection and clotting to be performed at 37°c before serum separation. if whole blood is allowed to clot at temperatures below this, the protein precipitates part iv • specific malignancies in the small animal patient in the clot and is lost. pure light-chain m component is rare but has been reported in dogs. 35 the pathology associated with mm is a result of high levels of circulating m component or of organ or bone infiltration with neoplastic cells, or both. associated pathologic conditions include bone disease, bleeding diathesis, hyperviscosity syndrome, renal disease, hypercalcemia, immunodeficiency (and subsequent susceptibility to infections), cytopenias secondary to myelophthisis, and cardiac failure. bone lesions can be isolated, discrete osteolytic lesions (including pathologic fractures [ figure 31 -25, a]) or diffuse osteopenias (figure 31-26 ). approximately one fourth to two thirds of dogs with mm have radiographic evidence of bony lysis or diffuse osteoporosis. 1, 3, 4, 36 skeletal lesions in cats with mm were rarely reported in the older literature, but more recent reports document lesions in 50% to 65% of cases. [6] [7] [8] 22 bones engaged in active hematopoiesis are more commonly affected, including the vertebrae, ribs, pelvis, skull, and proximal or distal long bones. 3 skeletal lesions are rare with igm (waldenstrom's) macrogammaglobulinemia, in which malignant cells often infiltrate the spleen, liver, and lymph tissue rather than bone. 4, 33, [37] [38] [39] bleeding diathesis can result from one or a combination of events. m components may interfere with coagulation by inhibiting platelet aggregation and the release of platelet factor 3; causing adsorption of minor clotting proteins; generating abnormal fibrin polymerization; and producing a functional decrease in calcium. 4, [40] [41] [42] approximately one third of dogs and one fourth of cats have clinical evidence of hemorrhage. 1, 7, 8 in dogs, nearly half have abnormal prothrombin (pt) and partial thromboplastin (ptt) times. thrombocytopenia may also play a role if bone marrow infiltration is significant (i.e., myelophthisis). hyperviscosity syndrome (hvs) represents one or a constellation of clinicopathologic abnormalities resulting from greatly increased serum viscosity. the magnitude of viscosity changes depends on the type, size, shape, and concentration of the m component in the blood. hvs is more common with igm macroglobulinemias because of the high molecular weight of this class of immunoglobulin. 37 iga myelomas, usually present as a dimer in the dog, may undergo polymerization, resulting in increased serum viscosity. 1, 4, 40 igg-associated hvs also can occur, albeit less frequently. high serum viscosity occurs in approximately 20% of dogs with mm and can result in bleeding diathesis, neurologic signs (e.g., dementia, depression, seizure activity, and coma), ophthalmic abnormalities (e.g., dilated and tortuous retinal vessels, retinal hemorrhage [ figure 31 -27], and retinal detachment), and increased cardiac workload with the potential for subsequent development of cardiomyopathy.* these consequences are thought to be a result of sludging of blood in small vessels, ineffective delivery of oxygen and nutrients, and coagulation abnormalities. hvs was reported less commonly in cats with mm in the older literature but has been reported in association with igg-, iga-, and igm-secreting tumors. 4, 6, 21, [23] [24] [25] in four of nine cats with mm, the relative serum viscosity was above control ranges. 8 renal disease is present in approximately one third to one half of dogs with mm, and azotemia was observed *references 1, 4, 37, 39, 40, and 43-45. in one third of cats in one report. 1, 3, 7 the pathogenesis of renal failure often is multifactorial. it can develop as a result of bence jones (light-chain) proteinuria, tumor infiltration into renal tissue, hypercalcemia, amyloidosis, diminished perfusion secondary to hyperviscosity syndrome, dehydration, or ascending urinary tract infection. 1, 4, [40] [41] [42] normally, heavy-chain and light-chain synthesis is well balanced in nonneoplastic immunoglobulin production. in mm, an unbalanced excess of light-chain products may result. 42 light chains have a low molecular weight and normally are filtered by the renal glomerulus; their presence can result in protein precipitates and subsequent renal tubular injury. the presence of light chains in urine without a concomitant monoclonal spike in serum, although rare, is indicative of pure light-chain disease. 35 tubules become obstructed by large laminated casts containing albumin, immunoglobulin, and light chains. 4, 35, [40] [41] [42] bence jones proteinuria occurs in approximately 25% to 40% of dogs with mm, and in two recent surveys, it occurred in approximately 50% of cases.* hypercalcemia is reported in 15% to 20 % of dogs with mm and is thought to result primarily from the production of osteoclast-activating factor by neoplastic cells. 1, 4, 46 other factors have been implicated in human mm, including elevated levels of various cytokines, tnf, il-1, and il-6. in two dogs with mm and hypercalcemia, serum elevations in circulating n-terminal pthrp were noted. 47 hypercalcemia may also be exacerbated by associated renal disease. initially thought to be a rare event in cats with mm, hypercalcemia was noted in 25% of recently reported cases. 7, 8, 48 susceptibility to infection and immunodeficiency long have been associated with mm and often are the ultimate cause of death in affected animals. 1, 4, 22 a b a, radiograph of a distal femur in a dog with severe osteolysis and a pathologic fracture secondary to a plasma cell tumor. b, radiograph of the same pathologic fracture after surgical repair with rush rods and bone cement. the local site was treated with adjuvant radiation. the dog was continued on chemotherapy for 2 more years and did well. infection rates in humans with mm are 15 times higher than normal and usually represent pneumonia or a urinary tract infection. 49 the response to vaccination also has been shown to be suppressed in humans with mm. 50 normal immunoglobulin levels often are severely depressed in affected animals. 4 in addition, leukopenias may develop secondary to myelophthisis. variable cytopenias may be observed in association with mm. approximately two thirds of dogs with mm have a normocytic, normochromic, nonregenerative anemia 1, 3, 4 ; this can result from marrow infiltration (myelophthisis), blood loss from coagulation disorders, anemia of chronic disease, or increased erythrocyte destruction secondary to high serum viscosity. in dogs with mm, similar factors lead to thrombocytopenia in nearly one third of the dogs and leukopenia in nearly one fourth. in cats, approximately two thirds are anemic, one half are thrombocytopenic, and one third are neutropenic. 7, 8 cardiac disease, if present, usually is a result of excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity. 40, 41 myocardial infiltration with amyloid and anemia may be complicating factors. in one report nearly half of the cats with mm had a cardiac murmur, for which the etiology was not established. 7 clinical signs of mm may be present up to a year before diagnosis in dogs (median, 1 month). in one cat, m-component elevations were detected 9 years before clinical presentation. 1, 7 in the latter case, the m-component elevation was consistent with monoclonal gammopathy of unknown significance (mgus). mgus (i.e., "benign," "essential," or "idiopathic" monoclonal gammopathy) is a benign monoclonal gammopathy that is not associated with osteolysis, bone marrow infiltration, or bence jones proteinuria. mgus also has been reported in dogs. 51, 52 signs of mm can vary, depending on the wide range of possible pathologic effects. tables 31-11 multiple retinal hemorrhages on the fundus in a cat with hyperviscosity syndrome secondary to multiple myeloma. several reports. 1, 4, 7, 8, 22 bleeding diathesis usually is represented by epistaxis and gingival bleeding. funduscopic abnormalities may include retinal hemorrhage (see figure 31 -27), venous dilation with sacculation and tortuosity, retinal detachment and blindness. 1, 4, 40, [43] [44] [45] cns signs may include dementia, seizure activity, and deficiencies in midbrain or brainstem localizing reflexes secondary to hvs or extreme hypercalcemia. signs reflecting transverse myelopathies secondary to vertebral column infiltration, pathologic fracture, or extradural mass compression also can occur. 1, 4, 32, 36, 53 one case of ataxia and seizure activity in a dog with extramedullary plasmacytoma (emp) secondary to tumor-associated hypoglycemia has been reported. 54 in addition, paraneoplastic polyneuropathy has been reported in a dog with mm. 55 a history of chronic respiratory infections and persistent fever may also be present in cats. hepatosplenomegaly and renomegaly can occur as a result of organ infiltration. bleeding diathesis due to hvs is less common in the cat, but epistaxis, pleural and peritoneal hemorrhagic effusions, retinal hemorrhage, and central neurologic signs have been reported. 4, 6, polydipsia and polyuria can occur secondary to renal disease or hypercalcemia, and dehydration may develop. hind limb paresis secondary to osteolysis of lumbar vertebral bodies or extradural compression has been reported in cats. 8, 56 the diagnosis of mm usually follows the demonstration of bone marrow plasmacytosis (see figure 31 -23), the presence of osteolytic bone lesions (see figure 31 -26), and the demonstration of serum or urine myeloma proteins (m component; see figure 31 -24). in the absence of osteolytic bone lesions, a diagnosis can also be made if marrow plasmacytosis is associated with a progressive increase in the m component. in the cat, because the degree of bone marrow infiltration may not be as marked, some have suggested consideration of plasma cell morphology and visceral organ infiltration in cases with demonstrable m-component disease in the absence of marked marrow plasmacytosis (less than 20%). 7 all animals suspected of having plasma cell tumors should receive a minimal diagnostic evaluation, including a cbc, platelet count, serum biochemistry profile, and urinalysis. particular attention should be paid to renal function and serum calcium levels. serum electrophoresis and immunoelectrophoresis are performed to detect a monoclonal spike (see figure 31 -24) and to categorize the immunoglobulin class involved. heat precipitation with electrophoresis of urine is performed to detect bence jones proteinuria, because commercial urine dipstick methods are not capable of this determination. definitive diagnosis usually is done by bone marrow aspiration. a bone marrow core biopsy or multiple aspirates may be necessary because of the possibility of clustering of plasma cells in the bone marrow. normal marrow contains fewer than 5% plasma cells, whereas in myelomatoid marrow, this level often is greatly exceeded. current recommendations require the presence of marrow plasmacytosis greater than 20%; however, a 10% cutoff in cats recently has been recommended, with special attention to cellular atypia. 7 patel and colleagues 7 comment that even the 10% threshold is problematic, and cellular atypia and visceral organ involvement should be considered equally important. routine thoracic and abdominal radiographs are recommended. occasionally, bony lesions can be observed in skeletal areas on these standard films, and organomegaly (liver, spleen, and kidneys) is observed in most cats (figure 31-28) . 7 abdominal ultrasound scans are recommended in all cats suspected of having mm, because they reveal one or more abdominal tumors in nearly all such patients. 7 these include splenomegaly with or without nodules, diffuse hyperechoic hepatomegaly with or without nodules, renomegaly, and iliac lymph node enlargement. skeletal survey radiographs are recommended to detect and determine the extent of osteolytic lesions, which may have diagnostic, prognostic, and therapeutic implications. nuclear scintigraphy (bone scans) for clinical staging of dogs with mm has been performed; however, because of the predominant osteolytic activity with osteoblastic inactivity, these scans seldom give positive results and therefore are not useful for routine diagnosis. 57 in physician-based oncology, bone mineral density analysis (dexa scan) to document osteoporosis and mri scans of bone marrow are commonly used for staging; however, neither of these has been applied consistently in the veterinary literature. in rare cases, biopsy of osteolytic lesions (i.e., jamshidi core biopsy; see chapter 23) is necessary for diagnosis. in one case of mm in a dog, splenic aspirates were diagnostically helpful. 58 if clinical hemorrhage is present, a coagulation assessment (e.g., platelet count, pt, and ptt) and serum viscosity measurements should be done. all animals should undergo a careful funduscopic examination. table 31 -13 shows the overall frequency of clinical diagnostic abnormalities in dogs and cats with mm, as compiled from published series involving at least five cases each. a clinical staging system for canine mm has been suggested 1 ; however, currently no prognostic significance has been attributed to it. molecular diagnostic techniques for mm have had limited use thus far in veterinary oncology; however, pcr techniques have been used to determine the clonality of the immunoglobulin heavy-chain variable region gene in feline plasmacytomas and myelomas (see section a of this chapter and figure 31-8) . 59 the use of this technology in cases in which the diagnosis is not straightforward awaits further investigation. disease syndromes other than plasma cell tumors can be associated with monoclonal gammopathies and should be considered in any list of differentials. these include other lymphoreticular tumors (lymphoma, extramedullary plasmacytoma, chronic and acute lymphocytic leukemia), chronic infections (e.g., ehrlichiosis, leishmaniasis, fip), and mgus.* therapy for mm is directed at both the tumor cell mass and the secondary systemic effects. all diagnostic procedures should be completed before primary therapy is started to ensure a complete diagnosis and to procure baseline values for monitoring response. in most dogs with mm, chemotherapy is effective at reducing the myeloma cell burden, relieving bone pain, allowing for skeletal healing, and reducing levels of serum immunoglobulins. it also can greatly extend both the quality and duration of most patients' lives. 1, 4 complete elimination of neoplastic myeloma cells is rare, but the chemotherapeutic drugs currently available can make mm a gratifying disease to treat for both the clinician and the client. however, eventual relapse is to be expected. only one half of cats with mm respond to chemotherapy, and most responses are short-lived. however, several long-term responses (i.e., longer than 1 year) have been reported, and treatment should be attempted when educated clients decide on a therapeutic option. † melphalan, an alkylating agent, is the chemotherapeutic drug of choice for the treatment of mm. 1, 4 in the dog, an initial starting dosage of 0.1 mg/kg is given orally once daily for 10 days and then reduced to 0.05 mg/kg given orally once daily continuously. addition of prednisone therapy is thought to increase the efficacy of melphalan therapy. prednisone is started at a dosage of 0.5 mg/kg given orally once daily for 10 days and is then reduced to 0.5 mg/kg given orally every other day until the drug is discontinued after 60 days of therapy. melphalan, however, is continued at 0.05 mg/kg/day until clinical relapse occurs or myelosuppression necessitates a dose reduction. most dogs treated with this melphalan/prednisone combination tolerate the regimen well. the most clinically significant toxicity of melphalan is myelosuppression, particularly a delayed thrombocytopenia. a cbc, including a platelet count, should be performed biweekly for 2 months of therapy and monthly thereafter. if significant myelosuppression occurs (usually thrombocytopenia or neutropenia), the dosage or the treatment frequency may need to be reduced. the author has successfully used an alternative, pulse-dosing regimen for melphalan (7 mg/m 2 given orally daily for 5 consecutive days every 3 weeks) in a small number of cases in which myelosuppression was limiting the more conventional, continuous low-dose therapy. the author now uses this pulse-dose regimen as a first-line treatment with the caveat that long-term response data are lacking. melphalan/prednisone therapy also can be used in cats with mm; however, the protocol appears to be more myelosuppressive in cats than in dogs, and careful monitoring is required. in the cat, a dosing schedule similar to that for the dog has been reported 8, 22 ; 0.1 mg/kg (approximately 0.5 mg, or 1 ⁄4 of a 2 mg tablet) is given orally once daily for 10 to 14 days, then every other day until clinical improvement occurs or leukopenia develops. one report has advocated long-term continuous maintenance (0.1 mg/kg given orally once every 7 days). 8 cyclophosphamide has been used as an alternative alkylating agent or in combination with melphalan in dogs with mm. 1, 4, 64 no evidence indicates that it is superior to melphalan therapy. in the author's practice, cyclophosphamide is limited to patients with severe hypercalcemia or widespread systemic involvement in which a faster acting alkylating agent may alleviate systemic effects more quickly. cyclophosphamide is initiated at a dosage of 200 mg/m 2 given intravenously once, at the same time oral melphalan therapy is started. because cyclophosphamide is less likely to affect thrombocytes, it may be substituted in patients in which thrombocytopenia has developed secondary to long-term melphalan use. chlorambucil, another alkylating agent, has been used successfully for the treatment of igm macroglobulinemia in dogs at a dosage of 0.2 mg/kg given orally once daily. 4, 37 few or no clinical signs of toxicity result from this dosing schedule. ccnu, yet another alkylating agent, has been used in a limited number of cats with mm, and a partial response has been reported with a dosing schedule of 50 mg/m 2 given orally every 21 days. 65 evaluation of the response to therapy evaluation of the response to systemic therapy for mm is based on improvement in clinical signs and clinicopathologic parameters and radiographic improvement of skeletal lesions. 1, 4 subjective improvement in clinical signs of bone pain, lameness, lethargy, and anorexia should be evident within 3 to 4 weeks after initiation of therapy. objective laboratory improvement, including reduction in serum immunoglobulin or bence jones proteinuria, usually is noted within 3 to 6 weeks. radiographic improvement in osteolytic bone lesions may take months, and only partial resolution may occur. ophthalmic complications (including longstanding retinal detachment) and paraneoplastic neuropathies can be expected to resolve along with the tumor mass. 45, 55 in one report on cats that responded to melphalan and prednisone, clinical improvement was noted in 4 weeks and serum protein and radiographic bone abnormalities were greatly improved by 8 weeks. 8 as previously discussed, mm does not resolve completely, and a good response is defined as a reduction in measured m component (i.e., immunoglobulin or bence jones proteins) by at least 50% of pretreatment values. 4 reduction in the serum immunoglobulin levels may lag behind reduction in bence jones proteinuria, because the half-lives are 15 to 20 days and 8 to 12 hours, respectively. 66 for routine follow-up, quantification of the elevated serum immunoglobulin or urine bence jones protein is performed monthly until a good response is noted and then every 2 to 3 months. repeat bone marrow aspiration for evaluation of plasma cell infiltration occasionally may be necessary. this is particularly prudent when cytopenias develop during chemotherapy and drug toxicity must be differentiated from marrow recurrence. long-term control of complications such as hypercalcemia, hvs, bleeding diathesis, renal disease, immunosuppression, ophthalmic complications, and pathologic skeletal fractures is achieved by controlling the primary tumor mass. however, therapy directed more specifically at these complications may be indicated in the short term. if hypercalcemia is marked and significant clinical signs are present, standard therapies, including fluid diureses with or without pharmacologic agents (e.g., calcitonin), may be indicated (see chapter 5) . moderate hypercalcemia typically resolves within 2 to 3 days after initiation of melphalan/prednisone chemotherapy. hvs is best treated in the short term by plasmapheresis. 4, 40, 62, 67, 68 whole blood is collected from the patient and centrifuged to separate plasma from packed cells. packed red cells are resuspended in normal saline and reinfused into the patient. bleeding diathesis usually resolves along with hvs, but platelet-rich plasma transfusions may be necessary with thrombocytopenia. renal impairment may require aggressive fluid therapy in the short term and maintenance of adequate hydration in the long term. careful attention to secondary urinary tract infections and appropriate antimicrobial therapy are indicated. ensuring an adequate water intake at home is important, and in some cases owners must be taught home subcutaneous fluid administration. continued monitoring of renal function is recommended, along with follow-up directed at tumor response. patients with mm can be thought of as immunologic cripples. some have recommended prophylactic antibiotic therapy in dogs with mm, 4 but in humans, no benefit for this approach has been observed over diligent monitoring and aggressive antimicrobial management when indicated. 41 cidal antimicrobials are preferred over static drugs, and nephrotoxic antimicrobials should not be used. pathologic fractures of weight-bearing long bones and of vertebrae, resulting in spinal cord compression, may require immediate intervention in conjunction with systemic chemotherapy. orthopedic stabilization of fractures should be performed and may be followed with external beam radiotherapy (see figure 31-25, b) . recently, inhibition of osteoclast activity by bisphosphonate drugs has been shown to reduce the incidence and severity of skeletal complications of mm in humans. 57 this class of drugs may hold promise for use in dogs and cats with various skeletal tumors. 69 rescue therapy may be attempted when mm eventually relapses in dogs undergoing melphalan therapy or in the uncommon patient that initially is resistant to alkylating agents. the author has had success with the vad protocol, a combination of doxorubicin (30 mg/m 2 given intravenously every 21 days), vincristine (0.7 mg/m 2 given intravenously on days 8 and 15), and dexamethasone sodium phosphate (1 mg/kg given intravenously once a week on days 1, 8, and 15); this regimen is used in 21-day cycles. although most dogs initially respond to this rescue protocol, the duration of response tends to be short, lasting only a few months. high-dose cyclophosphamide (300 mg/m 2 given intravenously every 7 days) also been has used as a rescue agent, although with limited success. liposomal doxorubicin produced a long-term remission in a dog with mm that had been resistant to native doxorubicin. 70 because mm ultimately is a uniformly fatal disease in most species, including humans, significant effort is being put into investigational therapies for this disease. currently, bone marrow ablative therapy and marrow or stem cell rescue, thalidomide (and other antiangiogenic therapies), bortezomib (a proteasome inhibitor), arsenic trioxide, the bisphosphonates, and several molecular targeting therapies are under investigation; however, their use in veterinary species is limited or completely absent at present. 57 nonetheless, the promise of molecular-targeted therapies is foreshadowed by a case of a dog with mm that was resistant to melphalan, prednisone, and doxorubicin 14 ; this dog achieved a partial response to tyrosine kinase inhibitor therapy (su11654; see chapter 14, section b) that was maintained for 6 months. the prognosis for dogs with mm is good for initial control of the tumor and a return to good quality of life. in a group of 60 dogs with mm, approximately 43% achieved a complete remission (i.e., normalization of serum immunoglobulins), 49% achieved a partial remission (i.e., immunoglobulin levels less than 50% pretreatment values), and only 8% did not respond to melphalan/prednisone chemotherapy. 1 long-term survival is the norm, with a median of 540 days reported (figure 31-29) . hypercalcemia, bence jones proteinuria, and extensive bony lysis are known negative prognostic indices in the dog. 1 the long-term prognosis for dogs with mm is poor, because recurrence of the tumor mass and associated clinical signs is expected. eventually, the tumor no longer responds to available chemotherapeutic drugs, and death follows from renal failure, sepsis, or euthanasia for intractable bone or spinal pain. 1, 4 the prognosis for mm in the cat is not as favorable in the short term as it is in the dog. 4, 7, 8, 22 although most cats (approximately 60%) transiently respond to melphalan/prednisone-or cyclophosphamide-based protocols, most responses are partial and not durable. typically, cats with mm succumb to the disease within 4 months. [6] [7] [8] 22, 24, 68 however, long-term survivors (longer than 1 year) have been reported. 7, 8, 22, 30, 48 one investigator grouped mm in cats into two prognostic categories, based on criteria known to predict disease behavior in dogs (table 31-14) . 8 although no rigorous statistical analysis was performed on this small group of cats (nine), the median survival times were 5 days for cats with disease categorized as "aggressive" and 387 days for those with disease categorized as "nonaggressive." experience in dogs with igm macroglobulinemia is limited. 4, 35 response to chlorambucil is to be expected, and in nine treated dogs, 77% achieved remission, with a median survival time of 11 months. 4 solitary collections of monoclonal plasmacytic tumors can originate in soft tissues (extramedullary plasmacytoma) or bone (solitary osseous plasmacytoma [sop]). a number of large case compilations of cutaneous plasmacytoma have been reported in the dog. 13, [71] [72] [73] [74] [75] [76] [77] plasmacytomas represented 2.4% of all canine tumor submissions in one large compilation of cases. 78 in this series of 751 cases, the most common locations in the dog were cutaneous sites (86%) (figure 31-30) , the mucous membranes of the oral cavity and lips (9%) (figure 31-31) , and the rectum and colon (4%). the skin of the limbs and head (including the ears) is the most frequently reported cutaneous site. 71, 72, 77 all other sites accounted for the remaining 1% of cases; these sites may include the stomach, spleen, genitalia, eyes, uterus, and liver. the american cocker spaniel, english cocker spaniel, and west highland white terrier (and perhaps yorkshire terriers, boxers, german shepherds, and airedale terriers) have an increased risk of developing plasmacytomas, and the median age of affected dogs is 9 to 10 years. 77, 78 cutaneous and oral emp in dogs typically manifests as benign tumors that are highly amenable to local therapy. the natural behavior of noncutaneous/nonoral emp appears to be somewhat more aggressive in the dog. gastrointestinal emp has been reported in a number of sites in the veterinary literature, including the esophagus, 79 stomach ( figure 31-32) , 64,80 small intestine, 81 and large intestine. 78, 80, [81] [82] [83] metastasis to associated lymph nodes is more common in these cases; however, bone marrow involvement and monoclonal gammopathies are less commonly encountered. in nine cases of colorectal emp, only two dogs experienced recurrence after surgical excision, and two cases involved multiple lesions. 78 emp of the trachea, liver, and uterus also have been reported in a dog, and all had a benign course after local resection. [84] [85] [86] most cases of sop eventually progress to systemic mm; however, the time course from local tumor development to systemic mm may be many months to years. 31, 87 sops reported in the dog have involved the zygomatic arch and the ribs. 31 plasmacytomas are less common in cats, and fewer reports exist in the literature. 18, [88] [89] [90] [91] [92] [93] [94] they occur in older cats (mean age, 8.5 years) with no significant gender predilection. the skin is the most common site; other sites include the oral cavity, eye, gi tract, liver, subcutaneous tissues, and brain. two reports exist of cutaneous emp in cats that progressed to systemic disease; one cat developed lymph node and distant metastasis, the other progressed to mm. 18 cutaneous plasmacytoma on the limb of a dog. clinical signs associated with solitary plasmacytomas relate to the location of involvement; in the rare cases involving high levels of m component, hvs may occur. most cutaneous plasmacytomas are solitary, smooth, raised pink nodules that measure 1 to 2 cm in diameter (see figure 31 -30), although tumors as large as 10 cm have been reported. combination of data from large series shows that more than 95% of the tumors occur as solitary masses, and fewer than 1% occur as part of a systemic mm process. 13, [71] [72] [73] 77 the cutaneous and oral forms of emp usually have a benign course and no related clinical signs. gastrointestinal emp, however, typically produces relatively nonspecific signs that may suggest alimentary involvement. colorectal plasmacytomas usually cause rectal bleeding, hematochezia, tenesmus, and rectal prolapse. 78 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 54 sop usually is associated with pain and lameness (if the appendicular skeleton is affected) or with neurologic signs (if vertebral bodies are involved). diagnosis of sops and emps usually requires tissue biopsy or fine-needle aspiration. the cells that make up a b solitary plasmacytic tumors in both cats and dogs have been classified histologically as mature, hyaline, cleaved, asynchronous, monomorphous blastic, and polymorphous blastic cell types. no prognostic significance has been observed with this classification system, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13, 77, 88 with poorly differentiated solitary plasmacytic tumors, immunohistochemical studies directed at detecting immunoglobulin, light and heavy chains, and thioflavine t may be helpful for differentiating the lesions from other round cell tumors. 31, 76, 77, [92] [93] [94] immunoreactivity has been demonstrated for canine igg f(ab) 2 and vimentin. 73 a variant characterized by an igg-reactive amyloid interspersed with the neoplastic cells also has been described. 74 in addition, pcr techniques can be used to determine the clonality of the immunoglobulin heavy-chain variable region gene in plasmacytomas and myelomas, which may be diagnostically useful in difficult cases. thorough staging is important in dogs and cats with plasmacytomas that are at higher risk for systemic spread. staging, which must be done before therapy is started, should include bone marrow aspiration, serum electrophoresis, and skeletal survey radiographs to ensure that the disease is confined to a local site. this is most important for sop and gastrointestinal emp because of these tumors' relatively high metastatic rate; it is less important for cutaneous and oral plasmacytomas because of their typically more benign behavior. in addition, endoscopic evaluation of the entire gi tract is recommended with gastrointestinal emp. cutaneous plasma cell tumors in the dog are almost always benign and have an excellent prognosis after conservative surgical excision. successful therapy with melphalan and prednisone has been rarely applied for a local recurrence or incomplete margins in dogs and cats. 53, 92 radiation therapy has been used infrequently for cases that are nonsurgical. surgery is recommended in combination with radiotherapy for sop when the lesion results in an unstable long bone fracture (see or the patient is nonambulatory because of neurologic compromise caused by a vertebral body sop. in the latter case, spinal cord decompression, mass excision, and possibly spinal stabilization may be necessary. 53 radiotherapy can be used without surgery, when fractures are stable, as a palliative measure for bone pain or, in the case of vertebral sop, if the patient is ambulatory and stable. good local control usually is achieved, but most patients eventually develop systemic mm. 31, 53, 87 sop of the axial skeleton can be managed by excision or radiotherapy alone. whether systemic chemotherapy should be initiated at the time of local therapy for sop when systemic involvement is not documented is the subject of debate. systemic spread may not occur for many months or even years beyond the primary sop diagnosis in humans and dogs, and studies in humans reveal no benefit from initiation of systemic chemotherapy before documentation of subsequent systemic spread. 42, 53 similarly, emp of the gi tract in humans most often is treated with surgical excision and thorough staging of disease. systemic therapy is not initiated unless systemic involvement is documented. systemic chemotherapy has been used following excision of a gastric emp in a cat, but the utility of adjuvant therapy in the species is unkown. 94 long-term follow-up of patients with a solitary plasmacytoma is indicated so that recurrence of disease and systemic spread can be recognized. in patients with sop, careful attention should be paid to the serum globulin levels, bone pain, and the radiographic appearance of bone healing. restaging of the disease, including bone marrow evaluation, is indicated if systemic spread is suspected. the prognosis for solitary plasma cell tumors generally is good. cutaneous and mucocutaneous plasmacytomas usually are cured by surgical excision. 13, 77 in large compilations of cases in dogs, the local recurrence rate was approximately 5%, and nodal or distant metastasis occurred in only seven of 349 cases (2%). 13, [71] [72] [73] 77 new cutaneous plasmacytomas at sites distant from the primary tumor developed in fewer than 2% of cases. one caveat: the author has encountered a handful of cases of aggressive multiple cutaneous plasmacytoma that eventual resulted in the death of the affected dogs. neither the tumor cell proliferation rate (as measured by ki-67 immunohistochemistry) in the dog nor histopathologic grading in dogs and cats was prognostic in large compilations of cases, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13, 77, 88 the presence of amyloid and overexpression of cyclin d1 (prognostic in human plasmacytomas) were not shown to be prognostic in dogs. 13 most dogs with emp of the alimentary tract and other abdominal organs (e.g., liver, uterus) that is treated by surgical excision alone or in combination with systemic chemotherapy (if metastasis is present) can enjoy long-term survival. 31, 64, [78] [79] [80] [82] [83] [84] [85] [86] in a compilation of nine dogs with colorectal plasmacytoma, two had local recurrence at 5 and 8 months after surgery; after surgery alone, the overall median survival time was 15 months. 78 dna ploidy and c-myc oncoprotein expression in biopsy samples were determined to be predictive for emps in dogs; however, tumors that were malignant all were from noncutaneous sites (i.e., lymph node, colon, and spleen), therefore location appears to be as predictive. 96 as previously discussed, lymphatic leukemia in dogs: an epizootiological clinical and haematological study survey of animal neoplasms in alameda and contra costa counties, california. ii. cancer morbidity in dogs and cats from alameda county immunohistochemical 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stimulating factors cloning and expression of murine thrombopoietin cdna and stimulation of platelet production in vivo preleukemic syndrome in a dog clinicopathologic aspects of acute leukemias in the dog myelodysplastic syndrome in two dogs use of human recombinant erythropoietin and prednisone for treatment of myelodysplastic syndrome with erythroid predominance in a dog thrombocytosis associated with a myeloproliferative disorder in a dog myeloproliferative disease in the dog and cat: definition, aetiology and classification the leukemia complex acute myelomonocytic leukemia in a dog acute myelomonocytic leukemia in a dog clinical-pathological findings and cytochemical characterizations of myelomonocytic leukaemia in 5 dogs acute myelomonocytic leukemia in a dog tumors of the lymphoid and hemopoietic tissues acute myelomonocytic leukemia manifested as myelophthisic anemia in a dog acute myelomonocytic leukemia in a dog unusual cytochemical reactivity in canine acute myeloblastic leukemia megakaryocytic leukemia in a dog myeloproliferative disorder involving the megakaryocytic line megakaryoblastic leukemia in a dog megakaryblastic leukemia in a dog platelet dysplasia associated with megakaryoblastic leukemia in a dog radiation-induced megakaryoblastic leukemia in a dog identification and characterization of megakaryoblasts in acute megakaryoblastic leukemia in a dog monocytic leukaemia in the dog erythroleukemia in a dog, pratique medicale and chirurgicale de l three cases of erythroleukemia in dogs blood cell markers current opinion in hematology polycythaemia vera in a bitch polycythemia vera in a dog polycythemia vera in dogs polycythemia vera in a dog diagnosis of canine primary polycythemia and management with hydroxy-urea polycythemia vera and glomerulonephritis in a dog greydanus-van-der-putten swm: polycythaemia vera in a dog treated by repeated phlebotomies polycythaemia vera in a dog polycythaemia vera in a dog chronic myelogenous leukemia in the dog blastic crisis in chronic myelogenous leukaemia in a dog chronic myelogenous leukaemia with meningeal infiltration in a dog chronic granulocytic leukaemia in a dog with associated bacterial endocarditis, thrombocytopenia and preretinal and retinal hemorrhages chronic granulocytic leukemia in a dog chronic myelogenous leukemia and related disorders treatment of basophilic leukemia in a dog basophilic leukemia in a dog basophilic leukemia in a dog eosinophilic leukemoid reaction in a dog chronic granulocytic leukaemia/eosinophilic leukaemia in a dog essential (primary) thrombocythemia probable essential thrombocythemia in a dog successful treatment of suspected essential thrombocythemia in the dog essential thrombocythemia in a dog: case report and literature review diagnostic and hematologic features of probable essential thrombocythemia in two dogs a review of myelofibrosis in dogs pathogenesis of myelofibrosis: role of ineffective megakaryopoiesis and megakaryocyte components 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stimulation of res the development of myeloma-like condition in mink with aleutian disease clinico-pathological aspects of canine cutaneous and mucocutaneous plasmacytomas phase i dose-escalating study of su11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies multiple myeloma and family history of cancer multiple myeloma: a case control study a case-control study of multiple myeloma in whites: chronic antigenic stimulation, occupation and drug use progression of a solitary, malignant cutaneous plasma cell tumour to multiple myeloma in a cat evolution of a b-cell lymphoma to multiple myeloma after chemotherapy histological classification of hematopoietic tumors of domestic animals immunoglobulin a myeloma in a cat with pleural effusion and serum hyperviscosity multiple myeloma in the cat hyperviscosity syndrome with igm monoclonal gammopathy and hepatic plasmacytoid lymphosarcoma in a cat serum hyperviscosity syndrome associated with multiple 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in two dogs macroglobulinemia in the dog, the canine analogue of gamma m monoclonal gammopathy macroglobulinemia with hyperviscosity syndrome in a dog serum hyperviscosity syndrome associated with iga multiple myeloma in two dogs plasma cell tumors plasma cell neoplasms ocular lesions in a dog with hyperviscosity secondary to an iga myeloma blindness in a dog with igaforming myeloma ophthalmic disease as the presenting complaint in five dogs with multiple myeloma bone destruction and hypercalcemia in plasma cell myeloma parathyroid hormone (pth)-related protein, pth, and 1,25-dihydroxyvitamin d in dogs with cancer associated hypercalcemia hypercalcemia in two cats with multiple myeloma infections complicating multiple myeloma and chronic lymphocytic leukemia infection, antibody response, and gamma globulin components in multiple myeloma and macroglobulinemia a benign hypergammaglobulinemia mimicking plasma cell myeloma idiopathic monoclonal (iga) gammopathy in a dog vertebral plasma cell tumors in 8 dogs hypoglycemia and polyclonal gammopathy in a dog with plasma cell dyscrasia multiple myeloma with associated polyneuropathy in a german shepherd dog plasma cell sarcoma in a cat plasma cell neoplasms fine-needle aspiration of the spleen as an aid in the diagnosis of splenomegaly characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of b-cell neoplasia use of plasmapheresis and chemotherapy for treatment of monoclonal gammopathy associated with ehrlichia canis infection in a dog monoclonal gammopathy associated with naturally occurring canine ehrlichiosis hyperviscosity syndrome associated with lymphocytic leukemia in three dogs monoclonal gammopathy in a dog with visceral leishmaniasis gastric extramedullary plasmacytoma in a dog hematological toxicity and therapeutic efficacy of lomustine in 20 tumor-bearing cats: critical assessment of a practical dosing regimen the treatment of multiple myeloma therapeutic plasmapheresis multiple myeloma in a cat response to liposome-encapsulated doxorubicin (tlc d-99) in a dog with myeloma extramedullary plasmacytomas in dogs: results of surgical excision in 131 cases mucocutaneous plasmacytomas in dogs: 75 cases cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study cutaneous plasmacytomas with amyloid in six dogs primary cutaneous plasmacytomas in the dog and cat an immunohistochemical study of canine extramedullary plasma cell tumours prognostic value of histopathological grading in canine extramedullary plasmacytomas colorectal plasmacytomas: a retrospective study of nine dogs esophageal plasmacytoma in a dog extramedullary plasmacytoma of the gastrointestinal tract in two dogs primary igg secreting plasma cell tumor in the gastrointestinal tract of a dog a solitary plasmacytoma in a dog with progression to a disseminated myeloma metastatic extramedullary plasmacytoma of the colon and rectum in a dog extramedullary plasmacytoma in the trachea of a dog uterine extramedullary plasmacytoma in a dog a primary hepatic plasma cell tumor in a dog part iv • specific malignancies in the small animal patient solitary plasmacytomas of bone and extramedullary plasmacytomas histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats intracerebral plasma cell tumor in a cat: a case report and literature review intraocular extramedullary plasmacytoma in a cat extramedullary plasmacytoma and immunoglobulin-associated amyloidosis in a cat immunohistochemical staining of neoplastic and inflammatory plasma cell lesions in feline tissues immunoglobulin-producing tumours in dogs and cats identification of immunoglobulin light chains in canine extramedullary plasmacytomas by thioflavine t and immunohistochemistry gastric extramedullary plasmacytoma in a cat analysis of dna aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry most patients with sop eventually develop systemic disease, but long, disease-free periods usually precede the event.the prognosis in cats is less well defined, owing to the scarcity of reported cases. if disease is confined to a local site and/or regional nodes, surgical excision and chemotherapy can result in long-term control; however, early, widespread metastasis and progression to mm is also reported in cats.* key: cord-018110-mcw4v13c authors: arnold, andreas; arnold, wolfgang; bovo, roberto; ganzer, uwe; hamann, karl-friedrich; iurato, salvatore; kiefer, jan; lamm, kerstin; livi, walter; martini, alessandro; o’donoghue, gerard m. title: inner ear date: 2010 journal: otorhinolaryngology, head and neck surgery doi: 10.1007/978-3-540-68940-9_6 sha: doc_id: 18110 cord_uid: mcw4v13c herpes zoster oticus, herpes zoster cephalicus, ramsay hunt syndrome. caused by reactivation of the varicella zoster virus at the level of the ganglion cells of cranial nerves vii and viii, herpes zoster oticus accounts for approximately 10-15% of acute facial palsy cases. one or 2 days of general malaise with fever. • burning earache followed by a vesicular eruption in-• volving the aperture of the external auditory canal and the auricle. the bluish-red vesicles eventually form crusts within a few days. facial palsy. • sensorineural hearing loss and vertigo (lesion of cra-• nial nerve viii). pain in the pharynx (lesion of cranial nerves ix and • x). facial pain due to involvement of cranial nerve v. • headache, neck stiffness, photophobia. • persistent complete hearing loss, persistent vertigo, postherpetic pain, persistent facial paralysis. inspection: varicella zoster vesicles • on the lateral surface of the auricle, concha and entrance of the auditory canal ( fig. 1.6 .1). vesicles may occur over the face and neck and may involve the buccal mucosa. warning: the vesicles may be small or may resolve before the patient is evaluated (pay attention to encrusted areas). otomicroscopy: vesicles or vesicle remnants in the • external auditory canal and on the tympanic membrane. audiometric evaluation: sensorineural hearing loss. • vestibular tests: spontaneous nystagmus towards the • affected side (at the beginning), later towards the opposite direction. serological confirmation: rarely utilized. • local treatment • of the vesicles with drying agents (e. g. 70% alcohol) and acyclovir cream. antiviral treatment with acyclovir (800 mg five times a • day orally). alternatively, brivudin or famciclovir may be given. prednisolone, 100 mg/day for 3-5 days together with • proton pumps blockers. alternatively, prednisone, 1 mg/kg of body weight daily orally for 7-10 days with tapering to zero over the following 10 days. in cases of persistent facial paralysis, gold weight implant in the upper eyelid, hypoglossal-facial anastomosis. the prognosis for recovery of the facial function is • less favourable than that of bell's palsy. incomplete recovery is frequent (only less than 50% of the patients recover satisfactorily). prognosis is worse in elderly patients. often persistent neuralgia (years). complete hearing loss and complete vestibular areflex-• ia are irreversible. the route of infection may be otogenic, meningogenic or haematogenic. the symptoms are: • during the course of a viral infection of the upper respiratory tract including the middle ear (picornavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, coronavirus, adenovirus). serous effusion of the middle ear, vestibular disturbances, combined or pure sensorineural hearing loss, tinnitus. no earache. • during the course of mumps, measles or parainfluenza meningitis. route of infection are the internal auditory canal and the cochlear aqueduct. protein deposits can be seen in the perilymphatic spaces ( fig. 1.6.2) . clinical signs of meningitis are fatigue, vomiting, headache, stiff neck, fever, and unilateral or bilateral deafness. purulent (suppurative) bacterial labyrinthitis may be secondary to acute otitis media or purulent meningitis. in acute otitis media, bacteria may enter the inner ear through the oval and round windows ( fig. 1.6 purulent labyrinthitis is frequently followed by meningitis as the microorganisms gain access to the subarachnoid space through the cochlea aqueduct or internal auditory canal. bacterial labyrinthitis can be a complication of cholesteatoma, spontaneous or acquired labyrinth fistula or may occur in malformations of the cochlea with enlarged perilymphatic spaces (mondini dysplasia). glucocorticoids, initially 500 mg intravenously for • 3 days followed by oral administration of prednisolone starting with 120 mg per day, then reducing the dosage by 10 mg per day treatment of the rhinogenic infection (nasal spray, • mucolytica) vestibular suppressant medications and antiemetics • antibiotics intravenously to avoid bacterial super-• infection (cephalosporine, aminopenicillin with or without a β-lactamase-inhibitor) high doses of antibiotics, e. g. cephalosporines (third • generation), chloramphenicole and aminoglycosides, according to the smear culture results vestibular suppressant medications and antiemetics • in lues iii, penicillin g or tetracycline • in tuberculosis, tuberculostatic therapy • in mucormycosis, amphotericine b, control of diabe-• tes infusions with electrolytes; antipyretica • each otogenic labyrinthitis caused by bacteria needs surgical intervention. myringotomy, insertion of ventilating tubes ( the most frequent bacteria causing meningogenic labyrinthitis are meningococci, pneumococci and hemophilus influenzae type b. : severe vertigo with nystagmus, vomiting, high fever. it invariably results in complete hearing loss and is often followed by facial paralysis. meningogenic labyrinthitis: • classic symptoms of meningitis, severe vertigo with nystagmus, vomiting, unilateral or bilateral, often fluctuating hearing loss or complete deafness. postinflammatory rapid ossification of the cochlear fluid spaces. meningitis, encephalitis, brain abscess, complete deafness, lethal outcome. : congenital or acquired syphilis (third stage). fluctuating hearing loss, endolymphatic hydrops, dizziness. hennebert sign positive. if untreated, it results in complete hearing loss. : formation of tuberculous granuloma along vascular spaces which spread into the labyrinth spaces. progressive sensorineural hearing loss. in most cases it is associated with a systemic haematogenic spreading of tuberculosis. • systemic, in many cases fatal mycotic sepsis (caused by, e. g., rhizopus, absidia) in immunodepressed patients (aids, leukaemia, diabetes): high temperature, meningitis, deafness, facial palsy, vertigo, purulent sinusitis. otoscopy or ear microscopy: serous or purulent otitis • media, pulsating tympanic membrane, cholesteatoma, bone fracture hearing examination: tuning fork, audiogram (mixed • or pure sensorineural hearing loss, deafness) frenzel glasses: at the beginning nystagmus in the • direc tion of the affected ear, later in the opposite direction high-resolution "emergency ct" • early onset of therapy (antibiotics, antimycotics) is • obligatory in this life-threatening infection of the temporal bone. deafness and long-lasting dizziness. lethal outcome • in most cases of generalized mucormycosis. in cases of bilateral complete deafness following bacte-• rial meningitis, early cochlear implantation is recommended before ossification of the cochlea occurs (see sect 1.6.12). concussion of the labyrinth. microinjuries of the vestibulocochlear organ (bleeding, membrane ruptures, microfractures) caused by a blunt concussion of the skull, with or without fracture of the skull base or contusio cerebri. sensorineural hearing loss affecting all frequencies or mainly the frequencies above 3 khz. in lateral (parietal) trauma, mainly the opposite ear is affected (contrecoup); when the blunt forces act from behind (occipital trauma) then both labyrinths can be damaged. vertigo and tinnitus are accompanying symptoms. luxation of the ossicles (e. g. incus), perilymphatic • fistula, slowly progressive sensorineural hearing loss, deafness, long-lasting vertigo, subdural bleeding, posttraumatic endolymphatic hydrops. postconcussion disequilibrium syndrome: patho-• mechanisms and symtoms are identical with those of benigne paroxysmal vertigo (see sect. 1.6.15) and cupolithiasis [1] . detailed history (of forensic importance) • inspection of the skull, searching for skin injuries, • haematoma inspection of the oral, nasal and nasopharyngeal cavi-• ties otoscopy or ear microscopy, searching for fracture • signs, haemotympanum, rupture of the tympanic membrane hearing examination: tuning forks, audiogram, tym-• panogram, stapedius reflexes frenzel glasses: to exclude vestibular irritation or loss • of function of one vestibular organ schüller-x-ray, high-resolution ct: to exclude frac-• tures examination of the vestibular function (electronystag-• mography, video-oculography) examination of the sense of smell to exclude rupture • of the fila olfactoria the recommended european standard is use of antioedematous principles (see sect. 1.6.5). if there is an additional conductive hearing loss caused by trauma of the middle ear structures, reconstruction of the ossicular chain is recommended some weeks following the injury. csf leak: e. g. complication of vestibular schwannoma surgery, middle and posterior fossa surgery, surgery for chronic ear disease (less frequently) a watery pulsating secretion when there is a tympanic membrane perforation or rupture. when the tympanic membrane is intact, there is clear fluid behind the tympanic membrane simulating a serous otitis media ( fig. 1.6.4) . outflow of watery fluid from the nose (otorhinoliquorrhoea). coughing at night. a sensation of salty fluid in the mouth. recurrent meningitis, meningoencephalitis. the prognosis is uncertain: in many cases complete restoration of the cochleovestibular deficit is possible; in some cases progressive hearing loss and/or long-lasting vertigo are possible. sudden deafness, sudden idiopathic sensorineural hearing loss. sudden sensorineural hearing loss is characterized by an acute in the majority of cases unilateral hearing loss originating in the inner ear of unknown pathogenesis and origin. hearing loss may be slight, moderate, profound or complete and concerns the high, middle, deep or all frequencies. the disease is accompanied by tinnitus in about 85% of cases and dizziness in about 30% of cases. the incidence ranges between 20 cases per 100,000 residents per year in austria and germany, eight to 13 cases per 100,000 residents per year in japan and about 11 cases per 100,000 residents per year in the usa. women are equally affected as men, most frequently at the age of 50 ± 5 years. however, there is an increasing incidence in younger people, whereas children are rarely concerned. the cause is still unknown. the following pathomechanisms are currently discussed: impairment of cochlear blood flow due to vasomotor according to the affected frequencies and severity the following types of sudden idiopathic sensorineural hearing loss can be distinguished ( fig. 1.6 .5): high-frequency idiopathic sensorineural hearing • loss, which may be due to cellular malfunction of outer (below 50 db hl) and inner (beyond 60 db hl) hair cells (fig. 1.6 .5a). low-frequency idiopathic sensorineural hearing • loss, which may be due to endolymphatic waterelectrolyte imbalance or even endolymphatic hydrops as a possible result of impaired blood flow within the vascular stria and subsequent hypoxic cellular damage ( fig. 1.6 .5b). middle-frequency idiopathic sensorineural hearing • loss, which may be due to impaired blood flow within the spiral lamina and subsequent hypoxic cellular damage of the organ of corti or, alternatively may be due to genetic defects ( fig. 1.6 .5c). all-frequency idiopathic sensorineural hearing • loss, which may be due to blood flow impairment in the spiral modiolar artery and/or upstream arteries resulting in hypoxic damage of cochlear tissues ( fig. 1.6 .5d). profound idiopathic sensorineural hearing loss and • deafness, which may be due to thrombotic or embolic blood flow obstruction in the arteria cochlearis communis or spiral modiolar artery resulting in hypoxic damage of cochlear tissues. perilymphatic fistula due to rupture of the round window membrane or lesion of the oval window which may result in acute profound hearing loss or even deafness is a disorder of a distinct origin and, therefore, cannot be referred to as idiopathic sensorineural hearing loss by definition ( fig. 1.6 .5e). , such as fluctuating hearing thresholds, progressive hearing loss in spite of current therapy etc. patients complain about symptoms in the following descending order of incidence: acute • unilateral subjective hearing loss, although this symptom may be not perceived in cases of slight hearing loss restricted to a few frequencies another complication may concern psychoemotional and psychosocial problems in cases of persistent hearing loss and/or tinnitus (see sect. 1.6.8). recommended european standard tus was not taken into consideration. patients would not regard themselves as fully recovered when their tinnitus is still persistent. however, in well-informed patients presenting with a slight hearing loss without previous ipsilateral or contralateral hearing loss and without tinnitus and/or dizziness, one may await spontaneous recovery for a few days. basic therapeutic interventions comprise normalization of systemic blood pressure, heart rate and haematocrit level (below 45). for patients complaining of moderate to severe psychoemotional ailments such as anxiety, fear, distress, restlessness, agitation, resignation and sense of guilt, a psychotherapist should be included. according to the aforementioned cause and pathogenesis possibly involved, therapeutic recommendations differentiate between the various types of sudden idiopathic sensorineural hearing loss. high-frequency idiopathic sensorineural hearing • loss. a daily dose of 250-500 mg prednisolone intravenously on three consecutive days is recommended. in the case of partial or no remission, prednisolone treatment should be continued orally for 16 days, starting with 100, 80, 60, 40, 20, 10, 5 and 2.5 mg each dosage for two days. prednisolone therapy should be accompanied by gastric proton pump inhibitors (40 mg omeprazole per day or 150 mg ranitidine per day or others). prednisolone is a synthetic analogue of endogenous corticosteroid hormones classified as glucocorticoids. besides anti-inflammatory effects, prednisolone possesses multiple other cellular effects. the rationale for administration of prednisolone is based on the consideration that inflammatory tissue alterations are also elicited by tissue ischaemia and hypoxia. in addition, prednisolone mobilizes amino acids for gluconeogenesis, alters glucose utilization and influences protein metabolism. finally, prednisolone binds with equal affinity to both glucocorticoid and mineralocorticoid receptors widely distributed in cochlear tissues, thereby contributing to restoration of cellular osmolarity, electrochemical gradients, transmembrane ion flux and neuronal conduction. in patients presenting with contraindications (e. g. diabetes, severe chronic gastritis, gastric or duodenal ulcera) for the treatment with prednisolone, intravenous infusion of a hyperoncotic hydrophilic haemodilutive plasma-expanding agent, such as hydroxyethyl starch or others (250-500 ml/day for 5-10 days) is a good alternative option. sudden idiopathic sensorineural hearing loss must be managed with care and as soon as possible concerning the diagnostic procedures and the mode and beginning of therapy. according to the categories of the oxford centre for evidence-based medicine (http://www.cebm.net), the evidence level of most clinical trials on therapy of sudden deafness is relatively low. in addition, the clinical trials considered in the cochrane international register of controlled clinical trials, e. g. prospective, randomized, placebo-controlled and double-blind conducted studies, are of relatively high level of evidence; however, the underlying study protocols vary tremendously. therefore, universally valid therapeutic options based on reproducible results are not available. instead, thera peutic recommendations are merely formed empirically. spontaneous recovery rates range from 31 to 68%; however, these data were revealed from retrospective, non-randomized and mostly non-controlled trials on a few patients only. furthermore, the term "recovery" was not exactly defined, e. g. partial remission was not distinguished from complete remission, and a persistent tinni-however, contraindications (e. g. arterial hypertension) should be noted, and potential side effects (e. g. temporary pruritus) should be considered. in the case of contraindications for haemodilution, another haemorheological active drug such as pentoxifylline (100 mg, equivalent to 5 ml/day dissolved in 100 ml 0.9% isotonic saline) may be intravenously infused for 5-10 days. additional administration of scavangers of toxic free oxygen radicals such α-lipoic acid (600 mg/day orally) during haemodilutive or haemorheological infusion therapy may be reasonable to prevent reperfusion injury within the cochlea. low-frequency and middle-frequency idiopathic • sensorineural hearing loss. a daily dose of 250-500 mg prednisolone intravenously on three consecutive days is recommended. in the case of partial or no remission, prednisolone treatment should be continued orally for 16 days together with gastric proton pump inhibitors as described above. additional osmotic infusion therapy using mannitol (15 g in 100 ml solution) and acetazolamide (500 mg) intravenously on three consecutive days may be administered. acetazolamide (250 mg/day) may be continued orally for 10 days. in patients presenting with contraindications (e. g. diabetes, severe chronic gastritis, gastric or duodenal ulcer) for the treatment with prednisolone, initial osmotic therapy is recommended. all-frequency idiopathic sensorineural hearing • loss. a daily dose of 250-500 mg prednisolone intravenously on three consecutive days is recommended. in the case of partial or no remission, prednisolone treatment should be continued orally for 16 days together with gastric proton pump inhibitors and additional haemodilutive/haemorheological infusion therapy together with α-lipoic acid should be administered as described for high-frequency idiopathic sensorineural hearing loss. in patients suffering from an elevated fibrinogen level (above 300 mg/dl) fibrinogen and low density lipoprotein apheresis may be a good alternative to haemodilutive or haemorheological infusion therapy. however, the expense is relatively high and the long-term outcome on hearing gain was proved to be equally effective as with conventional therapy as described above. profound idiopathic sensorineural hearing loss and • deafness. a daily dose of 500 mg prednisolone intravenously on three consecutive days together with haemodilutive/haemorheological infusion therapy and α-lipoic acid should be administered as described for high-frequency idiopathic sensorineural hearing loss. prednisolone treatment should be continued orally for 16 days together with gastric proton pump inhibitors as described, and haemodilutive/haemorheological infusion therapy and α-lipoic acid should be continued for another seven consecutive days. in patients suffering from an elevated fibrinogen level (above 300 mg/dl) fibrinogen and low density lipoprotein apheresis may be additionally tried (but see the comments for all-frequency idiopathic sensorineural hearing loss). in the case of minor or no remission at all, perilympatic fistula due to rupture of the round window membrane or lesion of the oval window may be considered and tympanoscopy should be performed preferably within 5-10 days after onset of hearing loss. if initial therapy with prednisolone and/or haemodilutive or alternative haemorheological agents is only partially or not effective at all, and this is the case in over 25% of patients, hyperbaric oxygenation therapy (ten to 15 sessions on ten to 15 consecutive days) should be started as soon as possible, preferably within 3-6 weeks after the onset of idiopathic sensorineural hearing loss. in the case of progressive hearing loss in spite of current therapeutic intervention or in the case of minor or no remission of profound hearing loss, tympanoscopy should be performed to exclude or seal a rupture of the round window membrane or a lesion of the oval window (see sect. 1.6.5.9). sudden sensorineural hearing loss may also be attributable to the following diseases: perilymphatic fistula • . perilymphatic fistulae of the oval or round window are rare clinical findings. they the prognosis is most favourable in low-frequency and middle-frequency idiopathic sensorineural hearing loss without previous hearing loss. however, recurrence rates of such hearing losses range up to 30%. patients with slight to moderate threshold shifts recover better than patients presenting with moderate to profound hearing loss. a relatively worse prognosis is expected in profound hearing loss or even sudden deafness. ménière's syndrome, idiopathic endolymphatic hydrops, morbus ménière. the syndrome is characterized by: recurrent spontenous attacks of vertigo, fluctuating hearing loss (at the early beginning mainly affecting low frequencies) tinnitus and aural fullness. according to the american academy of otolaryngology-head and neck surgery at least two attacks of objective rotational vertigo, each of 20-min duration or longer must occur to confirm the diagnosis of ménière's disease. unilateral sensorineural hearing loss may occur during skull trauma, following stapes surgery, middle ear surgery, after lifting heavy weights or spontaneously. perilymphatic fistulae in the area of the annular ligament of the stapes footplate cause sensorineural hearing loss and dizziness of different extent. they can occur after stapes surgery with insufficient sealing of the prosthesis. congenital perilymphatic fistulae of the stapes footplate occur in cases of malformation of the stapes and cause recurrent meningitis. they are usually detected during exploration of the oval window. perilymphatic fistulae of the round window membrane are rare findings in cases of sudden hearing loss. there are no clear clinical symptoms characteristic for round window membrane rupture. in some cases the history reveals strong physical exertion (explosion trauma after goodhill). all diagnosed perilymphatic fistulae should be completely sealed with connective tissue and fibrin glue to avoid labyrinthitis and/or meningitis. surgical sealing of the ruptured round window membrane or annular ligament with connective tissue sometimes results in partial or complete, but not predictable, restoration of hearing. haematological diseases (e. g. polycythaemia, poly-• globulia, leukaemia, exsicosis psychogenic hearing loss. • must be documented audiometrically on at least one occasion. the diagnosis "ménière's disease" always is a diagnosis "per exclusionem" since other diseases of the cochleovestibular system can mimic ménière's symptoms. temporal bone histopathology from ménière's patients are usually showing dilatations (hydrops), distortions and/or ruptures ( fig. 1.6 .6) of the delicate membranes separating the endolymphatic from the perilymphatic fluid compartements. hydrops can develop simultaneously as well in the cochlear as in the vestibular compartments, but also isolated in only one of the compartments (this may explain why in ménière the symptoms can only consist of attacks of vertigo or hearing loss with tinnitus). a malfunctioning spiral ligament and /or endolymphatic sac (disturbed resorption of the endolymph, immunologic factors causing "saccitis") seem to be involved in the pathophysiology of endolymphatic hydrops. recently the homing of herpes type i viruses has been demonstrated as well within the endolymphatic sac as in ganglion cells of scarpa's ganglion. reactivation of these viruses seems to be triggered by (immunologic) stress factors clinically causing the typical symptoms of the disease. there are many known causes of endolymphatic hydrops: viral labyrinthitis, autoimmune inner ear disease (e. g. cogan's syndrome), otosclerosis, leukaemia, otosyphilis, surgical trauma to the inner ear, temporal bone trauma, etc. when the cause cannot be identified, the term "ménière's disease" is used. the incidence is one in 8,000 individuals per year in europe. approximately 30% of patients will develop contralateral involvement over time (25% at 10-year follow-up). classic symptoms are: recurrent attacks of rotational vertigo (the most dis-• abling symptom for the patients) which last from several minutes to hours. vertigo is often associated with nausea and vomiting. fluctuating, unilateral low-frequency hearing loss of • the sensorineural type (upsloping audiometric pattern). in the late stages of the disease, there is flat or downsloping non-fluctuating sensorineural hearing loss (hearing threshold of 50-60 db, speech discrimination of 50-60%). during the attacks, subjective tinnitus and aural full-• ness/pressure are present. between attacks patients are not dizzy but aural fullness and tinnitus may persist. vertigo attacks can vary in frequency, intensity and duration. generally the attacks increase in severity and frequency with progression of the disease. the frequency and intensity of the vertigo attacks decrease after approximately 5-10 years. clinical history: extremely important. physical examination: normal findings. between the • attacks patients usually display horizontal, spontaneous nystagmus beating away from the affected ear (paretic nystagmus). electrocochleography • : a significant enhancement of the summating potential to action potential amplitude ratio (more than 30%) occurs in 60% of patients with ménière's disease. auditory brainstem response. • mri with contrast medium plays an important role in • excluding a retrocochlear lesion (vestibular schwannoma) in any patient with unilateral neurotologic symptoms! blood sedimentation rate, antinuclear antibody test • for autoimmune ear disorders (see sect. 1.6.7); fluorescent treponemal antibody absorption test to rule out syphilis. glucose tolerance and thyroid function tests are rec-• ommended as hypothyroidism and diabetes may be associated with the disease. lipid profile. caloric tests (with electronystagmography): reduced • caloric response on the affected side. intratympanic application of ganciclovir 50 mg/ml per • 10 days through a ventilating tube or a microwick inserted into the tympanic membrane [2] . endolymphatic glycerol test (1.2-1.5 ml/kg body weight of glycerol • mixed with lemon juice): frequently used in the past, but much less nowadays because of its side effects (headache, nausea, vomiting). note: the diagnosis is often by exclusion. vestibular schwannoma • sudden sensorineural hearing loss • autoimmune hearing loss • migraine • 1.6.6.8 therapy 1. in acute attacks bed rest, vestibular suppressant medication (diazepam) and antiemetics (transdermal scopolamine, thiethylperazine, prochlorperazine) are recommended 2. to prevent attacks, decrease the amount of fluid in the inner ear by diet and diuretics − diet: low salt intake (less than 3 g per day) and reduced water intake − diuretics: acetazolamide, chlortalidone, hydrochlorothiazide, furosemide. replace potassium if needed 3. in addition − vasoactive drugs (betahistidine) to improve blood flow in the inner ear − steroids to suppress inflammatory and/or allergic tissue reactions within the inner ear (endolymphatic sac) 4. avoidance of caffeine, alcohol, tobacco 5. antiviral approach [1] : oral acyclovir (800 mg 3 times a day) for 3 weeks; if the patient feels better, reduce the dose to 800 mg 2 times a day for 1 month and then to 800 mg daily for another month before ending the treatment. insertion of a • transtympanic ventilating tube (montandon) ( fig. 1 autoimmune hearing loss, immune-mediated inner ear disease. an underlying genetic predisposition results in auto-• immune disease expression following immunoregulatory defects in immune response to unknown environmental pathogens. today there is substantial evidence of autoimmune • mechanisms in relapsing polychondritis ( fig. 1. 3.2c), cochlear vasculitis (e. g. cogan's syndrome), progressive sensorineural hearing loss of both sides and some types of sudden deafness. with regards to ménière's disease, around 16% of bi-• lateral cases and 6% of monolateral cases may be due to immune dysfunction. autoimmune inner ear disease • represents less than 1% of all cases of hearing impairment or dizziness; nevertheless, the diagnosis might be overlooked because of the lack of a specific diagnostic test. the disease seems to be more common in females than in males; the first onset of symptoms generally occurs between 20 and 50 years of age. sympathetic cochleopathy: sudden sensorineural hear-• ing loss in the last hearing ear (similar to sympathetic ophthalmopathy). hearing loss: a rapidly progressive, often fluctuating, • bilateral sensorineural hearing loss over a period of weeks to months. the progression of hearing loss is too rapid to be diagnosed as idiopathic progressive sensorineural hearing loss or presbyacusis and too slow to conclude a diagnosis of sudden sensorineural hearing loss. tinnitus: 25-50% of patients also have tinnitus (ring-• ing, hissing, roaring) and aural fullness, which can fluctuate. vertigo and/or imbalance: generalized imbalance, • ataxia, motion intolerance, positional vertigo and episodic vertigo may be present in up to 50% of patients. occasionally only one ear is affected initially, but bilat-• eral hearing loss occurs in most patients (80%), with symmetric or asymmetric audiometric thresholds. currently, the diagnosis of autoimmune inner ear dis-• ease is based either on clinical criteria or on a positive response to steroids. there is seldom convincing evidence from broader laboratory tests indicating autoimmunity. detailed history: endocrine diseases? recurrent fe-• ver? otoscopy findings are usually normal; nevertheless external ear skin and/or cartilage inflammation and/or facial palsy may rarely occur (e. g. relapsing polychondritis), as well as tissue destruction at the level of the tympanic membrane, middle ear and mastoid (e. g. wegener's granulomatosis). there are no antigen-specific tests (migration inhibition test, lymphocyte transformation test and western blot analysis) that are commercially available and proven to be useful for the diagnosis of systemic autoimmune diseases. in clinical practice next to the indispensable blood sedimentation rate (bsr) a non-specific antigen screening test may be useful for evidence of systemic immunologic dysfunction; yet it does not strictly correlate with a diagnosis of immune-mediated inner ear disease. recommended tests are: blood tests for autoimmune disorders: levels of cir-• culating immune complexes, bsr, antinuclear antibodies, rheumatoid factor, complement c1q, smooth muscle antibody, tsh and antimicrosomal antibodies, antigliadin antibodies (for celiac disease), hla testing. blood tests for conditions that resemble autoimmune • disorders: fluorescent treponemal antibody absorption test (for syphilis), lyme titre, hba1c (for diabetes, which is often also autoimmune-mediated), hiv (hiv is associated with auditory neuropathy). note that a commercially available test, called "anti-68 kd (hsp-70) western blot" (otoblot tm ) was reported to deautoimmune inner ear disease is analogous to rapidly progressive glomerulonephritis. if not treated, the inner ear inflammation progresses to severe irreversible damage within 3 months of onset (and often much more quickly). on the other hand, steroid responsiveness is high and with prompt treatment the hearing loss may be reversible. nevertheless, several patients become steroiddependent. ringing in the ears (from latin tinnire meaning "ringing"). it is important to distinguish between objective and subjective tinnitus: patients affected by an • objective tinnitus notice a real existent endogenous acoustic source originating in the middle ear, eustachian tube, soft palate or extracranial or intracranial vessels. such acoustic phenomena may also be perceived by the non-affected fellow human being using a stethoscope; however, the incidence of this kind of tinnitus is relatively seldom. depending on the underlying disease, the patients notice intermittent clicks or crackles due to spasm of the middle tect a local autoimmune inner ear process in the absence of any systemic autoimmune process and to be correlated with steroid responsiveness. the test uses purified hsp-70 kda antigen from a bovine kidney cell line and is based on the assumption that the 68-kda protein is heat shock protein 70 (hsp-70). unfortunately, this assumption has now been refuted: in fact, there is mounting evidence that the target antigen of the 68-kda antibody is not hsp-70 (as was believed over the last 15 years), but the human choline transporter-like protein 2 (ctl2). furthermore, the sensitivity and specificity of this test are very low. large vestibular aqueduct syndrome • endocranic hypertension • prednisolone, 1 mg/kg per day for 4 weeks followed by • a gradual tapering over several weeks to a maintenance dose of 10-20 mg per day or every other day. shorterterm or lower-dose long-term therapy either has been ineffective or appears to increase the risk of relapse. patients often learn the necessary maintenance dose to preserve their hearing, as the disease activity often waxes and vanishes. if hearing suddenly worsens or tinnitus reappears in one or both ears during the tapering period, repetition of the initial high-dose treatment is indicated. oral as well as systemic steroid treatment over long • period of time should always be accompanied by proton pump inhibitors to avoid a gastric ulcer. in patients with no response to steroids within • 6-8 weeks, methotrexate and cyclophosphamide have been used over the long term. these agents are associated with considerable toxicity and the decision regarding when and how to use them should always be multidisciplinary. the normal oral dose of methotrexate is 7.5-20 mg weekly with folic acid. cyclophosphamide in addition to steroids has been used with the following regimen: cyclophosphamide 5mg/kg per day intravenously for 2 weeks, followed by a rest period of 2 weeks, and then a final period of infusions for 2 weeks. ear muscles or myoclonus of soft palate muscles, respiratory noise and breath, respectively, due to an abnormally wide eustachian tube, or a pulsatile noise due to intracranial hypertension, glomus tumour, angioma, aneurysm, arteriovenous fistula, stenosis or thrombosis of extracranial or intracranial vessels as well as due to systemic rheological diseases such as hyperglobulinaemia or anaemia. in contrast, a • subjective tinnitus is exclusively perceived by the affected patient. in most cases it consists in an intermittent or continuous whistling or fizzling, in broadband or narrowband noise, hum, ping or ringing, or even in pure tones of various frequency, intensity and duration. such auditory perceptions emerge from deficient neuronal plasticity within the central auditory system triggered by an auditory input failure. in this respect, a subjective tinnitus is a symptom of any disease of the peripheral and/or central auditory system associated with malfunction of hearing. in this particular context it should be emphasized that tinnitus is not a symptom of an organic or functional disease of the cervical spine, temporomandibular joint or any other orthopaedic or dental distress; likewise tinnitus is not caused by emotional, mental or physical distress, although the intensity and annoyance of tinnitus may be amplified by such problems. there are no epidemiological data available concerning the incidence and prevalence of an objective tinnitus. a transient subjective tinnitus is perceived by about 35-45% of the population of industrial nations at least once in their life; 13-17% have perceived tinnitus over a longer period in their life, with an annual incidence of 0.33% in germany. the point-prevalence of a constant chronic tinnitus (perceived as longer than 6 months up to years) averages about 4% of the population of industrial nations, of which about 0.5-1% regard themselves as severely psychoemotionally affected. of those patients who perceive tinnitus over a longer period in their life, 35-37% notice the tinnitus in a silent environment only (grade i); 44-51% perceive their tinnitus permanently, however it may be masked by moderate environmental noise (grade ii). in only 14-17% of cases is tinnitus perceived even in a relatively loud environment (grade iii). according to a recent evaluation of about 12,000 members of the german tinnitus support group (deutsche tinnitus-liga, http://www.tinnitus-liga.de) who perceived tinnitus for more than 6 months up to years, 45% localized their tinnitus in both ears, while a further 24% localized their tinnitus perception in the middle of the head. of the unilaterally affected, 29% perceived tinnitus in the left ear and 20% in the right ear. the high incidence of bilateral tinnitus averaging about 46% and the slight preference of the left ear is also known from former evaluations among non-selected populations. the prevalence of tinnitus is only somewhat higher (by 1-5%) in females than in males. respecting the age distribution, manifestation of tinnitus is most common between 40 and 60 years of age; however, there is an increasing incidence of noise-induced tinnitus in younger people owing to exposure to leisure noise such as noisy toys, amplified music, motorcycling and other loud recreational activities. data from long-term studies concerning the incidence and prevalence of a chronic tinnitus (for more than 6 months up to years) in this population are not available so far. concerning an objective tinnitus, there are no data available regarding the incidence of the underlying diseases, as mentioned already in sect. 1.6.8.2. the subjective tinnitus is a symptom of any diseases of the peripheral and/or central auditory system associated with malfunction of hearing in the following descending order of incidence: in 32% tinnitus is being caused by noise-induced damage of the inner ear due to single or repetitive exposure to industrial or leisure noise; in 12% by acute acoustic trauma of the inner ear due to single or repetitive sound impulses from pistols, revolvers, military rifles, sport guns, firecrackers, fireworks and others; in 8-10% by idiopathic sensorineural hearing loss (sudden deafness); in 8% by ménière's disease (morbus ménière); in 7% by age-related sensorineural hearing loss (presbyacusis); in 6% by toxic labyrinthitis due to an acute serous or purulent otitis media; in 4% by chronic otitis media inclusive of cholesteatoma; in 2-3% by otosclerosis; and in 1% by a vestibular schwannoma (acoustic neurinoma). in the remaining 20-23% tinnitus may be caused by obliteration of the outer ear canal with wax, exostosis or others; myringitis, rupture or perforation of the ear drum; dysfunction of the eustachian tube due to acute or chronic infections of the upper respiratory tract; barotrauma of the middle ear; tympanosclerosis; luxation of the incudomalleal or incudostapedial articulation; rupture of the round window membrane; perilymphatic fistula of the round or oval window; labyrinthine contusion or fracture of the temporal bone due to head trauma; meningitis or encephalitis; ototoxic medication (aminoglycosides, cisplatin, etc.); intoxication by alcohol or drugs; peridural in summary, there are almost 100 diseases of the peripheral and/or central auditory system which may cause tinnitus. in this respect, an accurate otoneurological diagnostic procedure is of prime importance. subjective tinnitus cannot be treated directly. in fact, treatment is targeted to the underlying disease of the peripheral and/or central auditory system to achieve elimination of auditory input failure, thereby correcting pathological neuronal plasticity within the central auditory system. it is important to explain these issues to the patient in the acute stage, e. g. first tinnitus counselling. 1. acute stage (less than 3 months' duration). acute tinnitus due to noise-induced damage or acoustic trauma of the inner ear, idiopathic sensorineural hearing loss (sudden deafness), acute attack of ménière's disease, toxic labyrinthitis, rupture of the round window, perilymphatic fistula of the round or oval window, labyrinthine contusion or fractures of the temporal bone due to head trauma should be treated with a daily dose of 250-500 mg prednisolone intravenously on three consecutive days. in the case of partial or no remission, prednisolone treatment should be continued orally for 16 days, starting with 100, 80, 60, 40, 20, 10, 5 and 2.5 mg each on two consecutive days. prednisolone therapy should be accompanied by gastric proton pump inhibitors (40 mg omeprazole per day or 150 mg ranitidine per day or others). in patients presenting with contraindications (e. g. diabetes, severe chronic gastritis, gastric or duodenal ulcer) for the treatment with corticosteroids classified as glucocorticoids such as prednisolone, infusion therapy as described below may be a good alternative option. in cases of severe hearing loss, additional intravenous infusion therapy using a hyperosmotic hydrophilic haemodilutive plasma-expanding agent, such as hydroxyethyl starch or others (250-500 ml/day for 5-10 days), would be reasonable to improve microcirculation. however, contraindications (e. g. arterial hypertension) should be noted, and potential side effects (e. g. temporary pruritus) should be considered. in the case of contraindications for haemodilution, another haemorheological active drug such as pentoxiphylline (100 mg, equivalent to 5 ml/day dissolved in 100 ml 0.9% isotonic saline) may be intravenously infused for 5-10 days. if this initial therapy with prednisolone and/or haemodilutive or alternative haemorheological agents is only partially effective or not effective at all, and this is the case in over 25% of patients, hyperbaric oxygenation therapy should be started as soon as possible (ten to 15 sessions on ten to 15 consecutive days). however, according to clinical trials therapeutic results on tinnitus are only available from patients suffering from acoustic trauma, noise-induced hearing loss and idiopathic sensory-neural hearing loss (sudden deafness); therefore, hyperbaric oxygenation therapy should be restricted to these three indications. for additional treatment of tinnitus due to an acute attack of ménière's disease, toxic labyrinthitis, rupture of the round window, perilymphatic fistula of the round or oval window, labyrinthine contusion or fractures of the temporal bone due to head trauma, see the specific sections. likewise, for treatment of all other diseases of the peripheral and/or central auditory system which may cause a subjective tinnitus, such as chronic otitis media, cholesteatoma, mastoiditis, otosclerosis and vestibular schwannoma (acoustic neurinoma), see the specific sections. 2. subacute stage (duration of more than 3 months up to 1 year) and chronic stage (duration of more than 1 year). in both, the subacute and the chronic stage a − patients presenting with tinnitus-associated and/ or other complaints, such as sleep disturbance, concentration and attention problems, psychoemotional and psychosocial problems, should be admitted to a cognitive tinnitus coping therapy including a multimodal behavioural treatment in a specialized psychotherapeutic-psychosomatic outpatient department, or in selected cases to a psychotherapeutic-psychosomatic inpatient clinic. − a promising novel therapeutic innovation is neurofeedback training, which has been shown to effectively relieve stress-associated symptoms and thereby annoyance of tinnitus. the outcome is dependent on the quality of the initial and the following medical attendance, e. second tinnitus counselling should be performed as follows: − tinnitus is an auditory phantom perception which emerges from deficient neuronal plasticity within the central auditory system triggered by an auditory input failure due to any disease of the peripheral and/or central auditory system. − it is ensured that all aforementioned initial therapies and/or other causal treatment of the underlying disease of the peripheral and/or central auditory system have failed to ameliorate or eliminate tinnitus at the latest by the end of the subacute stage. − the patient should be briefed that the following pharmaceuticals, natural remedies and other therapeutic strategies have been proven to have no persistent effect on subacute and/or chronic tinnitus in placebo-controlled clinical trials: intratympanal application of glucocorticoids, lidocaine, glutamate-receptor agonists or antagonists; systemic administration of antiarrhythmics such as lidocaine, anticonvulsive drugs such as lamotrigine or carbamazepine, antidepressives such as trimipramine, nortriptyline or amitriptyline, benzodiazepines such as diazepam or alprazolam, vasodilative drugs such as pentoxifylline, cyclandelate, prostaglandin e1, analogues of histamine such as betahistine, antagonists of histamine receptors such as cinnarizine, calcium antagonists such as flunarizine or trimetazidine, gaba agonists such as baclofen, antiphlogistic drugs such as azapropazone, diuretics such as dyazide, melatonin, zinc, vitamins, ginkgo biloba, laser therapy of any kind with or without ginkgo biloba, acupuncture of any kind, ultrasonic therapy and electromagnetic stimulation of any kind. − therefore, in the chronic stage correction of pathological neuronal plasticity within the central auditory system cannot be achieved directly by pharmaceuticals, natural remedies or other treatment strategies already mentioned, but can be achieved indirectly by compensation of the remaining hearing loss using hearing aids as soon as possible. however, in cases where hearing loss is less than 30 db hl at three or fewer frequencies (2 khz included), a broadband noise generator (formerly tinnitus masking device) may be helpful to defocus on tinnitus. − in addition, active listening to music of someone's own choice and/or to audiobooks are reasonable therapeutic auditory training strategies which may be helpful to direct the patient's attention to external auditory stimuli again. 1.6.9 noise-induced hearing loss 1.6.9 noise-induced hearing loss the disorder is defined as hearing loss caused by acute or chronic exposure to high-intensity sound. impulse sound exposure with an intensity above 150 db(a) spl peak equivalent with a peak sound pressure duration greater than 3 ms. explosion traumas are seen in explosives fabrication and the processing industry, in the military, in the gas, tyre and chemical industry as well as in motor vehicle accidents. additionally, they can occur from a physical injury to the ear, such as a blow to the side of the head, diving head first into water or deployment of an airbag. an explosion trauma of the ear generally leads to inner ear damage concomitant with a rupture of the eardrum and, most often, a disruption of the ossicular chain ( fig. 1.4.1b) . this leads to a unilateral or bilateral, moderate to severe combined (conductive and sensorineural) hearing loss with otalgia, tinnitus and, frequently, vestibular symptoms. at the time of examination, a serous otitis media has commonly developed. complete hearing loss and tinnitus are feared. further complications are long-lasting vertigo and rupture of the round window membrane. detailed patient history-important in the case of a video-oculography (vog) or electronystagmography • (eng). an eventual caloric testing must be performed with cold and warm air insufflation in the case of ear drum injury. tinnitus analysis with subjective loudness assessment • and testing of masking possibility ( fig. 1.6 .7). as in sudden hearing loss (see sect. 1.6.5) • initiate hyperbaric oxygen therapy as soon as pos-• sible! in the case of complete hearing loss and suspicion of as in sudden hearing loss (see sect. 1.6.5). with more severe inner ear damage, initiate additional • hyperbaric oxygen therapy as soon as possible. in the case of complete hearing loss and suspicion of • rupture of a cochlear window: tympanotomy and closure of a perilymphatic fistula (see sect. 1.6.5). minor high-frequency sensorineural hearing loss and a more or less disturbing tinnitus can persist in spite of rapid and correct therapy. the chances of spontaneous recovery without therapy • is low. sometimes untreated post-traumatic hearing loss is progressive. myringoplasty (see sect. 1.4.6) • tympanoplasty (see sect. 1.4.6) • with occupational explosion trauma, special compensation insurance must be addressed (berufsgenossenschaft in germany, suva in switzerland, inail in italy). shooting trauma, muzzle blast trauma. impulse sound exposure with an intensity above 150 db(a) spl peak equivalent with a peak sound pressure duration shorter than 3 ms (normally 0.4-1.5 ms). the main cause of shooting trauma is inappropriate use of a handgun, e. g. in the military, by police or by hunters. however, also guns firing blanks and toy guns produce sound pressures greater 100 db(a)! additionally, fireworks and airbags ( fig. 1.6 .8) can cause muzzle blast trauma. otomicroscopy shows no abnormalities. in audiometric testing, there is an acute, most often unilateral mild to moderate sensorineural hearing loss of around 4 khz (c 5 notch) and a positive recruitment. tinnitus in the damaged ear is common. sometimes otalgia or vestibular disturbance is present. in the case of repeated muzzle blast trauma in a short period of time (toy gun), a steep highfrequency hearing loss above 2 khz develops. persistent tinnitus. complete deafness or persistent vestibular vertigo is very rare. the surgical treatment is no replacement for conserva-• tive therapy. if needed, it is carried out additionally. derived from patient history, the event of accident and clinical findings any of the following other possible diagnoses should be considered: cochlear window rupture • contusio labyrinthi • distortion of the cervical spine (whiplash injury) • an initial good recovery of the high-frequency hear-• ing loss occurs in approximately half of the cases. after some weeks, a remaining hearing loss or a persisting tinnitus must be considered as permanent damage. a post-traumatic increase of hearing loss above 2 khz is possible. a spontaneous recovery of a high-frequency tinnitus is rare. if high-dose glucocorticoid, rheologic therapy and • hyperbaric oxygen therapy is administered as soon as possible, recovery to normal hearing is frequently achievable. with occupational blast trauma any special compensation insurance must be addressed (berufsgenossenschaft in germany, suva in switzerland, inail in italy). broadband sound exposure with an intensity above 100 db(a) spl peak equivalent for minutes to hours. exposure to non-impulse permanent sound causes metabolic as well as mechanical ultrastructurally visible damage at the level of the organ of corti (outer hair cells, stereocilia). excessive demand for oxygen and subsequent progressive ischaemia of the cochlea lead to production of free radicals, depletion of endogenous cellular antioxidants and, finally, apoptotic cell death. discotheques: averaged over 15 min, a music sound • pressure level of 100-110 db(a) with sound pressure peaks of up to 125-130 db(a) spl can be measured. acute, most often bilateral, mild to moderate high-frequency sensorineural hearing loss (c 5 notch) with positive recruitment. tinnitus is frequent, as is a temporary threshold shift. otalgias or vestibular symptoms occur rarely. permanent hearing loss and/or tinnitus. detailed patient history-important in the case of a • later lawsuit and for expert reports. complete ent examination. by definition, noise-induced hearing loss is an effect of working in noise and therefore occurs most frequently in metalworkers, mineworkers, airport workers, radio operators, disc jockeys and military personnel as well as construction workers and orchestra musicians, etc. because of extensive recruitment, this progressive binaural hearing loss leads to an important communication problem in noisy environments (conversation of multiple persons, theatre, restaurant). a pronounced hyperacusis is common. tinnitus is found in 70% of patients. social isolation following hearing loss, psychiatric impairment secondary to the tinnitus. detailed patient history, including professional and the prognosis is unfavourable despite correct therapy; progress is possible. bilateral, mostly symmetric sensorineural hearing loss following intermittent exposure to broadband and/or impulse sound with an intensity above 80-85 db(a) and a daily exposure of 6-8 h (work shift) over many years. therefore, it is a matter of chronic noise damage. chronic noise exposure causes metabolic as well as mechanic ultrastructural visible damage at the level of the organ of corti, initially causing a loss of outer hair cells, leading finally to neuronal degeneration. typically, hearing loss initially occurs as a sensorineural high-frequency notch, normally at 4 khz (c 5 notch). the middle frequencies, e. g. the main speech frequencies, are affected considerably later. the recruitment is always positive. in approximately 70% of cases, a bilateral tonal tinnitus exists. the extent and the progress of the hearing loss depend on the intensity, duration of exposure and frequency composition of the sound as well as the duration of recovery phases and the individual noise susceptibility. an individual noise susceptibility is suspected in genetically predamaged inner ears, after sudden hearing loss, after treatment with ototoxic medication and trauma. moreover, humans with blond hair, fair complexion and lightly coloured eyes seem to be especially endangered owing to a lack of melanin. in most cases, the hearing loss can be compensated for • with hearing aids. in selected cases, a tinnitus masker can be helpful. by avoidance of noise or use of effective ear protection when working in a noisy environment, the progress of the chronic noise induced hearing loss can be prevented. presbyacusis (see sect. 1.6.11), progressive idiopathic • hearing loss, hereditary sensorineural hearing loss. drug-induced or toxic sensorineural hearing loss. post-traumatic sensorineural hearing loss following • contusio or commotio labyrinthi, craniocerebral injury, distortion of cervical spine (whiplash injury). if evident asymmetry of hearing thresholds and/or • pathologic findings in vestibular testing or bera are present, the diagnosis of noise-induced hearing loss is unlikely and a search for a retrocochlear cause of hearing loss (tumour of the cerebellopontine angle, multiple sclerosis, etc.) is necessary. after cessation of activity in a noisy environment, • noise-induced hearing loss is not progressive anymore. this is the reason why routine use of noise protectors or a change to a less noisy work environment can stop the progression of hearing loss. if a sensorineural hearing loss shows progression de-• spite cessation of noise exposure, other or additional causes must be sought. adverse effect to the cochlear or vestibular portion of the inner ear caused by pharmaceutical agents. the most ototoxic compounds in clinical practice are aminoglycoside antibiotics (streptomycin, dihydrostreptomycin, neomycin-all routes of administration, kanamycin, gentamicin), loop diuretics, quinines and chemiotherapy agents (cisplatin) (table 1.6.1). the incidence of ototoxicity has not been accurately determined. risk factors are a decreased renal function, increased daily doses, extended duration, concomitant administration with more than one ototoxic drug and prematurity. the following symptoms may be temporary or permanent: high-pitched tinnitus (earliest sign of cochlear damage), hearing loss (with or without vertigo), nausea, dizziness. initially the loss of hearing affects the high frequencies. as damage progresses, the lower frequencies become involved. uwe ganzer and andreas arnold age-related hearing loss. presbyacusis describes the progressive sensorineural hearing loss nearly every human experiences starting in the fifth decade of life. it is more or less symmetric and begins in the higher-frequency range with or without tinnitus. degeneration of hair cells, cochlear neurons, stria vascularis, cochlear nerve and components of the central auditory pathway, e. g. cochlear nuclei. baseline of 15 db or more at both 6 and 8 khz, either unilaterally or bilaterally as assessed 5-7 weeks after beginning of the treatment. monitoring of the status of the cochlea with the acous-• tic emissions and high-frequency audiometry. discontinue or change the medication (if it is pos-• sible). antioxidant therapy (iron chelators, vitamin e, ascor-• bic acid). prophylactic treatment (aspirin). in a patient who has decreased renal function, dose • schedules should be adjusted. if the hearing is still serviceable, amplification with a hearing aid may be used. through a perforation of the tympanic membrane otic drops may enter into the round window niche, diffuse across the round window membrane and reach the membranous labyrinth. ototoxic preparations include alcohol, povidone iodide, gentamicin, neomycin, polymixin b, chloramphenicol and hydrocortisone. non-ototoxic preparations include amphotericin b, sulphacetamide, ciprofloxacin, triamcinolone and dexamethasone. the hearing loss is caused, on one hand, by the physi-• ologic processes of aging based on individual genetic predisposition and, on the other hand, by exogenous degeneration of parts of the inner ear (supporting cells, basilar membrane, outer hair cells) and central auditory pathway components. the exogenous degeneration is essentially the consequence of environmental influences, nutritional habits, toxicity of legal drugs, professional and recreational noise exposure, ototoxic medications, medical and neurological problems, etc. a hereditary component of presbyacusis has been demonstrated. worldwide, 400 million humans are affected. in the • uk 92% of persons older than 60 years show a hearing loss of more than 25 db. in denmark, the percentage of hearing loss per decade is 3 db up to the age of 55 years and 9 db for persons older than 55 years. progressive hearing loss with the greatest threshold shift in the high frequencies ( fig. 1.6.9 ). speech comprehension is reduced, mainly in ambient noise (party effect), loss of discrimination. discomfort in noisy environments, during phone calls. decline of directional hearing. tinnitus is common. psychosocial isolation und suspiciousness of the environment are caused by the loss of ability to communicate, secondary to the hearing loss. depressive crisis can be triggered by the tinnitus. detailed patient history, including professional and • recreational sound exposure as well as family history. complete ent examination. • otomicroscopy: without pathological findings. • hearing test battery: tuning fork, audiogram, speech • audiogram, tympanogram and stapedial reflex audiometry. symmetric sensorineural hearing loss limited to the higher frequencies, pantonal or gently declining from 1 khz. poor discrimination in the speech audiogram. mostly, a positive recruitment is seen. with pancochlear sensorineural hearing loss, recruitment can also be negative. tinnitus analysis with subjective loudness assessment • and testing of masking possibility (fig. 1.6.6 ). basic vestibular testing with frenzel glasses: exclusion • of spontaneous and provoked nystagmus, positional childhood deafness. hearing impairment in childhood • refers to any hearing loss, occurring from birth to late childhood, e. g. around the age of 16 years. it may be unilateral or bilateral. it may be present at birth, e. g. • congenital, or acquired after birth, either during the perinatal period of life or later during lifetime. as hearing impairment affects the acquisition of speech, its occurrence is also classified in relation to the stages of speech development as prelingual (0-2 years of age), perilingual (2-4 years) and postlingual (more than 4 years). it can be classified into four categories of severity: • mild (average hearing levels ranging from 10 to 39 db), moderate (average hearing levels from 40 to 69 db), severe (average levels ranging from 70 to 94 db) and profound (hearing loss that has average hearing levels greater than 95 db). in principle, hearing loss can be • conductive (due to malfunction of the outer ear or the middle ear) or sensorineural (due to malfunction of the inner ear or the auditory nerve), or mixed. its course may be temporarily, fluctuating, progressive • or permanent (permanent childhood hearing impairment, or pchi). normally, unavoidably slowly progressive hearing loss. the diagnosis of "presbyacusis" requires the exclusion of all other possible causes. not every hearing loss in the elderly is a presbyacusis! further causes of congenital hearing loss are: − infections during pregnancy (rubella, cytomegalovirus, toxoplasmosis). − inner ear malformations due to developmental arrest in embryonic stages such as mondini malformation or common cavity malformation. they may be related to syndromes that are associated with other symptoms or occur in isolated forms. 6. acquired hearing impairment: hearing loss may be acquired in the perinatal period owing to hypoxaemia, severe infections, prolonged newborn icterus and others. following the perinatal period, the commonest cause of acquired inner ear hearing loss in early childhood is bacterial meningitis, with permanent hearing loss complicating up to 30% of affected children, mostly mild or unilateral. however, about 2% of children affected by meningitis develop a bilateral permanent and profound hearing loss. vaccination against haemophilus influenzae type b and early vaccination against streptococcus pneumoniae reduce the risk of developing meningitis as well as the risk for acquired deafness due to meningitis. other causes include infections (e. g. measles, mumps, varicella), trauma, middle ear diseases and administration of ototoxic drugs. 7. progressive hearing impairment: progressive inner ear hearing loss during childhood can be associated with genetic mutations causing syndromes such as pendred syndrome (fig. 1.1.16 ) and the genetically associated syndrome of a large vestibular aqueduct ( fig. 1.1.26) , usher syndrome (retinitis pigmentosa and progressive hearing loss) and alport syndrome. other forms of genetically caused hearing loss occur as progressive hearing loss starting during adolescence. frequently, the cause of progressive hearing loss remains unknown. the most important symptoms of pchi are the absence of adequate reactions to environmental sounds and speech as well as the delay or absence of normal speech development, depending on the severity of the hearing impairment. additional symptoms may be poor general communication skills and behavioural difficulties out of frustration in communicational attempts. however, it is important to understand that these symptoms are not easy to recognize, even for professional child carers, and that diagnosis even of severe hearing impairments is often considerably delayed, even up to the age of 2-4 years, if based only on the recognition of these symptoms. in many cases, the parents' concern precedes professional diagnosis and should therefore be taken for serious and prompt further hearing assessments for the child. risk factors that are associated with pchi such as a family history of hearing impairment, infections during pregnancy (e. g. rubella, toxoplasmosis, cytomegalovirus), prematurity, low birth weight, necessity of admission to intensive care unit, prolonged newborn icterus, administration of ototoxic drugs (e. g. aminoglycosides), craniofacial abnormalities and hereditary syndromes should draw attention to possible hearing impairment. deficits in speech development include receptive skills of speech understanding as well as speech production. affected areas range from basic skills such as segmentation and analysis of phonemic structure, short-term auditory memory as well as vocabulary to higher levels of speech such as syntax and grammar. it is important to monitor the speech development of children, since some children may have progressive hearing loss occurring during childhood. the early auditory system is particularly receptive to • sounds and speech. sufficient auditory input is necessary to induce the maturation of the auditory system. failure to stimulate the auditory system during this period (referred to as the critical period) can have lifelong detrimental effects on the acquisition of spoken language. untreated hearing loss can also compromise a − reactions to sound and speech: does the child startle at loud sounds, does it react to voice when the speaker is not visible, e. g. calming or smiling? − language development: does the child vocalize, does it imitate (mama, papa), what is the range of vocabulary, are there articulation problems, are there problems with syntax or grammar? − behavioural abnormalities: e. g. aggression, low tolerance to frustration, communicative strategies. 1. inspection − general physical examination should pay attention to any signs of hereditary syndromes. − look at the facial features of the child and its parents: craniofacial abnormalities, e. g. outer canthi of the eyelid slant downwards in treacher collins syndrome or upwards in the branchio-oculo-facial syndrome. − ear anomalies such as hypoplasia or aplasia of the pinna, preauricular appendages and atresic ear canal draw attention to possible conductive hearing loss or associated inner ear malformations. − the neck should be evaluated for any branchial remnants (found in the branchio-oto-renal syndrome) or an enlarged thyroid gland (pendred syndrome and associated enlarged vestibular aqueduct). − blue sclerae are associated with osteogenesis imperfecta. − a white forelock and pigmental anomalies of the iris may indicate waardenburg syndrome. 2. otoscopy: note anomalies of the pinna and external auditory ear canal, check for the presence of a normal tympanic membrane, possible anomalies of the handle of the malleus, signs of ome (retraction, fluid behind the eardrum) or chronic otitis media. rarely, a whitish mass behind the eardrum may indicate congenital cholesteatoma ( fig. 1.4 .14). note that in sensorineural hearing losses, the tympanic membrane is normal. 3. rhinoscopy: to exclude nasal stenosis, choanal atresia, nasal infections. 4. pharyngoscopy: to exclude hyperplastic or infected adenoids or tonsils. we distinguish between subjective and objective auditory tests. subjective tests require some form of reaction of the subjects tested, whereas objective tests can be carried out without active feedback. even in small children and babies, age-adequate subjective tests are possible (e. g. behavioural response audiometry, conditioned response audiometry); however, they require special expertise. objective tests such as oae and bera can be performed at child's reading ability and educational attainment. this may limit access to further education, may restrict employment opportunity and lead to greater dependence on social services later in life. thus, permanent untreated hearing impairment can have far-reaching consequences for the child, its family and for the wider community. evidence suggests that early identification and treatment may significantly reduce the impact of pchi. fitting of hearing aids or cochlear implantation after the critical period of language development (from 0 to 4-6 years) will not be able to fully recover these effects. after meningitis • , labyrinthitis may develop to fibrosis and/or neo-ossification of the cochlear duct, making cochlear implantation difficult and less successful. early diagnosis via mri or ct after meningitis ( fig. 1.1.25) is recommended to detect possible early signs and proceed to implantation. recommended (european consensus conference 1998) since diagnosis of hearing impairment is often delayed and early intervention is of great importance. all newborns should be screened during their first days of life. screening methods should have high sensitivity and specificity, and should be objective as well as time-and cost-efficient. the following methods are available: − automated measurements of otoacoustic emissions (oae), such as transitory evoked oae (teoae) and distortion products of oae (dpoae) − automated brainstem evoked response audiometry (bera) − automated measurements of amplitude modulation following response (amfr) 2. if the child fails to pass the screening for hearing impairment, follow-up with eventual rescreening and more extensive auditory testing to confirm or exclude hearing loss is necessary. 1. risk factors that are associated with pchi such as a family history of hearing impairment, infections during pregnancy, prematurity, low birth weight, necessity of admission to an intensive care unit, prolonged newborn icterus and administration of ototoxic drugs should be investigated. 2. ask about: any age, sometimes requiring sedation. diagnosis should only be based on the combination of subjective and objective tests, and should be reevaluated at subsequent developmental stages of the child to reach a higher degree of exactitude, to distinguish between transitory, permanent or progressive problems and to account for maturation and developmental processes. subjective tests are: behavioural response audiometry • (age range 0-2 years): spontaneous responses to sounds such as calming, blinking and startling are watched for by experienced examiners. in visual reinforcement audiometry, reactions of the child such as head turning are reinforced by attractive visual stimuli. bilateral freefield testing is possible; thresholds found are generally 20-30 db above real auditory thresholds. performance test and play audiometry • (age range 2-5 years): this test requires that a child can be actively involved in a task. it uses a conditioned response (e. g. stacking cubes) to evaluate auditory thresholds in bilateral free-field conditions. pure tone audiometry • (age range from 3.5-4 years upwards): testing side specific pure tone thresholds (see sect. 1.1.5.2). the child must be able to wear headphones and cooperate in the task. : various speech tests with ageappropriate language material are available. results are influenced by auditory thresholds, capacity of auditory speech analysis as well as general speech development. tests for • central auditory processing disorders: special tests such as the dichotic listening test or hearing in noise are used to diagnose central auditory processing dis orders such as in auditory attention deficit syndrome. screening tests • : they are designed to detect hearing losses greater than 30-40 db, in general without giving detailed thresholds. tympanometry • : to detect middle ear problems (e. g. ome). stapedial reflex measurements are useful to estimate thresholds (see sect. 1.1.5.2). oae • : oae reflect the normal activity of outer hair cells. they are generally present when hearing loss does not exceed 30-40 db. however, they do not reflect the function of inner hair cells and the auditory nerve; therefore, they might be present in cases of auditory neuropathy or neural hearing loss. teoae are click-evoked and have a broad response spectrum over the whole frequency range. dpoae are evoked by continuous two tones and are frequency-specific. by determining growth functions of dpoae, one can obtain an approximation of auditory thresholds. : the synchronized neural activity of the auditory pathway (spiral ganglion and cochlear nerve, cochlear nucleus, lateral lemniscus, superior olive and inferior colliculus) is recorded in response to click stimuli (broad frequency response) or tone bursts (limited frequency specificity). auditory thresholds can be approximated. the method may require sedation in children. : like for auditory brainstem response, the synchronized neural activity is measured but is elicited by sine waves that are amplitude-modulated. higher frequency specificity can be achieved. as one of the main symptom of pchi is delayed speech and language development, it is important to include assessment of speech and language development by psychologists, speech and language therapists or teachers of the deaf in a multidisciplinary approach. many children with pchi have additional neurological and vision deficits. neurological examination and vision screening should be carried out if indicated. high-resolution, thin-section ct is the modality of • choice to visualize the bony structures of the outer ear, the middle ear, the mastoid, the inner ear and the internal acoustic meatus (figs. 1.1.23, 1.1.24). soft tissue masses or fluid in the middle ear or mastoid can also be detected. special attention has to be paid to detect possible malformations of the ossicles, of the inner ear, e. g. mondini malformation ( fig. 1.2. 2), common cavity, labyrinthine malformations, enlarged vestibular aqueduct ( fig. 1.1.26) , or in postmeningitis cases, neo-ossifications of the scala tympani, media or the labyrinth ( fig. 1.1.25 ). mri is indicated to visualize the fluid content of the • inner ear, the auditory and vestibular and facial nerves in the inner acoustic meatus, and to detect central nervous system abnormalities. it is of special importance to detect early signs of fibrosis and neo-ossification of the cochlea after meningitis. genetic testing may be useful in syndromic as well as non-syndromic hearing loss for diagnostic purposes and counselling of patients and parents. specific genes associated with syndromes such as waardenburg, pendred and usher have been identified. in non-syndromic hearing hearing aids are possible surgical options (see sect. cochlear implants replace the function of the inner ear in transferring acoustic sounds into neural excitation patterns. unlike hearing aids, which amplify sounds acoustically, cochlear implants convert the sounds into electrical stimulation patterns, which electrically stimulate fibres of the cochlear nerve and thus elicit hearing sensations ( fig. 1.6.10) . a cochlear implant system consists of two parts: the external speech processor and the implant itself ( fig. 1.6 .11). the speech processor picks up external sounds, analyses them for frequency and time content and generates instructions for stimulation. together with the necessary energy, the information is sent to the implant via a short high-frequency radio connection. the sender is centred over the implant with a magnetic link. the implant receives the instructions and generates electric pulses. these are delivered by the intracochlear electrodes (currently between 12 and 22) that follow the tonotopic organization of the cochlea. electrodes at the base (near the round window) elicit high-pitched auditory sensations; electrodes near the apex elicit low pitches. for profoundly deaf children (typically those with • hearing losses greater than 100 db) and those chil-loss, the gjb2 gene encodes for the connexin 26 molecule. it can be tested in many centres and may account for approximately 50% of cases of presumed non-syndromic genetic deafness. however, the number of gene mutations associated with deafness continues to increase; more then 100 mutations have been described. therefore, negative findings in genetic testing do not preclude the genetic origin of a hearing loss. early intervention • is a key factor to prevent sequelae of hearing impairment. even children as young as 3-6 months can be fitted with hearing aids; however, special expertise is needed to fit very small children. : the first step in therapy of pchi is providing adequate amplification by means of hearing aids. they should be fitted on the basis of subjective and objective measures, and bilaterally, if the hearing loss is bilateral. hearing aids have to be maintained and ear moulds have to be adjusted regularly to fit the ear canals, which typically enlarge with age. in conductive hearing loss, e. g. in ear malformations, that is not ready to be corrected surgically, bone-conduction hearing aids are the treatment of choice ( fig. 1.2.4) . reactions to sound and speech as well as speech and language development of children fitted with hearing aids have to be monitored by the children's parents, paedaudiologists, teachers and therapists to make sure that amplification is adequate and optimal benefits are obtained. training of communicative skills and counselling of parents is of great importance and should start as soon as possible. : if hearing capacities as well as speech and language development remain insufficient in patients with severe or profound hearing impairment, despite optimally fitted hearing aids, a cochlear implantation has to be considered. : reconstruction of the outer ear canal and ossicular chain, implantation of active middle ear implants and placement of bone-anchored fig. 1.6 .10 cochlear implant dren with severe to profound hearing loss, who do not obtain sufficient benefit from powerful hearing aids to develop their speech and language skills, cochlear implants are extremely valuable in providing access to speech signal and sounds. cochlear implantation can be performed as early as • 8-12 months if indicated. early intervention, e. g. implantation before the age of 2 years, is best to make use of critical periods in hearing as well as speech and language development. therefore, early diagnosis and hearing aid trial periods are of importance. bilateral implantation is possible and beneficial in • adults and in children, allowing for better speech understanding under difficult listening conditions and partial development of spatial and directional hearing. children with multiple handicaps in addition to pro-• found hearing loss will obtain significant profit from cochlear implantation in most cases, even if receptive and expressive language development may not be expected owing to, e. g., intellectual handicaps. for malformations of the inner ear or neo-ossifications • after meningitis, special surgical techniques have been developed. cochlear implantation is not possible if the auditory • nerve is absent. in these cases, brainstem implants may be an alternative approach. cochlear implantation is contraindicated if sufficient • rehabilitational and/or technical support for maintenance of the device function cannot be ensured. additional speech and language therapy is necessary • in most patients with severe and profound hearing impairment. general support, careful choice of educational settings • and counselling of parents is of importance. children with a cochlear implant require regular pro-• gramming and control of the speech processor, which is best ensured in multidisciplinary cochlear implant rehabilitation programmes. differential diagnosis between conductive and sen-• sorineural hearing loss is an essential prerequisite. pchi has to be differentiated from central auditory • processing disorders that may be mistaken for peripheral hearing loss. non-organic hearing loss (psychogenic) should not be • missed, occurring most often in teenage children. unilateral hearing loss is often overlooked in child-• hood. progressive loss may pass unrecognized; the same is • true for hearing loss affecting only part of the frequency range as the findings of the initial objective hearing screening can be normal. walter livi a hearing aid is a miniature electronic instrument that detects, amplifies, elaborates and transmits sound to the hearing impaired patient's ear. its basic components are the microphone (input), the amplifier (elaborator) and the receiver (output). hearing aids can be classified into three groups depending on the technology used: 1. analogue: the microphone converts sound waves to a continuous electrical signal that is similar to the stimulus in intensity, frequency and time. the amplifier then amplifies the electrical signal, which can be modified by manual controls (trimmers), and then transmitted to the receiver that reconverts the elaborated electrical signal to a sound wave. 2. digitally programmable analogue: they represent an evolution of analogue hearing aids that differ only in the phase of amplification. the amplified electrical signal is not modified by a manual trimmer but is amplified electronically by the computer. the elaboration of the signal remains an analogue process. the microphone converts sound waves into an analogue electrical signal. the analogue-todigital converter transforms the continuous electrical signal into a series of binary numbers (0-1). the digital sound processor digitally elaborates the numerical signal according to algorithms contained in the program. the analogue-to-digital converter transforms 1.6.12.9 prognosis evidence confirms that early identification and treatment, coupled with sustained, appropriate habilitation and educational support can achieve excellent outcomes. development of spoken language can proceed at rates similar to that for normal-hearing children even in profoundly deaf children, provided that early identification and cochlear implantation are achieved. they can achieve impressive competence with oral communication, and can often attend mainstream schools (with varying degrees of assistance), achieving their full educational potential. negative prognostic factors include late age at diagnosis, the presence of other cognitive disabilities, inappropriate communication strategies, inadequate educational and rehabilitational support and poor socioeconomic status. fig. 1.1.21) . the series of numbers into an electrical signal. the receiver then reconverts the electrical signal into sound waves. types of hearing aids are shown in fig. 1.6 .12. depending on the place in which they are worn, hearing aids can be classified in the following way: behind the ear (bte), in the ear (ite), body aid, and eyeglass aid (spectacles). the choice is influenced by the type and entity of the hearing impairment and by the needs of each patient: 1. bte hearing aids − can be used for all types of hearing impairments. − are composed of a plastic shell that contains the microphone, amplifier, receiver, volume control (or other manual controls or switches) and battery. − they are quite small and are placed bte (pinna). − they are connected to the flexible tube of the ear mould by a plastic hook or by a fine wire to a receiver positioned directly in the ear canal (receiver in the ear, rite) ( fig. 1.6 .13). the ear mould is composed of biocompatible material that is made to measure for the ear canal of the patient. 2. ite hearing aids − they can be placed completely inside the canal (cic) or mainly in the external ear canal (ite) or in the concha of the external ear canal (itc) (fig. 1.6 .12). − they are made of biocompatible material and are well accepted by patients because they are small and practical. − they are not a good choice in cases of severe to profound hearing loss because they do not provide sufficient amplification. − they are not often prescribed to children, because of the limited size of the child's ear canal and its continuous change in size. 3. body worn pocket aids: the plastic case of the aid contains all components except for the receiver, which is placed in the ear canal. they are no longer in use because they are not very practical. 4. eyeglasses/spectacles: all components of the hearing aid are in the arm of the glasses. they can transmit sounds by: − air conduction: air-conduction spectacles are practically obsolete. − bone conduction: bone-conduction spectacles contain the vibrator at the end of the arm and transmit the vibrations to the mastoid. they may be prescribed in cases of mild to moderate conductive loss and of mixed loss up to 35 db. note that a much better type of bone-conduction aid is the bone-anchored-hearing aid. 5. implantable hearing devices: a conventional hearing aid takes sound and makes it louder. the amplified sound is conducted to the ear canal either via an ear mould or directly via the hearing aid. − a device that is semi-implantable (retrox) has been classified by the fda as a transcutaneous airconduction hearing aid system (tachas). this is a conventional hearing aid, where the sound-transmitting silicon tube is placed from behind through the skin and cartilage of the auricle to direct the sound into the outer ear canal. − in the vibrant soundbridge implantable hearing system a tiny magnet (floating mass transducer, fmt) is directly attached to the ossicular chain (during surgery) and amplifies the natural vibrations of the ossicles. many patients report that "direct" coupling leads to improved hearing quality and improved speech understanding. the system consists of external and internal parts. the external part, called the audio processor, is worn underneath the hair and held in place with a magnet. it contains a microphone, a battery and electronics. the audio processor converts environmental sounds into signals that are transmitted to the implanted internal coil of the soundbridge. the implanted part consists of the internal coil, magnet, conductor link and the fmt. the signal from the audio processor is transmitted across the skin to the internal coil, which relays the signal down the conductor link to the fmt. the fmt is attached either to the incus or to the round window membrane (fig. 1.2.5 ). the fmt converts the signal into vibrations that directly drive and move the ossicles or via the round window the perifig. 1.6.13 the application of modern design to advanced electronic technology. a hearing aid that implements the receiver in the ear (rite) solution fit binaurally to guarantee the best result for speech discrimination (interpersonal communication). the patient must be motivated to correctly use both hearing aids. three phases should be respected for an optimal result in fitting a hearing aid: 1. prescription: in this phase the medical specialist is involved and he/she must carry out the testing necessary and an otomicroscopic objective examination. the testing includes subjective and objective tests; that is pure tone audiometry using earphones and free field. impedance testing with particular attention to the stapedial reflex and in some cases, especially with children, the study of evoked potentials (auditory evoked brainstem responses). : this is carried out by the audiologist/dispenser on the basis of the results from the testing and diagnosis. in this phase the hearing aid is chosen along with any assistive listening devices (if necessary) to satisfy the individual needs of the patient. to obtain the maximum benefit from hearing aids it is necessary that the patient and audiologist/ dispenser work together closely. after approximately 2 months, the plastic processes are completed. in this phase the medical and paramedical competences (audiologist/dispenser, ent specialist, speech therapist, psychologist) converge to obtain the best result. although hearing aids from a technological point of view rely on extremely sophisticated technologies, they are "obsolete" from the cosmetic point of view and this is usually the reason why many patients refuse hearing aids. for this reason hearing aids today have been restyled and special attention is given to their "design". if a hearing aid is to be accepted it should be perceived as a modern assistive device for communication, an extension of the patient's body, eliminating the sense of shame that the patient feels by wearing a hearing aid. there is a kind of tabu that is linked to dentures, hearing aids, cosmetics for men and in the past to eyeglasses. but today these negative connotations are decreasing and that changes the perception of the abovementioned items. they are no longer disturbing; they may become a part of fashion trends! according to the audiological classification of hearing loss there is a distinction on the basis of the average tone threshold into mild (threshold between 20 and 40 db), moderate (40-70 db), severe (70-90 db) and profound (90-120 db) hearing loss. the choice of the hearing aid with respect to the above classification takes into account the audiometric curve (flat, symmetrical, asymmetrical, lymph and amplify their natural movement. these vibrations then conduct the sound to the basilar membrane and the organ of corti. this system is designed for mild to severe sensorineural hearing loss (fmt attached to the incus) or for moderate or severe mixed hearing loss (fmt attached to the round window), e. g. cochlear otosclerosis, malformations of the ear. another middle ear implant is the middle ear transducer, an implantable hearing device where the sound is transmitted by a similar vibrating driving system attached to the head of the malleus. − a bone-anchored-hearing aid is a hearing aid fixed to a bone-anchored titanium screw, in which bone conduction is used to transmit sound directly via the skull into the cochlea (fig. 1.2.4) . − in deaf patients cochlear implants are used (see sect. 1.6.12). sound is transformed by the so-called speech processor to electric signals which are sent to a retroauricular subcutaneously implanted receiver. the receiver is connected with a stimulating electrode which is inserted into the cochlea. it is well known that hearing impairment can negatively influence interpersonal relations and create difficulties in everyday tasks. the fitting of a hearing aid is necessary for patients who cannot benefit from pharmacological therapy and/or surgical procedures, and in some cases may be of support to the latter. patients with a bilateral hearing loss with a loss in the better ear of at least 30 db for at least one of the frequencies examined (from 0.5-3.0 khz) and when the speech discrimination for monosyllabic words in the better ear is 80%. in cases of monolateral hearing loss, the loss should be 30 db or more at 2.0 khz or at two frequencies between 0.5 and 3.0 khz. make certain that the patient can properly use the hearing aids after a period of training with the hearing healthcare professional. the patient must also be motivated to use the hearing aid all the time. when deciding on hearing aids, the professional and the patient must evaluate subjective, social, cognitive and lifestyle aspects. stereophonic hearing is necessary for good speech discrimination, especially in noisy surroundings. if both ears can benefit from amplification, the rule today is to tests. a vestibular schwannoma, which in very rare cases is mimicked by the same symptoms, can be excluded by mri. treatment is exclusively conservative. in the early stage of the disease sedating drugs such as h1 antagonists (50 mg dimenhydrinate 2-3 times a day) are recommended, but only for a short time, not longer than 2 days. as soon as possible vestibular habituation training should be started to induce a rapid vestibular compensation. corticosteroid treatment is recommended during the first 14 days (start with 500 mg intravenously with decreasing doses to 0 mg within 10 days). to accelerate vestibular compensation active movements (sports), stimulating agents such as caffeine and avoidance of calming procedures such as bed rest seem useful therapeutic options. differential diagnosis of vestibular neuritis is simple, because the duration of vertigo for some days is very characteristic; therefore, benign paroxysmal positioning vertigo, vestibular paroxysmia, ménière's disease or vestibular migraine can be excluded by a careful questionnaire. the fact that vestibular neuritis is monosymptomatic facilitates the differential diagnosis in patients with additional hearing problems. although vestibular schwannoma only very seldom becomes apparent by vertigo complaints, mri allows a clear differential diagnosis. the prognosis is generally favourable. if there are additional factors which can inhibit vestibular compensation, such as sedating drugs, old age or additional abnormalities in the cns, the complaints can continue for a long time. acute unilateral, partial or complete loss of peripheral vestibular function, caused probably by a viral inflammation. current findings point to a viral origin (herpes simplex virus) similar to facial palsy. owing to immunologic deficiencies, herpes simplex viruses, which were already present in the patient as a result of an earlier infection, are reactivated and destroy vestibular sensory fibres. vestibular neuritis is one of the most frequent peripheral vestibular disorders (about 25% of vertigo patients seen in an ent vertigo unit suffer from this disease). the symptoms are marked by the acute appearance of severe vertigo, mostly purely rotatory, sometimes accompanied by vomiting, nausea and ataxia. in the acute state a violent horizontal-rotatory nystagmus, beating towards the intact side, is always present. hearing impairment or tinnitus do not belong to vestibular neuritis. in the acute state a tendency to fall is obvious, so a prevention against falls is necessary, to avoid orthopaedic sequelae. not seldom benign paroxysmal positioning vertigo follows a vestibular neuritis within a short delay, this is named "lindsay-hemenway syndrome". a careful questionnaire reveals the sudden onset of the vertiginous complaints, which decrease within a period of some days. the only objective sign is a strong horizontal-rotatory nystagmus, beating to the intact side. the caloric test proves the hypofunction of the lesioned side, which is in the beginning not compensated in the rotatory test. vestibular spinal tests show a marked deviation and a tendency to falls directed to the lesioned side. a lesion in the auditory system, which does not belong to the vestibular neuritis, can be excluded by audiological karl-friedrich hamann benign paroxysmal positional vertigo. benign paroxysmal positioning vertigo (bppv) is a mechanically induced vertigo caused by a canalolithiasis or a cupulolithiasis. normally otoliths are fixed in the otolithic membrane. by head traumatism, in old age or idiopathically, otoliths can be loosened and travel in one or some of the semicircular canals. bppv is one of the most frequent kinds of vertigo, mainly in the elderly. the different semicircular canals are not affected equally. in 96% of cases the posterior vertical canal is concerned, the horizontal canal in 3% of cases and the anterior vertical semicircular canal only in 1% of cases. the vertigo attacks have a short duration, only a few seconds, typically triggered by certain head movements, for example by head turning in the morning for a look at the alarm clock. bppv never occurs when the head is not moved. complications in the real sense of the word do not exist. as for all forms of vertigo, falls can occur. the questionnaire reveals that vertigo appears only during head movements and lasts only for some seconds. apart from the characteristic complaints, the diagnosis is made by nystagmus observation under frenzel's glasses. by specific positioning of the head in the plane of one of the semicircular canals (hallpike manoeuvre), one can prove a bppv if a typical nystagmus appears. the involved semicircular canal can be identified by analysis of the nystagmus, because the pattern of eye movements for each semicircular canal is known. other neurotological tests such as caloric or rotatory tests do not show pathological findings; the auditory system is not involved as well. additional diagnostic procedures are not necessary. imaging techniques are only suitable for exclusion of possible central abnormalities. owing to the mechanical pathophysiologic nature of bppv only a mechanical treatment is reasonable. the goal of a rational treatment of bppv is to liberate the semicircular canals from the dislocated otoliths. this can be carried out by liberatory manoeuvres of semont [2] (fig. 1.6 .14) or epley [1] (fig. 1.6.15 ). both have principally the same intention, namely to bring the dislocated otoliths by specific movements of the head to a "neutral point" in the vestibular apparatus nearby the utriculus. for the treatment of a canalolithiasis or a cupulolithiasis of the horizontal canal, a barbecue rotation or brandt-daroff exercises can be recommended as for a prophylaxis of bppv. only in extremely rare cases (less than 1%) surgical treatment can be indicated. two procedures exist: (1) neurectomy of the posterior canal nerve, (2) plugging of the affected canal. because of the typical clinical signs (vertigo only in combination with head movements, duration of vertigo never more than 60 s, provocation of typical nystagmus by specific positioning), normally the differential diagnosis does not present a problem. one of the rare differentialmotion sickness is a special kind of physiological vertigo, induced by an unusual stimulation of the multisensory system, which normally guarantees adequate orientation in space. diagnostic possibilities is the vestibular paroxysmia, which is characterized by vertigo attacks of a few seconds. but these attacks are not correlated typically with certain head movements which trigger the bppv. another differential diagnosis, but less frequent, is the possibility of a vestibular migraine, which, in contrast to bppv, should be accompanied by headaches. the prognosis of bppv is very favourable. after one liberatory manoeuvre about 70% of patients no longer have complaints. in the remaining 30%, repetitive liberatory manoeuvres lead to a complete cure. if necessary, brandt-daroff exercises must be continued. the rate of recurrences is relatively high. in a period of 2 years after a liberatory manoeuvre, about 20% of patients complain about vertigo again; in a period of 8 years, the rate of recurrences reaches 55%. only in extremely rare cases does a surgical treatment become necessary (see sect. 1.6.15.8). fig. 1.6 .14 a first movement of a liberatory manoeuvre (semont) for treatment of a canalolithiasis of the left posterior canal: starting from a sitting position the patient is positioned to the left side, the head turned 45° to the unaffected right side. b second movement of the liberatory manoeuvre (semont): the head and trunk of the patient were thrown from the left side to the right side without changing the position of the head relative to the body it is generally accepted that motion sickness develops when a sensory conflict between the different sensory systems, responsible for the orientation in space, occurs. both intrasensory mismatch (within the vestibular system) and intersensory mismatch (between the vestibular and visual system for example) can trigger the symptoms of motion sickness. the crucial condition is that different sensory receptors give different signals about the passive motion of an individual in space. so the perceived pieces of information do not correspond amongst themselves nor to the expected perception pattern, previously adapted by experience. principally in all people with an intact sensory system motion, sickness can be induced, if certain conditions which can create a sensory conflict are fulfilled. fig. 1.6.15 a-e epleys manoeuvre. a first step: the patient is sitting in the upright position, the head is 45° turned to the concerned side. b second step: the patient is in the lying position, the head still turned to the concerned side. c third step: the head is turned 180° to the contralateral side. d fourth step: the head and trunk of the patient are turned 90° to the contralateral side. e fifth step: the patient is returned in the upright position. intervals between each step of 3-5 min increases. the medical treatment consists in the uptake of an h1 histamine antagonist such as dimenhydrinate or meclozine. it must be pointed out that all h1 antagonists have sedative side effects. interestingly, ginger root, given in a pulverized form, has significantly favourable effects on motion sickness symptoms. a differential diagnosis of motion sickness does not exist. the only exception is that a real vestibular disease can be triggered also by a specific movement or a sensory conflict. the prognosis of motion sickness is very favourable. at the latest in the moment when the inducing motion ceases, a rapid decrease of the uncomfortable symptoms begins. after some hours, the symptoms of motion sickness disappear. motion sickness is clinically characterized by nausea, pallor, yawing, vomiting and mainly by a feeling of severe discomfort. these symptoms last not only for the time of conflict stimulation but also for a certain period afterwards, when motion stimulation had stopped. complications in the real sense of the word do not exist. falls and aspiration caused by vomiting are sequelae of the symptoms themselves. motion sickness is diagnosed very simply, because the coincidence of the inducing motion and the typical symptoms is pathognomonic. further diagnostic procedures are not necessary. a prophylactic therapy can be useful if it is predictable that a motion sickness inducing sensory conflict will occur. the best prevention consists in a vestibular habituation training with the intention to prepare the orientation system for a conflict stimulation. as a medical prophylactic treatment, scopolamine used in the form of a transdermal skin patch can be recommended. when the symptoms of motion sickness appear, one can try to break free from the sensory conflict situation. in the case of seasickness the suffering person has to leave the cabin and should go on the ship's deck. then he/she should fixate on an object not too far away. in this way there the correspondence between visual and vestibular information deafness and vertigo from head injury evidence for a viral neuropathy in recurrent vertigo intratympanic application of an antiviral agent for the treatment of ménière's disease suggested reading checkliste: hals-nasen-ohren-heilkunde. thieme herpes simplex virus antibodies in the perilymph of patients with ménière's disease peripheral vestibular disorders guidelines for the diagnosis and evaluation of therapy in menière's disease menière's disease: a review herpes simplex virus and ménière's disease schuknecht: presbyacusis. laryngoscope a new semiimplantable hearing system device-retrox l'adattamento degli apparecchi acustici. oticon, 3rd edn. arti grafiche reggiani totally implantable hearing aids: the effects of skin thickness on microphone function hearing aids. boomerang current status in the development of implantable middle ear hearing aids vestibular neuropathy, "vestibular neuronitis" (wrong term, because one neuron cannot be inflamed). 1.6.15 benign paroxysmal positioning vertigo vertigo-its multisensory syndromes training gegen schwindel vestibular exercises improve central vestibulo-spinal compensation after vestibular neuritis suggested reading benign positional vertigo. clinical and oculographic features in 240 cases vertigo-its multisensory syndromes a positional manoeuvre for treatment of horizontal canal benign positional vertigo eye movements from single utricular nerve stimulation in the cat the canalith repositioning procedure: for treatment of benign paroxysmal positioning vertigo curing the bppv with a liberatory manoeuvre drug effectiveness on experimental optokinetic and vestibular motion sickness vertigo-its multisensory syndromes postural behaviour in motion sickness the prevention and treatment of motion sickness motion sickness, ginger, and psychophysics after tympanoplasty open or closed, radical cavity − implantable bone-conduction aids (bone-anchored hearing aids) in adults in some cases and in children (over the age of 6 years). − bone-conduction vibrators mounted on a headband for a child. − bone-conduction spectacles for adults. 5. prelingual profound hearing loss in adults who have never used hearing aids. all hearing aids, including cochlear implants give unsatisfactory results. 6. prelingual profound hearing loss in adults who have always used analogue hearing aids. in many cases digital hearing aids with custom-made ear moulds can be recommended and when possible cochlear implants.the most frequent complaints that arise from patients using traditional hearing aids are the hearing aid whistles (feedback), loud sounds are uncomfortable, unsatisfactory speech discrimination in noisy surroundings and the perception of the person's own voice altered owing to the occlusion effect of the external auditory canal. to solve these problems today there are digital ite and bte hearing aids with artificial intelligence that use technologies capable of improving speech discrimination in noise, eliminating feedback and with the "open fitting" system the problems connected to the occlusion effect are resolved. fitting hearing aids in children is difficult both for the diagnosis and in the actual fitting. it is essential that children are fitted with hearing aids at a very early stage (within 6-12 months). to obtain the best results, the family must be actively involved in the process, the child must be placed in an adequate scholastic and social environment and followed closely by a speech therapist. to evaluate the results of the fitting, objective audiometric testing (impedance testing, auditory evoked brainstem response, electrocochleography, otoacoustic emissions) is essential. the testing and the evaluation of the hearing aid fitting must be carried out in a medical environment with the cooperation of the audiologist/dispenser. when the child is in kindergarten it is important to use a personal fm system to eliminate any interference from background noise present in classrooms. most hearing-impaired children suffer from moderate hearing loss and in those cases a hearing aid is an adequate solution. in cases of profound hearing impairment, after approximately 6 months of hearing aid use and an accurate evaluation of the entity of the hearing loss and after a careful psychological evaluation and speech evaluation, the possibility of a cochlear implant may be considered and must be carried out before the child is 18 months old. key: cord-270805-o6rbfmie authors: hussein, osama title: second wave of of covid-19 is determined by immune mechanism date: 2020-09-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110238 sha: doc_id: 270805 cord_uid: o6rbfmie a second wave of new severe acute respiratory syndrome coronavirus 2 (covid-19) cases is widely feared. in fact resurgence of cases has been clearly observed in several countries that had seen flattening of the epidemic curve. in general, relaxation of community control measures is almost always blamed for the resurgence of cases. in this letter, the author describes an immunological explanation for the double-peaked epidemic curve of new viral diseases including covid-19. according to this hypothesis, a second wave of cases is due to the effective innate immunity in some of the population. these individuals may later develop clinical disease upon repeated exposure. this theory claims that a double-peaked pattern of new cases in a new viral epidemic is intrinsically determined by the pattern of pathogen interaction with the host. according to this hypothesis, relaxation of the community control measures is not responsible; at least in part, for resurgence of cases. countries. in japan, a crisp two-peak incidence curve is clear(2). similar trends are reported from other countries. the notion of epidemics that normally have two peaks of new cases over time is widely accepted(3); although epidemiologists have not characterized this pattern as an established model. in general, the resumption of international travel or the relaxation of the community control measures are are almost always blamed for the resurgence of the disease incidence. in this letter, this author suggests that a double-peaked epidemic curve is an intrinsic feature of viral outbreaks. according to this hypothesis, immune response to virus exposure in different individuals determines the spread pattern in the community. the two principal divisions of the immune response to pathogens are the innate and the adaptive immunity. both divisions play a major role in the body response to viral exposure. together with the development of clonal virus-specific cd8 + t lymphocytes, these individuals will experience subclinical or clinical viral illness and are responsible for the early rise in the epidemic curve. assuming the above hypothesis is true; a viral epidemic curve will show an early upstroke corresponding to persons who pass into viremia upon initial exposure to the virus followed by a second slower rise due to cases who develop the disease after repeated process of inoculation and clearance at the point of entry. it is fairly reasonable to assume that the latter group of patients may be the healthier individuals who will have lower case-fatality rate. together with the notion of the covid-19 cases getting milder with time, objective evidence from japan indicates that the second peak in incidence is not accompanied with a corresponding peak in fatality. the implications of this hypothesis are medically and socially relevant. according to this theory, resurgence of a viral outbreak is intrinsically determined by the nature of the virus interaction with the host. relaxation of the community measures or "reopening" of the economy may not be blamed for a second rise in incidence rates. the theory also provides one explanation for the possible observation of decreased case-fatality over time. in conclusion, the author describes an immunological explanation for a double-peaked epidemic curve of covid-19 and other new viral diseases. e64-e7. 2. who coronavirus disease (covid-19) dashboard. geneva: world health organization beware of the second wave of covid-19 pathogen recognition and innate immunity activation and evasion of type i interferon responses by sars-cov-2 human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the author has no potential conflict of interest to declare. key: cord-260299-0blol7to authors: karadag, engin title: increase in covid‐19 cases and case‐fatality and case‐recovery rates in europe: a cross‐temporal meta‐analysis date: 2020-06-02 journal: j med virol doi: 10.1002/jmv.26035 sha: doc_id: 260299 cord_uid: 0blol7to the new coronavirus (covid‐19) infection reported in china in december 2019 has become a pandemic in a few weeks, affecting the entire world. in this respect, it is crucial to determine the case‐increase, case‐fatality, and case‐recovery rates to control covid‐19. in this study, the case‐increase, case‐fatality, and case‐recovery rates of covid‐19 in 36 european countries were analyzed with the meta‐analysis method using data released by the health organizations and who. the data were obtained from the website of health organizations of 36 european countries and the website of who until 11 may 2020. the analyses were carried out on 1 744 704 covid‐19‐diagnosed cases in 36 european countries. the case‐increase, case‐fatality and case‐recovery rates of covid‐19 were calculated using 95% confidence intervals (95% ci), single‐arm meta‐analysis, cross‐temporal meta‐analysis, and meta‐regression random‐effects model. the standardized case‐increase rate of covid‐19 is 5% (95% ci [0.040, 0.063]) and the average case‐increase rate in european countries has started to decline by around 3% (95% ci [0.047, 0.083]) weekly. the countries with the highest rate of case increase are belgium, sweden, russia, the netherlands and the united kingdom. although the case‐fatality rate of covid‐19 patients was 4.5% as of may 11 (95% ci [0.037‐0.055]), this rate is 6.3% (95% ci [0.047, 0.083]) in standardized time (6th week). the case‐recovery rates of patients are 46% (95% ci [0.376‐0.547]). this study presents important results regarding the covid‐19 pandemic in europe. although the rate of increase in new covid‐19 cases has dropped, there is not much decline in the case‐fatality rates and no increase in case‐recovery rates. the case‐fatality rate of covid‐19 in europe was estimated to be in the range of 4% to 4.5% and a minimum of 4 weeks (as of 11 may) is expected to have the figure below 1% in a country with an average case‐increase rate. monitoring case fatalities in belgium, the netherlands and sweden, and treatment successes in germany and austria play a role of utmost importance. italy have taken the second and third places in the world with the most number of covid-19 cases. these countries have also a very high case-fatality rate. 3 it has been assumed that the virus has been circulating within the european population since january based on the number of cases and the advanced stage of the disease. 4 estimating the rates of case increase, case fatality, and case recovery are key parameters for understanding the basic epidemiological characteristics of the pandemic. 5 although several studies have been conducted to determine the case-increase, case-fatality, and case-recovery rates at various stages of the outbreak, studies involving reliable rates of case increase, case fatality, and case recovery on the basis of metaanalysis in the european specific for covid-19 are limited. 6, 7 the available evidence on the contagiousness, case fatality, and case recovery of covid-19 is focused on china. 8, 9 in initial studies, the case-fatality rates were reported as 15%. 2 however, this figure was calculated with a small group of inpatients. afterward, as more data found, the case-fatality rate dropped to 11.0% and to 4.3% and then to 3.4%. serious attention has not been paid to these basic epidemiological characteristics in europe, 1 and considering the rapid increase in covid-19 in europe, it is of paramount importance to understand the rate of the case increase, case fatality and case recovery in europe to guide the applications of prior prevention and control measures. therefore, the epidemiological characteristics, case-increase, casefatality, and case-recovery rate of covid-19 in europe using the data of a 3-month period from 24 january until today were found using the method of meta-analysis. data were analyzed using microsoft excel and comprehensive meta-analysis 3.3 ® . the health organizations of european countries and who conducted a comprehensive systematic literature review in their online databases on 11 may 2020 to provide the data required for meta-analysis. the search terms are covid-19; 2019-ncov; the number of daily cases, deaths, and discharges. in addition, laboratory-approved covid-19 cases were included in the analyses to guarantee the comprehensiveness and accuracy of the research. all search results were evaluated ( figure 1) according to the preferred reporting items for systematic reviews and meta-analyses (prisma) and the final analysis was conducted on 1 744 704 covid-19 diagnosed cases in 36 countries. to minimize the potential for biases such as sampling or measurement bias in undertaking a meta-analysis study, rigorous methods should be used to locate, select, and aggregate the results of individual studies. incorporating an assessment of the risk of bias of these studies is essential in interpreting their results and may help to avoid underestimating or overestimating the parameter of interest. 10 in this study, ahrq's guidance on constructs to include or exclude from risk-of-bias assessment 11 was taken into account in evaluating the risk of bias. as the country dataset was used instead of research data, the guideline was adapted according to the study. therefore, the risk of bias of each country's data included in this study were evaluated as follows: ▪ in terms of the risk of precision, the laboratory-confirmed covid-19 cases, deaths, and recovered cases in each country were taken into account. the cases which were not laboratory-confirmed were excluded. ▪ the number of cases, deaths, and recoveries announced by the health organizations of the countries were confirmed by data from who and other health institutions. unconfirmed data were excluded from the analysis because of the risk of biased, bad, or inadequate reporting. ▪ a standard calendar has been created for covid-19 cases, deaths, and recovered cases in countries for applicability risk (see section 2.3 for details). ▪ publication bias was assessed using the funnel chart and the trim and fill test. no evidence of bias was observed in the data included in the meta-analysis according to funnel graphs. in addition, there was no difference between the size of the effects observed in the trim and fill test 12 conducted according to 95% ci in the random-effects model and the size of the virtual effects generated according to the randomeffects model to correct the effects caused by bias. there are different methods to calculate the case-increase rate. however, as the covid-19 outbreak only has a 5-month period, the rate of new cases diagnosed daily in this study was calculated as the ratio ofthe new cases to the total number of cases. the first covid-19 cases were not detected on the same date in the european countries and the rate of new cases/total cases was very high in the first days of the pandemic, therefore, the analyses were standardized as day 0 on the 100th day in all countries. in addition, case rate analyses were standardized by population, as the populations of case countries were not similar. the weighted average of the case-increase rate calculated for each day of the week was used for the case-increase rate of each week. the case-fatality rate was determined as the ratio of those who died from covid-19 disease to the patients with covid-19. in calculating weekly case-fatality rates, the average of fatality rates calculated for each day of that week was used for the fatality 2 | karadag rate of each week, starting from the day following the first death. the use of epidemiological velocity terms interchangeably in the literature has become a common nomenclature error. therefore, the fatality rate should not be confused with the mortality rate. a mortality rate-often confused with a case-fatality rate-is a measure of the number of deaths (in general, or due to a specific cause) in a population scaled to the size of that population per unit of time. 13 a case-fatality rate, in contrast, is the number of dead among the number of diagnosed cases. 14 the case-recovery rate was determined as the ratio of the number of patients recovering with the diagnosis of covid-19 disease. in the calculation of the case-recovery rates, the day of the 100th incident was accepted as the 0th day and calculated as the ratio of the total number of case recovery to the relevant day for the case-recovery rate on the 1st, 8th, 15th, 22nd, 29th and 36th days. meta-analyses were carried using microsoft excel and comprehensive meta-analysis 3.3® software. heterogeneity between the studies was tested using the cochran χ 2 test and i 2 , when i 2 was below 50%, a stabilization model was used. when i 2 was over 50%, a random effect model was selected. the combined case-increase rate and 95% confidence intervals (95% ci) single-arm meta-analysis and the cross-temporal meta-analysis random-effects model (weighting, case sizes of each country were taken into account) weighted effect size were calculated. to investigate the heterogeneity, the percentage of population the over 70-year-olds, and the population growth rate to the rate of increase in cases, the rate of case fatality and case recovery were tested using the meta-regression random-effects model. p < .05 was considered statistically significant. the study examined the increase of covid-19 cases in european countries using cross-temporal meta-analysis. 1 therefore, analyses were standardized to the 100th day, on which the case was spotted, as the zeroth day. the reason for starting the analyses from the 100th case is that the increase rate below 100 cases is very high due to the small numbers. in addition, case increase analyses have been standardized by the population due to the fact that the populations of case countries are not similar. in addition, the meta-regression analysis shows no statistically significant effect of the population rate above 70-year-olds on average case-increase rates (p > .05). considering the day on which the 100th case was seen as the zeroth day, the weekly case-growth rate, increase difference, standard deviations, and effect size (es) were used to define d and r 2 of the difference. ( ) firstweek secondweek (1) considering the day, on which the first case-fatality was seen as the zeroth day, the weekly case-fatality rate, case-increase difference, standard deviations, and effect size (es) were used to define d and r 2 of the difference (equations 1 and 2) . the case-fatality rate in europe was found to be 1% higher in the second week (d = .307, r 2 = .023) compared to the first week. the case-fatality rate was 0.5% higher in the third week compared to the previous week (d = .327, r 2 = .026) and the case-fatality rate was 0.8% higher in the fourth week compared to the previous week (d = −.847, r 2 = .152). the daily case-fatality rate was 0.5% higher in the fifth week (d = −0.422, r 2 = .043) compared to that of the fourth week (table 4 ). in the meta-regression analysis, no statistically significant effect was found in countries with the population rate of the above 70-year-olds and the population density on the case-recovery rates (p > .05). considering the day, on which the 100th case was seen as the zeroth day, the weekly case-growth rate increase difference, standard table 6 ). this meta-analysis includes laboratory-approved case data obtained between january 2019 and 11 may 2020 to analyze the epidemiological characteristics of covid-19 in europe. this study, covering 36 european countries and 1 744 704 cases, reflects the latest data since the emergence of covid-19. the results showed relatively low heterogeneity in terms of single-arm meta-analysis. the sensitivity analysis also showed that the results were not affected by countries' data and that there was no bias. furthermore, meta-analyses of randomized controlled data were not found to be superior to nonrandomized data in terms of evidence level. 15 the cases included in the analysis were between 24 january and 11 may. the meta-analysis based on the random effect model showed that covid-19 had a case-increase rate of 5%. it was also noted that the discharge rate of patients was 17%, and the casefatality rate was 4%. the results of the cross-temporal meta-analysis in europe showed a decline in the case-increase rate of cases. a country with a european average case-increase rate was estimated to have a minimum of 4 weeks (as of 11 may) to experience a decline below 1%. there is not much variation in weekly case-fatality rates at covid-19 according to the results of a cross-temporal meta-analysis. the analysis estimated that the case-fatality rate of covid-19 in europe would range between 4% and 4.5%. the case-fatality rate of sars-cov, which was a similar outbreak, was 10%, while the casefatality rate of mers-cov was over 35%. 16, 17 in contrast to the two viruses, covid-19 has a lower case-fatality rate. according to the clinical trials, it has been observed that the elderly (>60 years) were reported to be in the majority of deceased patients 18 in conclusion, while there is a negative trend in the increased rate of cases in european countries, differences in population characteristics, treatment methods and health infrastructure affect case-fatality and case-recovery rates. even though the first 3 months of the pandemic has ended, the case-recovery rate of patients is relatively low. although the clinical effect of some drugs has been discussed in recent days, it has been reported that plasma therapy had a definite healing effect for survivors, especially in severe, cases. 21 this indicates that countries having a case increase in the number of patients recovering with plasma treatment will recover faster from the pandemic. in addition, while italy, spain, and the united kingdom are more involved in the number of deaths in research, reports and other studies, belgium, the netherlands, and sweden, where case-increase and case-fatality rates are high and the rate of case-recovering patients is relatively low, should be followed carefully. some limitations of the study should be taken into consideration. first of all, our results are limited to the early term of the pandemic on a country basis. in addition, a country is an extremely rough unit of analysis. also, the number of confirmed cases may be biased for various factors, whereas these factors were probably stable in our case over a short period of time when they were analyzed. engin karadag http://orcid.org/0000-0002-9723-3833 monitoring transmissibility and mortality of covid-19 in europe clinical features of patients infected with 2019 novel coronavirus in wuhan coronavirus disease 2019 (covid-19) in italy case-fatality rate and characteristics of patients dying in relation to covid-19 in italy different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study imperial college london mrc centre for global infectious disease analysis. news/wuhan coronavirus the reproductive number of covid-19 is higher compared to sars coronavirus covid-19 patients' clinical characteristics, discharge rate,and fatality rate of meta-analysis assessing risk of bias in prevalence studies: modification of an existing tool and evidence of interrater agreement assessing the risk of bias of individual studies in systematic reviews of health care interventions. agency for healthcare research and quality methods guide for comparative effectiveness reviews trim and fill: a simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis a dictionary of epidemiology epidemiology in medicine meta-analysis of well-designed nonrandomized comparative studies of surgical procedures is as good as randomized controlled trials from sars to mers, thrusting coronaviruses into the spotlight sars and other coronaviruses as causes of pneumonia clinical features of three avian influenza h7n9 virus-infected patients in shanghai clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province covid-19 in colombia endpoints. are we different, like national health commission of the people's republic of china's main website how to cite this article: karadag e. increase in covid-19 cases and case-fatality and case-recovery rates in europe: a cross-temporal meta-analysis key: cord-263508-row2mn17 authors: chan, jasper fuk-woo; lau, susanna kar-pui; woo, patrick chiu-yat title: the emerging novel middle east respiratory syndrome coronavirus: the “knowns” and “unknowns” date: 2013-07-21 journal: j formos med assoc doi: 10.1016/j.jfma.2013.05.010 sha: doc_id: 263508 cord_uid: row2mn17 a novel lineage c betacoronavirus, originally named human coronavirus emc/2012 (hcov-emc) and recently renamed middle east respiratory syndrome coronavirus (mers-cov), that is phylogenetically closely related to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5, which we discovered in 2007 from bats in hong kong, has recently emerged in the middle east to cause a severe acute respiratory syndrome (sars)-like infection in humans. the first laboratory-confirmed case, which involved a 60-year-old man from bisha, the kingdom of saudi arabia (ksa), who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the world health organization (who) on september 23, 2012. since then, a total of 70 cases, including 39 fatalities, have been reported in the middle east and europe. recent clusters involving epidemiologically-linked household contacts and hospital contacts in the middle east, europe, and africa strongly suggested possible human-to-human transmission. clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of mers-cov, and the optimal laboratory diagnostic options and potential antiviral targets for mers-cov-associated infection. introduction: the "new sars"? ten years after the devastating epidemic of severe acute respiratory syndrome (sars) caused by sars coronavirus (sars-cov), which resulted in a total of 774 deaths among more than 8000 confirmed cases in over 30 countries, the world is facing a new challenge posted by a "sars-like" infection caused by another novel coronavirus emerging from the middle east, which was originally named human coronavirus emc/2012 (hcov-emc) and recently renamed by the coronavirus study group of the international committee for taxonomy of viruses as middle east respiratory syndrome coronavirus (mers-cov). 1e8 the complete genome of the virus was sequenced and released in october 2012 after the isolation of the virus from two patients with severe community-acquired pneumonia in bisha, the kingdom of saudi arabia (ksa), and doha, qatar, first announced by the world health organization (who) on 23 september 2012. 9 as of may 12, 2013, the total number of laboratory-confirmed cases of mers-cov infection has increased to 34 with 20 deaths, including two cases confirmed retrospectively from a jordanian cluster of severe respiratory disease reported by the ministry of health of jordan in april 2012 (table 1) . 6,7,10e14 although the number of laboratory-confirmed cases remains limited, the severe clinical manifestations with an unusually high mortality rate of over 50%, the spread of the infection beyond the geographical confinement in the middle east, and the epidemiological evidence of human-to-human transmission arising from the recent clusters of cases in a family in the united kingdom (cases 10 to 12), and in hospitals in ksa (cases 18 to 30, 32 and 33) and france (cases 31 and 34), have raised significant concerns on the possible emergence of another sars-like epidemic in the near future. in anticipation of the potential spread of this highly pathogenic virus from the middle east and europe to other parts of the world, especially the densely populated southeast asia, an updated review of the latest research findings on mers-cov and their implications on the clinical management of mers-cov infection is essential. viral genomic studies reveal the first lineage c betacoronavirus associated with human infection mers-cov belongs to the genus betacoronavirus in the family coronaviridae under the order nidovirales. coronaviruses are enveloped viruses with positive-sense singlestranded rna genomes. studies in their biodiversity, comparative genomics and phylogeny in the past 10 years have improved our understanding of this family of viruses. 15e30 according to the most recent classification by the coronavirus study group of the international committee for taxonomy of viruses, there are four genera in coronaviridae, namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. 28, 31 the genus alphacoronavirus contains two human coronaviruses, hcov-229e and hcov-nl63, which are associated with the common cold. no human coronavirus has been discovered in gammacoronavirus and deltacoronavirus, which mainly contain avian coronaviruses with just a few mammalian coronaviruses. the genus betacoronavirus is comprised of four lineages (a, b, c and d) which contain four coronaviruses associated with human infections: hcov-oc43 and hcov-hku1 (lineage a), sars-cov (lineage b), and mers-cov (lineage c). 15 mers-cov is the first known lineage c betacoronavirus associated with human infection and is phylogenetically closely related to the other lineage c betacoronaviruses including tylonycteris bat cov hku4 (ty-batcov-hku4) and pipistrellus bat cov hku5 (pi-batcov-hku5), which were discovered in lesser bamboo bats (tylonycteris pachypus) and japanese pipistrelle bats (pipistrellus abramus), respectively, captured in hong kong, china. 9, 22, 32 analysis of the genome of mers-cov revealed that it has a genome size of 30,106 bases with the rdrp and s genes having over 90% and around 70% amino acid identities with those of ty-batcov-hku4 and pi-batcov-hku5. 9,32 molecular clock analysis using the rdrp gene showed that mers-cov might have diverged from the most recent common ancestor of lineage c betacoronaviruses in year w941 ad (529 bc to 1878 ad). 32 it is postulated that the emergence of mers-cov represents another series of interspecies transmission events in coronaviruses, from bats to possibly other animals and then to humans, a scenario similar to the sars epidemic. epidemiology reports and cell line susceptibility studies suggest possible animal reservoirs and human-to-human transmissions epidemiological linkage with animals, including camels, goats, sheep, and farm animals or their caretakers before symptom onset in some of the reported cases supported the hypothesis of mers-cov being a zoonotic agent (table 1) . furthermore, in vitro data from cell line susceptibility studies showed that the virus had a broad species tropism and was able to replicate in bat, primate, porcine, rabbit, and civet cell lines. 33, 34 as in the case of sars-cov, which likely emerged from its natural animal reservoir the horseshoe bats (rhinolophus sp.), and jumped to other mammals during their caging in the wild life markets of south china and then to humans, mers-cov might have also originated from bats to these susceptible animal species before adapting to humans. 16, 35, 36 in addition to pipistrellus bats, which are the natural hosts of the closely related pi-batcov-hku5 and are also found in the middle east, rousettus, rhinolophus, myotis, and carollia bat cell lines are also susceptible to mers-cov infection in vitro. 34 active surveillance of different bat and animal species predominantly found in the middle east would help to delineate the natural bat reservoir and the evolutionary pathway of mers-cov among other susceptible animal species. indeed, a recent study showed that a number of different bat species in ghana and europe are infected with coronaviruses, which also share close homologies with mers-cov. 37 determination of the virus' animal hosts might in turn facilitate the control of the outbreak as in sars, where closure of the wet markets likely contributed to the cessation of the epidemic. human-to-human transmission represents a new stage in the evolution of mers-cov infection. there are, so far, six 12 the seroprevalence and transmissibility of mers-cov remain undetermined, although animal-to-human and human-to-human transmissions both appear to be limited at this stage (fig. 1) . among 2400 persons seeking medical attention in a hospital in jeddah, ksa, none had mers-covspecific igg detectable by indirect immunofluorescence assay, suggesting that the current situation is likely to be different from those of other human coronaviruses which are endemic in humans. 6, 38 the lack of secondary cases among nearly 200 contacts of cases 2 and 5 who did not practice optimal infection control measures before the diagnosis of mers-cov infection was confirmed, implied that this novel virus might be less efficient than sars-cov in human-tohuman transmission. 7, 13, 39 however, the findings of these studies should be interpreted with cautions for two reasons. first, only a relatively small number of subjects (10/64 in case 2 and 85/123 in case 5) were tested by reliable laboratory tests. second, the timing of testing was not clearly described and might be suboptimal for the purpose of excluding the diagnosis. it has been proposed that a second test should be performed in symptomatic patients with initially negative results by reverse transcriptionpolymerase chain reaction (rt-pcr), within the first 10 days of symptom onset, based on the observation in sars where viral load peaked at day 10 of symptom onset. 5,40e42 therefore, the apparently limited spread of mers-cov at present might be an underestimation and ongoing transmission of the virus should be cautiously monitored. unlike its close relatives in bats, mers-cov is highly pathogenic in humans. the most common clinical presentation among the 34 laboratory-confirmed cases of mers-cov infection is acute severe community-acquired pneumonia with acute renal failure following an estimated incubation period of 1e9 days (table 1) . 12, 43 this is unusual among human infections caused by coronaviruses, in which severe pneumonia with respiratory failure is seldom seen, except in sars. the other human coronaviruses, namely hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1, predominantly cause acute self-limited upper respiratory tract infections, and only occasionally cause lower respiratory tract infections in the elderly and immunocompromized populations. only two of 34 patients (cases 12 and 16) had a self-limiting, mild, influenza-like illness not requiring hospitalization. asymptomatic or mild infections have otherwise not been detected among the other contacts of confirmed cases by rt-pcr of upper respiratory tract specimens and/or serological tests, 2400 saudi residents in jeddah by indirect immunofluorescent antibody testing of archived sera, and 169 french hajj pilgrims with upper respiratory symptoms and rt-pcr of prospectively collected nasal swabs. 6, 7, 13, 44 the other 32 (94.1%) patients, many of whom had no underlying medical condition, developed rapid clinical deterioration with lower respiratory tract involvement and respiratory failure requiring ventilator support within a few days to 1 week after initial systemic symptoms of fever, myalgia, and malaise, and upper respiratory tract symptoms such as rhinorrhea and sore throat. this correlated with the finding in our recent cell line susceptibility study, which showed that mers-cov replicated much better in the lower respiratory tract cell lines including calu-3 (polarized airway epithelium), a549 (lung adenocarcinoma), and hfl (embryonic lung fibroblasts), than the upper respiratory tract cell line hep-2 (laryngeal epidermoid carcinoma). 33 another in vitro study also showed that pseudostratified human bronchial epithelium cultures are highly permissive to mers-cov infection. 45 in ex vivo organ cultures, mers-cov productively replicated in both human bronchial and lung tissues, whereas sars-cov only productively replicated in lung tissue. 46 recently, a macaque model showed that mers-cov caused acute, localized to widespread pneumonia, resulting in mild to moderate clinical disease resembling the illness observed in humans. 47 radiologically, pneumonia is evident by focal consolidations involving single or multiple lobes, with progressive involvement of bilateral lung fields, especially the lower zones. in case 1, thoracic computed tomography scans revealed mediastinal hilar lymphadenopathies, airspace opacities with air bronchograms, scattered ground-glass opacities, interstitial septal thickenings, and nodularities in the upper lobes without significant pleural and pericardial effusions. 6 acute renal failure was the other dominant clinical feature in mers-cov infection and is seen in at least six of the 34 reported cases. many of the remaining 28 severe cases probably also developed renal impairment, although their clinical details were not available. this was unusual, even when compared to sars in which 28.8% of the patients had abnormal urinalysis and detectable viral load by quantitative rt-pcr in urine, but only 6.7% developed acute renal failure with histological evidence of acute tubular necrosis and most did not require renal replacement therapy. 48 this clinical presentation correlates with the in vitro finding of efficient replication of mers-cov in kidney cell lines, including hek 293, vero, llc-mk2, and 769p. 33, 34, 49 more importantly, the presence of renal involvement appeared to be a poor prognostic factor, as those with renal failure either died or required renal replacement therapy, while two cases without renal impairment survived ( table 1 ). the lack of extrapulmonary lesions observed in the macaque model of mers-cov infection suggested that acute renal failure was more likely due to hypoxic damage than a direct viral cytopathic effect. 47 other clinical, laboratory and microbiological findings reported in mers-cov infection included pericarditis, disseminated intravascular coagulation, leukocytosis with neutrophilia and lymphopenia, thrombocytopenia, anemia, hyponatremia, hypoalbuminemia, elevated liver enzymes, lactate dehydrogenase, c-reactive protein, and procalcitonin levels, possible secondary bacterial pneumonia caused by klebsiella pneumoniae, staphylococcus aureus, and acinetobacter sp., and coinfection with other respiratory viruses, including influenza a(h1n1)pdm09 and type 2 parainfluenza virus. 6,7,10e13,50 it is interesting to note the absence of watery diarrhea in the acute phase of mers-cov infection, despite the in vitro finding of viral replication in the colonic cell line caco-2 (colorectal adenocarcinoma), in contrast to sars in which around 20% of patients developed enterocolitis. 1, 40 it remains to be seen in future case cohorts whether this is due to under-reporting, or a genuine difference in clinical presentations between the two diseases. together with severe acute respiratory and renal failure, these clinical features underscore the success of the innate immune evasion mechanisms of mers-cov in humans, leading to overwhelming infection and possible cytokine dysregulation, as reflected by the lack of interferon through inhibition of interferon regulatory factor family 3 (irf-3). 45, 49 the discovery of dipeptidyl peptidase 4 (dpp4), a multifunctional 766-amino-acid-long type-ii transmembrane glycoprotein exopeptidase expressed on human non-ciliated bronchial, renal, enteric, hepatic and prostatic epithelial cells, which is important in the regulation of hormone and chemokine bioactivity, glucose metabolism, t-cell activation, chemotaxis modulation, cell adhesion, apoptosis and regulation of tumorigenicity, as a functional receptor for mers-cov, provides further insights into the unique pattern of organ involvement and unusually severe clinical presentation of this emerging infection. 51 clinical utility and practical concerns of published laboratory diagnostic options several laboratory methods are available for establishing a virological diagnosis of mers-cov infection. the most definitive tests are viral culture from respiratory, fecal, urine, or tissue specimens, and/or a fourfold rise in the serum neutralizing antibody titers taken at 14 to 21 days apart. the first clinical isolate of mers-cov was cultured on monkey kidney cells, like vero and llc-mk2, which showed cytopathic effects of syncytium formation, rounding and detachment of cells. 6 subsequent studies have identified various human cell types, including bronchial epithelial, colonic, hepatic, renal, and neuronal cells, monocytes and histiocytes that support the replication of mers-cov. however, the use of viral culture is limited by the requirement of a biosafety level three setting, which is not available in most clinical microbiology laboratories. a serum neutralizing antibody test has the disadvantage of requiring convalescent sera and is therefore mainly used for diagnosis in the convalescent instead of acute phase. 13, 52 furthermore, the sera of sars patients may contain lowtiter cross-reactive neutralizing antibodies against mers-cov, which may lead to the wrong serodiagnosis, especially in countries where the general population had previous exposure. 53 it remains to be seen whether neutralizing antibodies against mers-cov might also crossreact with other closely related lineage c betacoronaviruses, like ty-batcov-hku4 and pi-batcov-hku5. 54 in most of the laboratory-confirmed cases, diagnosis was established by the detection of nucleic acid by rt-pcr of respiratory tract samples, including combined nose and throat swab, sputum, tracheal aspirate, bronchoalveolar lavage, and/or urine taken between day 5 (cases 3 and 11) and day 20 (case 5). 51 two highly sensitive real-time rt-pcr assays targeting regions upstream of the e gene (upe), with sensitivity of up to 3.4 rna copies per reaction and within an open reading frame (orf) 1b, with sensitivity of up to 64 rna copies per reaction, have been proposed for screening and confirmation of mers-cov infection, respectively, and are available in about half of the countries in the who european region. 55, 56 additional testing of other gene targets with partial or whole genome sequence analysis may also be used for confirmation. 55, 56 for example, pan-coronavirus rt-pcr targeting the rdrp gene was used in case 2 and a real-time quantitative rt-pcr assay targeting the n gene has been used in in vitro studies, and may also be useful for clinical diagnosis, although further evaluations are needed. 33 other potential diagnostic options which might be used in areas without rt-pcr are n or s protein-based enzyme-linked immunosorbent assays, which were shown to be highly sensitive and/ or specific for the diagnosis of sars. of note, the detection of other respiratory viruses in the respiratory tract specimen does not preclude the need for specific mers-cov testing in patients with epidemiological risk factors, or unusually severe disease, despite antiviral treatment as exemplified by the presence of coinfections in cases 10e12. the exceptionally high crude mortality rate of 60% in mers-cov infection is partly due to the lack of specific anticoronavirus treatment and effective vaccine. in most of the cases, intensive supportive treatment with extracorporeal membrane oxygenation, renal replacement therapy, and empirical broad-spectrum antibacterial and antiviral agents were used. case 2, who is still in a critical condition nearly 6 months after symptom onset, also received a corticosteroid in the initial phase of treatment, but its efficacy is unknown. its use might be limited by serious side effects and the availability of ecmo for organ support during the critical phase. alternatively, type i interferons appear to be promising therapeutic options, as mers-cov has been shown to be much more sensitive than sars-cov to the antiviral action of interferon in vitro. 49 the replication of mers-cov was shown to be reduced in human lung ex vivo organ cultures treated with type i interferons. 46 a recent study showed that while both interferon-a2b and ribavirin reduced the replication of mers-cov in vero and llc-mk2 cells, the combination of the two drugs achieved the same endpoints at a much lower concentration, which might facilitate their clinical applications. 57 other potential specific antiviral targets include type ii transmembrane serine proteases (tmprss2) and endosomal cathepsins, which are responsible for mers-cov s protein activation required for virus-cell fusion and entry of the virus into host cells. 54 the identification of dpp4, but not angiotensinconverting enzyme 2 (ace2), aminopeptidase n, and carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1), as a functional receptor for mers-cov implies that in vivo manipulation of dpp4 levels and development of inhibitors against the s1 domain-dpp4 interface might have potential therapeutic roles in mers-cov infection, as with ace2 analogues in the case of sars. 51 finally, the recognition of the predicted receptor-binding domain /critical neutralizing domain at residues 377 to 662 in the mers-cov s protein might facilitate vaccine development for this emerging infection. 58 in the past few months, following the who's announcement of the first two cases of mers-cov infection on september 23, 2012, clinicians and scientists worldwide have collaborated closely in an attempt to prevent a sars-like epidemic from happening again. the discovery of certain important characteristics of mers-cov including its genome arrangement, phylogenetic relatedness with other coronaviruses, in vitro tissue and species tropism, and functional receptor, enhanced our understanding of the clinical presentation, pathogenesis, epidemiology, and design of diagnostic and therapeutic options of this emerging novel human coronavirus. however, key questions concerning the definitive animal reservoirs of the virus, evolutionary process, transmissibility, and the prognostic factors and optimal treatment modalities of the infection, remain elusive. more importantly, the increasing number of laboratory-confirmed cases in the middle east, and the recent evidence of human-tohuman transmission in europe are suggestive of continuing viral adaptations in humans, which might precede a largescale epidemic. indeed, as of 23 june 2013, after the acceptance of the article, the total number of laboratoryconfirmed cases have increased to 70 with 39 fatalities. 59 additional clusters of cases involving household and/or hospital contacts were reported in ksa, italy, and tunisia. collaborations between global and local health authorities and their sustained support for further research on mers-cov are crucial to control this "new sars". severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans latest outbreak news from promed-mail: novel coronavirus e middle east recovery from severe novel coronavirus infection evidence of person-to-person transmission within a family cluster of novel coronavirus infections contact investigation of a case of human novel coronavirus infection treated in a german hospital world health organization. global alert and response: novel coronavirus infectioneupdate. geneva: who coronavirus diversity, phylogeny and interspecies jumping severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia molecular diversity of coronaviruses in bats coronavirus hku1 and other coronavirus infections in hong kong comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits recent transmission of a novel alphacoronavirus, bat coronavirus hku10, from leschenault's rousettes to pomona leafnosed bats: first evidence of interspecies transmission of coronavirus between bats of different suborders taxonomy of viruses. virus taxonomy: 2012 release (current genetic relatedness of the novel human lineage c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications on disease pathogenesis and clinical manifestation human coronavirus emc does not require the sarscoronavirus receptor and maintains broad replicative capability in mammalian cell lines bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia the united kingdom public health response to an imported laboratory confirmed case of a novel corona viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome viral loads in clinical specimens and sars manifestations clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures pneumonia from human coronavirus in a macaque model acute renal impairment in coronavirus-associated severe acute respiratory syndrome human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus world health organization. global alert and response: novel coronavirus infectione update. geneva: who dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections crossreactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests the spike-protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2 and is targeted by neutralizing antibodies detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction laboratory capability for molecular detection and confirmation of novel coronavirus in europe inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update key: cord-271862-jk37ej4c authors: qian, hua; miao, te; liu, li; zheng, xiaohong; luo, danting; li, yuguo title: indoor transmission of sars-cov-2 date: 2020-04-07 journal: nan doi: 10.1101/2020.04.04.20053058 sha: doc_id: 271862 cord_uid: jk37ej4c background: by early april 2020, the covid-19 pandemic had infected nearly one million people and had spread to nearly all countries worldwide. it is essential to understand where and how sars-cov-2 is transmitted. methods: case reports were extracted from the local municipal health commissions of 320 prefectural cities (municipalities) in china, not including hubei province, between 4 january and 11 february 2020. we identified all outbreaks involving three or more cases and reviewed the major characteristics of the enclosed spaces in which the outbreaks were reported and associated indoor environmental issues. results: three hundred and eighteen outbreaks with three or more cases were identified, involving 1245 confirmed cases in 120 prefectural cities. we divided the venues in which the outbreaks occurred into six categories: homes, transport, food, entertainment, shopping, and miscellaneous. among the identified outbreaks, 53.8% involved three cases, 26.4% involved four cases, and only 1.6% involved ten or more cases. home outbreaks were the dominant category (254 of 318 outbreaks; 79.9%), followed by transport (108; 34.0%; note that many outbreaks involved more than one venue category). most home outbreaks involved three to five cases. we identified only a single outbreak in an outdoor environment, which involved two cases. conclusions: all identified outbreaks of three or more cases occurred in an indoor environment, which confirms that sharing indoor space is a major sars-cov-2 infection risk. in less than 4 months, sars-cov-2 has rapidly spread to nearly all countries worldwide and by 3 april 2020 it had infected more than a million people, and killed nearly 50,000 people. 1 understanding where and how sars-cov-2 is transmitted from an infected person to a susceptible person is essential for effective intervention. the once-in-a-century covid-2019 pandemic occurred right in the age of artificial intelligence and big data. many clusters/outbreaks were identified via contact tracing by local health authorities in china and elsewhere using both traditional and new technologies. the identification of these clusters allowed the health authorities to quarantine close contacts for effective intervention and provided an opportunity to study the characteristics of where and how these clusters occurred. the first covid-2019 patient was identified in wuhan in december 2019, and the largest number of confirmed chinese cases occurred in hubei province, of which wuhan is the provincial capital. 2 since 20 january 2020, the local health authorities of cities outside hubei have reported online the details of most identified cases of infections. in this study, we identified the outbreaks from these case reports from the local municipal health commissions of 320 prefectural cities (municipalities) in china, not including hubei province, between 4 january and 11 february 2020 and reviewed the major characteristics of the enclosed areas in which these outbreaks were determined to have occurred and associated indoor environment issues. we collected descriptions of each confirmed case from the local municipal health commission website of 320 prefectural cities in mainland china, not including hubei province. each local municipal health commission announced a description of the confirmed cases each day. the case descriptions generally included age, sex, venue of infection, symptoms, date of symptom onset, hospitalisation, and confirmation and history of exposure. many described cases also included the individual trajectory and relationship with other confirmed cases, and quite often clusters had already been identified. we consulted the websites nationwide except for those of cities in hubei province and collected all available data up to 11 february 2020. data from a few major cities -beijing, shanghai, and guangzhouwere not included in our analysis due to insufficient case descriptions. case descriptions from hong kong, macao, and taiwan were collected from their health authorities. we input the data into a database in a unified format and conducted crossvalidation to ensure data reliability. a total of 7324 cases with the minimum required descriptions (i.e., the information listed above) were found; these accounted for 66.7% of the 10,980 confirmed non-hubei cases in china by 11 february 2020. 3 in this study, we defined a cluster as an aggregation of three or more cases that appears to be linked to the same infection venue (e.g., an apartment, an office, a school or a train) during a sufficiently close period. we defined an outbreak as a cluster in which a common index patient is suspected, and we excluded tertiary and highergeneration infections in counting the number of cases involved. we also excluded outbreaks that involved only two cases to exclude possible spouse-to-spouse transmission and to reduce the workload due to the large number of clusters or outbreaks with two cases. we also identified the index patient(s) of the identified outbreaks and their date of symptom onset. we divided the identified outbreaks into categories for further analysis. first, six categories of infection venues were considered: homes (apartments and villas), transport (train, private car, high-speed rail, bus, passenger plane, taxi, cruise ship, etc.), restaurants and other food venues, entertainment venues (gyms, mahjong, cards, tea houses, and barbershops) and shopping venues (shopping malls and supermarkets), with an additional miscellaneous venue (hospitals, hotel rooms, unspecified community, thermal power plants, etc.) . second, four categories of infected individuals were considered based on their relationship: family members, family relatives, socially connected and socially non-connected. the funding bodies had no such involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. the corresponding authors also confirms that they had full access to all the data in the study and had final responsibility for the decision to submit for publication. we identified 318 outbreaks involving 1,245 infected individuals in 120 cities. the top three cities (table s2) were shenzhen, guangdong (24 outbreaks, 7·5%; 84 cases, 6·7%), chongqing (16 outbreaks, 5·0%; 61 cases, 4·9%) and bozhou, anhui (nine outbreaks, 2·8%; 35, 2·8%). the average number (sd) of cases per outbreak was 3·921·65. among the 318 identified outbreaks, more than half (171; 53·8%) involved three cases, more than a quarter (84; 26·4%) involved four cases, and only five (1·6%) outbreaks involved ten or more cases. table s1 briefly describes four outbreaks, including the largest outbreak in a shopping mall in tianjin (21 cases). among the 318 outbreaks, 129 involved only family members, 133 involved family relatives, 29 involved socially connected individuals, 24 involved socially non-connected, and only three involved multiple relationships. in addition to family members, family relatives and socially connected individuals constituted a large proportion of the infected cases ( figure 1a ). eighty-three of the 318 identified outbreaks had multiple possible venues, which means either that the exact venue of infection cannot be identified or that more than one venue was involved in the infection. if we double-or triple-count these venues, we have a total of 416 infection venues for 318 outbreaks ( figure 1b ). among the 318 outbreaks, 254 (79·9%) occurred in a home (one in a villa; all others in apartments), 108 (34·0%) in transport, 14 at a restaurant or other food venue, seven at an entertainment venue, and seven at a shopping venue (shopping mall and supermarket), with an additional 26 at a miscellaneous venue (e.g., hospital, hotel room, unspecified community, and thermal power plant). most of the 254 home outbreaks included three to five cases (145 with three cases, 66 with four cases, and 25 with five cases). the average number of cases was 3·7 for the home outbreaks, 3·8 for transport, 4·9 for food venues, 3·6 for entertainment venues, 8·7 for shopping venues, and 4·4 for miscellaneous venues. the proportion of large outbreaks was high for shops and food venues, possibly because more susceptible individuals were present in these venues than in homes. shopping and entertainment venues were each associated with only seven outbreaks. this seems to suggest the difficulty of implementing preventive measures in places with large numbers of susceptible individuals. between 29 december 2019 and 31 january 2020, we also identified 231 outbreaks with known start and end dates of the suspected infectious period (figure 2a ). the identified outbreaks peaked between 23 and 28 january (figure 2a ), which coincides with the celebration period of the chinese new year (cny). cny 2020 lasted from new year's eve on 24 january to the lantern festival (i.e., the 15 th of lunar january) on 8 february. the official holiday in mainland china was from 24 to 30 january 2020. the peak date for the number of transport outbreaks was 1 to 2 days earlier than that for the home outbreaks as people travelled home for cny. because home outbreaks dominated, the changes in the temporal profile of the number of cases ( figure 2a ) closely follows that of the home outbreaks ( figure 2b ). however, for outbreaks with more than six cases, no particular pattern was identified over time, which suggests a sporadic nature. among the 231 outbreaks with a known suspected infectious period, 126 included a known date of symptom onset for the index patient ( figure 3 ). we further divided those 126 outbreaks into two subgroups according to the index patient's symptom-onset date: on or before 28 january (96 outbreaks) and after 28 january (30 outbreaks). the average time from symptom onset to the ending infectious date was 3·76±4·42 days for those on and before 28 january and 0·87±2·80 days for those afterward. the first salient feature of the 318 identified outbreaks that involved three or more cases is that they all occurred in indoor environments. although this finding was expected, its significance has not been well recognised by the community and by policy makers. indoors is where our lives and work are in modern civilisation. the transmission of respiratory infections such as sars-cov-2 from the infected to the susceptible is an indoor phenomenon. the emergence of homes as the most common covid-19 outbreak venue in china is not surprising. during the covid-19 epidemic in mainland china, homes became temporary quarantine places. our estimated home dominance of 79·9% is close to the official estimate of 83% of the so-called household clusters among the nearly 1000 clusters (not outbreaks) defined by the china national health commission. 4 after wuhan announced its city lockdown on 23 january, the warning message spread throughout the country. people in provinces outside hubei also began to stay at home. most chinese families have one child, and some families may also include grandparents. the relatively low number of cases in these home outbreaks might be considered an advantage of compulsory home quarantine because transmission was limited to the small number of family members. similar stay-athome policies have now been implemented elsewhere during the pandemic. the rising trend shown before the peak period in figure 2 was probably due to the introduction of imported cases due to the spring festival travel season (chunyun in chinese), a period around cny during which many people leave cities in which they work to visit their rural families. the 2020 chunyun brought people from the epicentre wuhan to their home all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.04.20053058 doi: medrxiv preprint cities before wuhan's lockdown on 23 january. social and family gatherings continued after 23 january in most cities outside hubei. interventions such as contact tracing and confinement of estates, villages, and individual buildings were implemented gradually in most cities outside hubei immediately after cny, which explains the sharp declining curve after 28 january. our study does not rule out outdoor transmission of the virus. however, among our 7,324 identified cases in china with sufficient descriptions, only one outdoor outbreak involving two cases occurred in a village in shangqiu, henan. a 27-year-old man had a conversation outdoors with an individual who had returned from wuhan on 25 january and had the onset of symptoms on 1 february. the second salient feature of the 318 identified outbreaks is the relatively small number of outbreaks that involved 10 or more cases. the largest outbreak occurred in a tianjin shopping mall and involved 21 cases, although wu et al. 5 reported that 25 cases were involved (table 1) . this feature contrasts with the 2003 sars-cov epidemic, during which 7 major super-spreading events in hong kong and singapore alone were identified to involve as many as 329 cases, and super-spreading events dominated the epidemic. 6 the occurrence of many small outbreaks (in number of cases) in the covid-19 pandemic suggests a different transmission pattern from that of the 2003 sars-cov epidemic. some virus, epidemiological, and environmental factors could have contributed to this observed difference between the 2003 sars-cov epidemic and the current covid-19 pandemic. we cannot pinpoint the exact transmission routes from these identified outbreaks. most health authorities advised that the covid-19 virus is transmitted mainly by close contact and via the fomite route (e.g., china nhc 7 and cdc 8 ). the china nhc also suggested that longrange aerosol transmission may occur when certain conditions are met, such as in crowded enclosures or spaces with poor ventilation. frequent close contact occurs and high touch surfaces exist in buildings. 9-12 we do not have data on the hygiene conditions and human density of the infection venues of the 318 outbreaks studied here. the exact location of the infection venues and the necessary parameters such as the floor area or the number of occupants were not provided in the case reports. instead, we reviewed the current design standards of thermal and ventilation conditions, occupant density and close contact behaviour in the various indoor environments discussed here (table s3 ). the required ventilation rates vary significantly among homes, offices, trains, and buses. for example, the required ventilation rate is only 3·9 l/s per person in shopping malls and 2·8 l/s per person in public buses, whereas a ventilation rate of 8 to 10 l/s is required for good indoor air quality. 26 an international systematic review showed that a rate as high as 25 l/s per person may be needed. 13 many existing buildings are crowded, poorly ventilated, and unhygienic. a comprehensive review of ventilation conditions in chinese indoor environments by ye et al. 14 showed that the co2 concentration can reach 3,500 ppm in some buildings. the design and operation of buildings have also been under pressure to reduce energy use 15 and increase human productivity. balancing the need for energy efficiency, indoor environment, and health in both urban planning and building design has not been easy. 16 the quality of indoor environments might be sacrificed by putting a greater focus on cost than on health. this study has limitations. we only studied outbreaks in china, where very strict intervention measures were implemented. we relied fully on the case reports of the local health authorities in each city, and variation exists in the details and the quality of their original epidemiological investigations. we also made no attempt to access any of the infection venues, and the details of each of these indoor spaces remains unknown. this study shows that the individual indoor environments in which we live and work are the most common venues in which the virus of the once-in-a-century-pandemic is transmitted among us. an individual infected in one building may infect others in the building(s) that he or she later visits. people are in constant contact as they move from one indoor space or building to another, which creates an indoor contact network through which a virus can spread. 17 the buildings and transport cabins in various parts of the world are thus connected and facilitated the spread of the covid-19 pandemic virus. the association between crowding and infection has been known since pringle. 18 the most dramatic example might be in the cruise ship outbreak on the crowded diamond princess, of which the peak basic production number was predicted to be 11 19 or 14·8 20 before quarantine and much higher elsewhere. the world's first statutory housing policy, the artisans and labourers dwellings act 1875 21 was developed following 19 th century empirical evidence that crowding led to a high incidence of infectious disease. a recent systematic review by the world health organization also found an association between crowding and infection. 22 a lancet editorial in 2018 declared '[t]he right to a healthy home'. 23 one who meeting also declared that 'everyone has the right to breathe healthy indoor air' 24 and that 'the provision of healthy indoor air should not compromise global or local ecological integrity, or the rights of future generations'. 25 we hope that in the post-pandemic future, mankind will reflect deeply on the need for a healthy indoor environment. all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.04.20053058 doi: medrxiv preprint the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.04.20053058 doi: medrxiv preprint (a) (b) all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.04.20053058 doi: medrxiv preprint (c) figure s1 . two hundred and thirty-one outbreaks with known starting and ending dates of suspected infectious period, arranged by (a) starting date of suspected infectious period, (b) ending date, and (c) median date. all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 figure s4 . provincial distribution of the identified outbreaks and number of cases involved in these outbreaks. table s1 . brief summary of 4 outbreaks with 10 to 21 cases. shop (10), liaocheng, shangdong between 30 january and 7 february, 10 workers from the zhenhua supermarket (振华量贩聊城闸口店) were confirmed to be infected. seven of them worked in the 1f shopping area. six other secondary infections were reported (five family members of the two cases and one close contact of the third case). local health authorities identified 169 close contacts by 11 february, and a free check and test were offered to all customers who visited the shop from 16 to 30 jan. based on our collected data, 10 cases were involved. according to news reports (wenzhou news network, 2020), a total of 17 cases were involved in this outbreak, including 5 administrators, 5 shopping assistants or cleaners, and 7 customers of wenzhou intime department store (温州银泰百货商场). the patient with the earliest onset of symptoms was a female cleaner (16 january) and the last was a female customer (1 february). the first case was identified on 20 january. the mall was closed on 22 january. the local health authority traced more than 100,000 people who visited the mall or nearby for 15 minutes or more, including 1,330 workers in the mall and nearby residents. the mall was opened again on 5 march. (21), tianjin based on our collected data, 21 cases were involved. according to wu et al. (2020) , 25 cases were involved. six shop workers and nine customers in a shopping centre were infected, with onset dates between 20 january (a shop assistant) and 3 february (a customer). the shopping complex was built in 1981, with a 4,000 m 2 shopping area, mainly on the first and second floors. there were 213 workers including shop assistants. people were purchasing clothes and gifts before cny, so the shop was very crowded with at least 10,000 visitors to the venue during the possible infectious periods. the outbreak mainly occurred on the first floor, where clothes, shoes, jewellery, and small appliances are sold. the building was closed on 26 january. all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.04.20053058 doi: medrxiv preprint 88 宁夏 ningxia 1 5 table s3 . characteristics of indoor environments as required by standards where covid-19 clusters occurred. all rights reserved. no reuse allowed without permission. the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is in general, we tried to review the chinese standards/studies of chinese buildings and cited international standards or studies elsewhere if needed. who, 2020a. coronavirus disease (covid-2019) situation reports. situation report -68 by air infiltration rates in the bedrooms of 202 residences and estimated parametric infiltration rate distribution in guangzhou air change rates in urban chinese bedrooms patterns of human social contact and contact with animals in shanghai social mixing patterns in rural and urban areas of southern china preferred interpersonal distances: a global comparison hou et al. (2019) stated 'the result is that in approximately 54% of chinese bedrooms, regardless of climate region or season, the only outdoor air entering bedrooms is by infiltration the authors are grateful to shengqi wang, xiaoxue cheng, luping ma, ya lei, siying mei, ziying zhou, yiran lu, mengjie duan, yifan li, qionglan he and ziang xi, who helped collect the original data. the authors declare no conflict of interest.h. qian, t. miao, and y. li contributed equally. y. li and h. qian contributed to study design, hypothesis formulation, and coordination. l. liu, x. zheng and d. luo contributed to data collection and initial data analyses. h. qian and t miao contributed to major data analyses. y. li wrote the first draft of the paper, and h. qian and t. miao contributed to major revision. all of the other authors contributed to revision.all of the authors approved the submitted version and have agreed to be personally accountable for their own contributions. key: cord-266077-l13wv186 authors: shenoy-bhangle, anuradha s; putta, niharika; adondakis, michael; rawson, james; tsai, leo l title: prospective analysis of radiology resource utilization and outcomes for participation in oncology multidisciplinary conferences date: 2020-07-01 journal: acad radiol doi: 10.1016/j.acra.2020.05.036 sha: doc_id: 266077 cord_uid: l13wv186 rationale and objectives: radiology participation is necessary in oncology multidisciplinary conferences (mdcs), but the resources required to do so are often unaccounted for. in this prospective study we provide an analysis of resource utilization as a function of outcomes for all mdcs covered by an entire radiology section and provide a time-based cost estimate. materials and methods: following institutional review board approval, prospective data on all mdcs covered by abdominal radiologists at a single tertiary care academic center were obtained over nine weeks. a predefined questionnaire was used by a single observer who attended every imaging review and recorded the total time spent by the radiologists and several outcome measures. the total time recorded was used to provide a time-based cost estimate using a national salary survey. results: six radiologists participated in a total of 57 mdcs, with 577 cases reviewed and discussed. 181 (31%) cases were performed at outside facilities requiring full reinterpretation. clinically significant revisions to original reports were recorded in 107 (18.5%) cases. radiologist input directly resulted in alteration of cancer staging in 65 (11%) patients and specific recommendations for follow-up diagnostic workup in 280 (48%) of cases. the mean total time devoted by the staff radiologist per week to mdcs was 18.7 hours/week, nearly a half of full-time effort, or 8% of total effort per radiologist. the total annual projected cost of radiology coverage for each weekly mdc was $26,920. conclusion: section-wide radiologist participation in mdcs directly resulted in change in clinical management in nearly half of reviewed cases. this was achieved at a notable time cost, highlighting the need for efficient integration of radiology mdc participation into radiologist workflow and compensation models. m ultidisciplinary conferences (mdcs) are an increasing part of radiologists' workflow not only at major academic and tertiary referral centers but also in the community setting (1) . the american college of surgeons first established the commission on cancer accreditation program to provide standards for the establishment of mdcs (2) . now there are over 1500 certified medical centers in the us, with each site required to register the composition of their mdcs with minimum attendance rates for participants, including radiologists. the importance of a multidisciplinary approach to management of oncology patients and the importance of a radiologist's presence and contribution to improved clinical decision making has been studied and validated in the context of breast cancer, hepatocellular carcinoma, pancreas cancer, gastrointestinal tumors, and gynecologic cancers (3à9) among others. a majority of these papers focus on a multidisciplinary approach to patient management in an oncology setting or the added value of reinterpretation of imaging studies by specialized radiologists. for example, pawlik et al. reviewed 203 cases in one multidisciplinary pancreas clinic and demonstrated 38 (19%) cases with altered clinical stage and overall 48 (24%) cases with altered management as a result of the discussion at the mdc that also included input from radiologists (10) . mdc impact on radiology and pathology workflow was previously studied in a single conference setting, highlighting time demands, but without a translation into cost, or reference to outcomes (11) . since that study, the radiologist's role in mdcs has been shaped by technical improvements such as the introduction of online transfers of outside hospital (osh) studies directly to picture archiving and communication systems (pacs), as well as the overall increasing number of mdcs, necessitating a more current evaluation. furthermore, there has not been a study that has examined the impact of mdc coverage across an entire section. the purpose of this study is to fill these knowledge gaps by collating both resource and basic outcome data from a consecutive set of mdcs currently supported by an entire imaging section, and by calculating the associated cost. this prospective study was approved by our institutional review board and was healthn insurance and n portability accountability act compliant. the study was conducted at our 673-bed hospital which also serves as a tertiary referral oncology center with specialized oncology care, serving approximately 725,000 outpatients a year. our hospital network includes a network of 18 satellite hospitals and partnered health centers that refer oncology patients for specialized care. this study encompasses all (a total of 7) weekly mdcs covered by the abdominal section at our tertiary referral center: gastrointestinal oncology; genitourinary oncology; gynecologic oncology; liver tumor board, liver tumor multidisciplinary clinic, pancreaticobiliary tumor board, and pancreaticobiliary multidisciplinary clinic. the description of conference style and participants is provided in table 1 . although all of these conferences are focused on oncology patients, non-oncology cases referred for second opinion to our clinical colleagues from the hepatobiliary or pancreas center are also included. the mdcs are staffed by six fellowship-trained abdominal radiologists (out of a total of 15 members of the section) who subspecialize in the conference focus areas, with one faculty member covering two of the conferences. there are backup radiologists from the same group who attend the mdc if the primary radiologist is unavailable. an abdominal imaging or body mri clinical fellow typically participates in each of the conferences. the radiologists' workflow for mdcs is comprised of two parts: a review of the cases prior to the conference and presentation of cases at the conference. the initial report of these cases is provided either by any of the radiologists from the same group, but not necessarily those reviewing cases for the mdc, or by radiologists at outside institutions. centers outside our institution that are affiliated with our network have separate radiology departments and their studies need to be imported into our pacs, and are thus considered outside studies. reviews are usually performed within 24 hours prior to each conference after a final list of cases is sent by the mdc coordinators to all participants. a clinical fellow usually previews the cases, followed by a review with the staff (attending) radiologist, who makes all final decisions regarding image interpretation and recommendations. the findings are then presented at the mdc; at five of the mdcs, the fellow is the presenter, while the attending presents at the two pancreaticobiliary mdcs. however, the corresponding attending is present at every conference for any additional questions or review of addon cases. data was collected from mdcs conducted consecutively from june 20, 2018 to august 24, 2018. a single researcher (n.p.) attended all review sessions with the clinical fellow and attending radiologist. the researcher then attended all of the mdcs, with the exception of genitourinary oncology, which overlapped with the liver tumor conference. for the genitourinary oncology conference, the attending radiologist covering the mdc (l.t.) recorded the data from that conference and emailed the results to the researcher at the conclusion of the conference. the attending radiologists were all fellowship-trained in abdominal imaging with 3 to 23 years of faculty experience. all studies were reviewed on a pacs workstation. any studies from an osh were required to be imported into our pacs system before being reviewed for an mdc. the data collected by the observer was obtained using the same standardized questionnaire for every mdc. the data included logistical information, including the time spent by the fellow and the attending previewing and reviewing the cases, respectively, the number of osh versus in-network cases that were reviewed, the time spent reviewing osh studies, and the number of "add-on" cases (less than 12 hours before the mdc or cases added on at the mdc without prior notice) that were requested after the final mdc lists were sent. all radiology findings and recommendations discussed at review and mdc were recorded for each case, along with management decisions reached by the multidisciplinary group. the data from each case was then measured for four basic outcomes that highlighted contributions directly attributable to the radiologist: 1) any changes from the original imaging report that the attending radiologist deemed clinically impactful to warrant presentation at mdc, 2) changes in cancer staging for oncology cases, and 3) instances when the radiologist either recommended a new diagnostic imaging study at the mdc, or 4) recommended cancelling a planned follow-up imaging study for lack of added value. examples for each are provided in table 2 . all data were transferred immediately to an anonymized master spreadsheet for tracking and statistical analysis. categorical variables were described by counts and percentages while continuous variables were described by mean and standard deviations. conversion of radiology time to full time equivalents (ftes) was based on a standard 40-hour work week. a cost conversion was calculated using the us national median salary obtained from medscape's 2018 compensation overview (12) . data were collected over a total of 57 mdcs over nine weeks. means are presented as mean § standard deviation. there was a mean of 8.1 § 0.7 and median of eight mdcs for each conference type. a total of 577 cases were reviewed, with a mean of 10.1 § 3.9 and median of nine cases reviewed per conference. key results are presented below, with summary data shown in table 3 . clinical fellows spent a mean of 2.2 § 1.5 hours and median of 2.5 hours per mdc, or 13.7 hours/week. attending radiologists spent a mean of 2.0 § 0.5 hours and median of 2.0 hours per mdc, or 12.4 hours/week. these times did not include the time spent by the attending radiologist and the fellow presenting at the conferences, which were each an hour long. when taking this into account, clinical fellows and attending radiologists spent a total of 20.0 hours/week and 18.7 hours/week, respectively, exclusively on mdcs. . 65 cases (11.2%) were add-ons. these included cases that were emailed to the radiologist after the official final list was emailed to the mdc group, as well as cases added during the mdc. these cases were typically reviewed during or immediately after the conference directly with the mdc team, and the time spent could not be reliably quantified to include in the total measured review times. revisions to the original radiology report were presented at mdc in a total of 107 (18.5%) cases. of these revised reports, more than half (65 cases, or 60.7 %) involved alterations (both upstaging and downstaging) in cancer staging (11 % of total cases). in 280 (48.5 %) of cases, the radiologist provided direct guidance for diagnostic follow-up at the mdc. the us national median radiology salary in 2018 was $401,000 (12) . in our study, a total of 18.7 hours/week were spent by six staff members, or 3.1 hours/week per attending. this corresponds to a yearly cost of $32,080 per physician or $188,470 for the section, and an annual cost of $26,920 for each weekly mdc. mdcs are a critical component of the clinical and educational missions of medical centers. the benefit of radiology input in mdcs is widely accepted, however, the costs associated with this have not been previously studied. our study is the first to provide an estimate of such resources while also sampling across a diverse mixture of mdc types covered by an entire radiology section. we also focused on the time spent by clinical fellows and attending radiologists previewing studies and participating at each mdc and translated it into fte for attending radiologists to assign an estimated cost per physician, per section, and per mdc. these observations have implications on clinical workflow and compensation models as there continues to be an increasing overall demand for radiology support in mdcs as well as emphasis on valuebased healthcare as highlighted by the american college of radiology (acr) imaging 3.0 initiative (13). the time spent by faculty radiologists on mdc conferences averaged 18.7 hours/week, or 0.47 fte. when divided across the six participating faculty members, this represents 3.1 hours/ week/radiologist, or 8 % of total effort per fte. this is slightly increased in comparison to a study a decade ago that calculated 2 hours preparation time per meeting hour spent by the radiologist for one specific mdc involving both imagers and pathologists (11) . our calculated time of 3.1 hours/week/radiologist represents a lower limit and likely an underestimation, as other mdc-related consultations routinely occurring outside of the review period, in particular the time spent on add-on cases (11.2% of total cases) could not be accurately recorded. although more add-on cases may have a benefit in that the radiologist takes less time to review such studies, this has been discouraged by our practice as such cases are not reviewed to the same depth as others, nor are they viewed properly on a pacs station, increasing the risk of errors or misses. additionally, our department benefits from having clinical fellows help reduce the faculty preparation time. our abdominal fellows participate in mdcs as part of a dedicated conference rotation, and we consider this activity an essential part of our academic mission (14) . we calculated a yearly cost of $32,080 for each radiologist to participate in the mdcs in our study. this translates to $188,470 per year total for coverage of all 7 mdcs, or an annual cost of $26,920 for each weekly mdc. if we extrapolated this nationwide using conservative estimate, accounting only for 1500 -commission on cancer accredited sites and assuming only a single weekly multidisciplinary conference at each site and a minimum of 80% participation rate as required by commission on cancer (2), the cost of radiology effort would be approximately $38.4 million per year in the us. nearly one-third of reviewed cases (31.4 %) and the total time spent on review by attendings (28.5 %) were osh studies, reflecting a large community referral pattern driven mostly by cancer care. while osh cases presented at the two liver mdcs always resulted in an official second opinion report issued by our radiologists, this was not performed for the remaining 5 mdcs, where the findings were instead summarized on an online medical note generated by the conference coordinators. this meant that only 41 out of 181, or 22.6 % of imported osh scans were issued full radiology reports following mdc review. multiple studies have stressed the value of official second opinion reads by subspecialists (7,15à21), highlighting a potential area of practice improvement. the increased number of second opinion reports may also generate additional revenue in the form of professional fees, though at our institution a portion of our osh volume comes from our network affiliates which, in our state, precludes additional billing. thus, the move towards requiring an official second opinion read on all osh studies would likely add to total time costs with a partial increase in revenue. in 48.5% of cases the radiologist provided direct recommendations for follow-up or next imaging studies. much of this likely stemmed from additional clinical information discussed live at the mdcs and participation by subspecialized radiologists. these results are concordant with the results from the study by chingkoe et al. (7) where a change in management was seen in 38.5% of patients following review by the radiologist for a single pancreas mdc. this change in management encompassed changes in diagnosis; in clinical stage; in treatment as well as any additional workup that might be required. changes to the original report were presented by radiologists in about a fifth of all cases. of these, more than half (11% total) were changes to oncologic staging, similar to that observed at a pancreas-specific mdc (8.7%) (7) . this was in part due to subspecialty staff reviewing or reinterpreting inhouse and osh studies, thereby providing second opinion expert reads. we also attribute some of these discrepancies to additional clinical history or supplementary studies obtained during mdc review that lead to new observations or interpretations. discrepancies resulting from interpretive errors or misses are placed in our quality review database for review and education. in many radiology departments there is no time reserved specifically for mdc review. this is currently the case at our institution, where there is no dedicated time allotted to the staff for reviewing cases for mdcs and the effort required is diverted from clinical or personal time. this can present a challenge in sections where the existing clinical workload may be at capacity and mdc participation may not be captured in traditional rvu-based compensation models. given the trend towards increased utilization of subspecialized mdcs for patient management, there may be a need to reconsider workflow designs, particularly at a time when burnout is highly prevalent (22, 23) . indeed, since this study, new lymphatic, pelvic floor dysfunction, and prostate mdcs have been added, with coverage provided by the same group of six radiologists. with the incorporation of these new mdcs, we estimate the total effort per covering radiologist to be increased from 8% to 10%. there may be several solutions to address the increased radiology demand for mdc participation. the creation of protected time to allow for uninterrupted mdc reviews would provide some separation from concurrent clinical demands and would likely improve the educational component for the fellow. a centralized and streamlined approach to importing osh studies and assigning them earlier to the reviewing radiologist for official second opinion reads would save time and provide more consistent documentation. with the recent rise of telemedicine in response to the covid-19 pandemic, virtual readouts and conference presentations offer greater convenience as mdcs can be performed remotely, potentially expanding the number of participating radiologists and reducing preparation time related to travel (24) . regardless of these changes, there will be an associated cost based on radiologist time, as shown in this study, which would need to be taken into account, whether it be borne by the department, the institution, or the radiologists themselves. there are limitations to this study. first, we were limited to observing radiologists during scheduled reviews and at mdcs; additional input or decisions made outside of these times, for example, on follow-up emails or dictation of second opinion reports, were not recorded, resulting in an underestimation of the review/consultation time and some outcome measures. also, not every osh original report was available, leading to an underestimation of discrepancy rates. we did not stratify the severity of discrepancies found in original radiology reports which would have allowed deeper insight into the overall impact of radiology input. however, we did establish a baseline threshold, based on the clinical decisions of the attending radiologists, to only include findings that were significant enough to warrant discussion at the mdc; thus, discrepancies or new findings that were deemed irrelevant to patient care and management were excluded. this study focused on a cost analysis specific to radiology. the outcomes observed in our study, for example the 11 % changes in oncologic staging, suggest a clear benefit from radiologist participation in mdcs, as validated by prior studies. a more comprehensive cost-benefit analysis would be desirable though challenging due to the wide range of different mdc subspecialties, each requiring disease-specific measures. such studies focusing on the general benefit of mdcs, including long-term clinical impact, patient satisfaction, and quality of life, are still needed (25) . the rise of online medical records and accessibility of records via patient portals also poses unique challenges, for example in cases of discrepancies reported in second opinion reports (26, 27) . indeed, the results of this study will form the basis of future investigation focusing on value analysis. take home points 1. a total of 0.5 fte, or 8% effort per radiologist, was required for mdc participation, corresponding to a yearly total cost of $32,080 per radiologist. this translates to an annual total radiology cost of $26,920 for each weekly mdc. 2. changes to the original reports were made in approximately one-fifth of cases. this highlights the value of radiology participation in mdc and an established peer learning process. 3. a third of cases and attending review time were dedicated to review of outside studies. professional fees from second opinion reports would help offset these costs. 4. the trend towards increased radiology participation in mdcs warrants consideration of required time and costs when incorporating this into institutional workflows. implementing a multidisciplinary tumor board in the community practice setting cancer program standards: ensuring patient-centered care 2016 edition the effect of a radiology conference consultation on cancer patients management improving patient care by incorporation of multidisciplinary breast radiology-pathology correlation conference the impact of a multidisciplinary breast cancer center on recommendations for patient management: the university of pennsylvania experience the requirements of a specialist breast unit subspecialized radiology review at multidisciplinary pancreas conference: impact on patient management efficacy of the multidisciplinary tumor board conference in gynecologic oncology: a prospective study utility of a multidisciplinary tumor board in the management of pancreatic and upper gastrointestinal diseases: an observational study evaluating the impact of a single-day multidisciplinary clinic on the management of pancreatic cancer multidisciplinary team meetings and their impact on workflow in radiology and pathology departments medscape radiologist salary survey supporting the academic mission second-opinion interpretations of gynecologic oncologic mri examinations by sub-specialized radiologists influence patient care breast imaging second opinions impact surgical management hepatopancreaticobiliary imaging second-opinion consultations: is there value in the second reading? clinical impact of second opinion radiology consultation for patients with breast cancer specialized second opinion interpretations of breast imaging: impact on additional workup and management quality and value of subspecialty reinterpretation of thoracic ct scans of patients referred to a tertiary cancer center clinical impact of second-opinion musculoskeletal subspecialty interpretations during a multidisciplinary orthopedic oncology conference burnout in radiology burnout in academic radiologists in the united states virtual read-out: radiology education for the 21st century during the covid-19 pandemic the impact of multidisciplinary team meetings on patient assessment, management and outcomes in oncology settings: a systematic review of the literature implications of direct patient online access to radiology reports through patient web portals online portals: gateway to patientcentered radiology key: cord-273573-a9inlk96 authors: jaeger, gry; skogmo, hege kippenes; kolbjørnsen, øyvor; larsen, hans jørgen søiland; bergsjø, bjarne; sørum, henning title: haemorrhagic pneumonia in sled dogs caused by streptococcus equi subsp. zooepidemicus one fatality and two full recoveries: a case report date: 2013-09-11 journal: acta vet scand doi: 10.1186/1751-0147-55-67 sha: doc_id: 273573 cord_uid: a9inlk96 in spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. these infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like streptococcus equi subsp. zooepidemicus to create serious disease. early recognition and treatment are tremendously important for a successful outcome in these cases. a polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. coughing was noticed among the dogs four days after immunisation. three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. streptococcus equi subsp. zooepidemicus was isolated in pure culture from all three cases. they were treated and rehabilitated successfully, and won a sledge race three months later. this paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease. contagious upper airway infections in dogs occur regularly and are most commonly caused by canine parainfluenza virus (cpiv) or bordetella bronchiseptica, amongst other agents [1] . this clinical syndrome has also been named infectious tracheobronchitis (itb), canine infectious respiratory disease (cird), "kennel cough" or "kennel croup", so named due to its occurrence in environments where many dogs live or stay close together for shorter periods of time. characteristic clinical signs include a self-limiting paroxysmal cough lasting for up to two weeks, which usually resolves without treatment. in norway, immunisation against cird is performed using live attenuated viruses, annually with cpiv and every third year against canine adenovirus type 2 (cav-2) [1] . however, in spite of vaccination, outbreaks of cird remain common. some dogs with cird will develop serious pneumonia due to an immature immune system or other causes of immunodeficiency. occasionally, bacteria such as streptococcus equi subsp. zooepidemicus can cause fatal pneumonia [2] [3] [4] [5] [6] [7] [8] . this case-report describes the first canine outbreak of haemorrhagic pneumonia in the nordic countries caused by s. equi subsp. zooepidemicus. most of the animals in the pack of athletic sled dogs showed symptoms of cird with three dogs demonstrating symptoms of severe peracute infection. one sled dog died while two were successfully treated, rehabilitated and returned to competition. to the author's knowledge, this is the first report documenting the chronology from onset of clinical signs, through convalescence to complete recovery for peracute haemorrhagic pneumonia in dogs. the vaccination regimen related to season and extreme training will also be discussed. the affected animals belonged to a pack of 26 racing sled dogs with one owner. twenty-two dogs where in active competition/training at an international level, competing annually in the norwegian, european and world championships. the dogs were a mix of pure bred german shorthaired pointer (gsp), mixed-bred alaskan husky/gsp or mixed-bred gsp/greyhounds. the dogs were mainly kept as outdoor dogs in standard heated kennels, but at times allowed indoor as family pets. in october 2008 they participated in the norwegian national championship in 4-dogs and 8-dogs classes. prior to the race all dogs were healthy with no signs of infection or disease. three days after the race the 22 competing dogs were vaccinated with duramune dappi + lc vet. fort dodge containing attenuated distemper virus (cdv), strain onderstepoort, attenuated adenovirus type 2 (cav-2), attenuated parvovirus (cpv), attenuated parainfluenza virus (cpiv), inactivated enteric coronavirus (ccov) as well as inactivated leptospira interrogans serovar canicola and serovar icterohaemorrhagiae. after vaccination the dogs rested for two days with no exercise or training. on the third day the dogs continued regular training. following training on the 4th day post-vaccine, several dogs in the kennel started to cough, and 24 hours later there were more animals in the pack with an intensive cough. all animals had normal body temperature and showed no other clinical signs. six days after the vaccine two of the dogs became anorectic and depressed. one of these dogs stayed overnight in the shelter and was found dead the next morning (case 1). the second dog was taken into the owner's family house for the night due to anorexia, intensive coughing and salivation (case 2). according to the owner this dog had been vomiting blood during the night and demonstrated a rectal body temperature of 39.7°c. after a telephone consultation with the local veterinarian the owner administered 500 mg (19 mg/kg) of oxytetracycline (oxytetral; alpharma) orally and thereafter brought the dog to the small animal hospital at norwegian school of veterinary science (nsvs) 140 km away, together with case 1 for post-mortem examination. the next day a third dog was found anorectic and depressed (case 3) and was brought immediately to the nsvs. the rest of the kennelled dogs continued to cough, though no other animals developed further clinical signs. cases 2 and 3 were more intensively trained than the other dogs on day 3 and 4 postvaccination. post-mortem examination revealed epistaxis and haemorrhagic frothy fluids in the trachea and bronchial airways on cut sections. haemorrhages were present in the thymus, epicardium, intercostally, and in the pleural space 200 ml of uncoagulated blood were present. the lungs were congested, wet, consolidated and diffusely to cavernous haemorrhagic, these changes being more severe in the left lung lobes (figure 1) . histopathology of the lungs revealed a subacute necrotising suppurative pneumonia, with haemorrhagic, often cavernous areas in the lungs and intra-lesional gram-positive cocci. a large number of macrophages with phagocytosed erythrocytes were present (figures 2 and 3) . a subacute pleuritis was also seen. streptococcus equi subsp. zooepidemicus was isolated in pure culture from the lung tissue with identification based on morphology, microscopy, lancefield grouping (streptococcal grouping kit, oxoid, basingstoke, hampshire, england) and biochemical testing. properties included βhaemolytic colonies on bovine blood agar, gram-positive, katalase negative cocci belonging to lancefield group c. glucose, lactose, and sorbitol were fermented, trehalose was not. the isolate was also tested by using api20strep (biomérieux®, lyon, france) with the same conclusion. toxicological diagnostic screening of the liver tissue showed no evidence of anticoagulant poisons. clinical data for cases 2 and 3 are present in table 1 . on presentation, the dog was depressed, dehydrated, shivering, hypersalivating with blood stained saliva, and coughed spontaneously with haemorrhagic expectorate. the neck was slightly stretched and auscultation of thorax revealed increased vesicular sounds. thoracic radiographs showed moderately increased attenuation of the ventral part of the right middle lung lobe, moderately to severely increased attenuation of the ventrocaudal part of the right caudal lung lobe as well as air bronchograms (figures 4 and 5 ). these changes are consistent with acute pneumonia. a faint soft-tissue opacity was seen in the lung fissures, interpreted as a possible low amount of free pleural fluid. the dog was treated with intravenous (iv) ringer acetate 100 ml/kg/hr for eight hours, thereafter 50 ml/kg/hr, enrofloxacin (baytril, vet. bayer; 5 mg/kg bodyweight (bw) iv once daily), ampicillin (pentrexyl, bristol-myers squibb; 35 mg/kg bw iv three times daily) and buprenorfin (temgesic, schering-plough; 0.02 mg/kg bw iv three times daily). all medications were given for four days. the dog was hospitalised in an oxygen cage. simultaneously as the treatment was initiated an expectorate sample was sent for routine bacteriological cultivation. s. equi subsp. zooepidemicus was isolated in pure culture and directly demonstrated to belong to the lancefield group c of streptococci. complete blood count with serum biochemistry analysis was normal except for a mild leucopenia ( table 2 ). the coagulation profile was normal. on the second day of hospitalisation the dog showed substantial clinical improvement and was normothermic (38.1°c), less dyspneic, less tachypneic (respiration rate (rr) = 44/min), and had reduced salivation and coughing. there was no longer blood in the saliva nor epistaxis. the result from the bacteriological investigation of the expectorate demonstrated sensitivity against penicillin, tetracyclin, cefalexin, ampicillin, amoxicillin/ clavulanate, enrofloxacin and linkomycin. the antibiotic regimen was switched to phenoxymethylpenicillin (apocillin; actavis) 660 mg per os (po) three times daily for 14 days. no diagnostic tests for respiratory viruses were performed. clinical signs gradually resolved over the next few days and the dog was sent home seven days after hospitalisation. control radiographs before departure from the clinic revealed absence of air bronchograms, though a mild to moderate increased attenuation with an interstitial pattern was still present. this animal was presented to nsvs one day after case 1. the dog had been coughing for several days and gradually worsened with reduced appetite and depression developing on the day of presentation. on physical exam there was moderate dyspnoea with abdominal respiration and increased vesicular sounds, slight neck extension, blood stained saliva as well as fever (table 1) . haematology showed moderate leucocytosis due to neutrophilia and monocytosis ( table 2 ). radiography of the thorax showed the same changes as described for case 2. the dog was hospitalised and medically treated in the same way as for case 2. clinical progression was similar to case 2, with normalisation of temperature (38.8°c), respiration rate (28 breaths/min), heart rate (100 beats/ minute) and appetite on the second day of hospitalisation. no salivation or spontaneous coughing was observed unless whilst excited after visiting the exercise figure 4 left lateral radiograph of the lung of case 2, first day of admission to hospital. the radiograph reveals marked increased attenuation of the ventral part of the right caudal lung lobe and lobus accessorius, and moderate increased attenuation of the right middle lunglobe, with air bronchograms (white arrows). pen. repeat thoracic radiographs on the seventh day of hospitalisation revealed air bronchograms in the right middle lung lobe, but reduced consolidations. the dog was sent home on phenoxymethylpenicillin (apocillin; actavis) 660 mg po three times daily for another 14 days. follow-up hospital care of both dogs included radiographs of the thorax after one, three, five and eight weeks (figures 6 and 7) together with complete blood counts (table 2) . after thoroughly scrutinising the last taken radiographs together with assessing their clinical condition they started a step-wise training program. the radiographs taken during recovery revealed a very mild interstitial attenuation of the lung lobes that had been most severely affected and mild to faint visualisation of fissure lines which was interpreted as either mild amount of free fluid or mild fibrosis. these minor findings were gradually reduced, but faint fissure lines could still be seen after five weeks for case 3 and after eight weeks for case 2 (figures 6 and 7) . the dogs were kept confined for one week after they were released from the hospital, and did run free on a large (2-3 acres) fenced yard for two weeks. case 2 was in full training eight weeks post infection and case 3 was slightly behind. early in january the following year both dogs participated in a sled race resulting in a time track record, and medal placements in various championships were achieved the following season. the dogs in this highly trained sled-dog pack competed in a sledge dog race after prolonged transportation and were additionally moderately exercised before the race. three days after the sled race the dogs were given a commercial polyvalent canine vaccine containing cdv, cav-2, cpv, cpiv, ccov and leptospira. clinical signs of respiratory disease were observed four days after vaccination. immunosuppression in dogs immunised with a similar polyvalent vaccine was reported by phillips and others [9] . they demonstrated that the absolute lymphocyte count in blood was suppressed on day five and seven following immunisation and that one group of dogs immunised with cdv and cav-2 also had decreased lymphocyte count on day three post vaccination. all the dogs produced neutralising antibodies to canine distemper virus and some parts of the natural immunity were not influenced by the vaccination. however, the immunisation with polyvalent vaccines significantly suppressed the lymphocyte response to phytohaemagglutinin, a mitogen that induce proliferation in tlymphocytes in vitro on day 5, 7 and 11 post inoculation, but returned to normal response level by day 14 following immunisation. the combination of cdv/canine adenovirus type 1 (cav-1) or cdv/cav-2 was believed to be responsible for the suppression in lymphocyte responsiveness. the duration of the suppressed lymphocyte response was at least seven days and was most pronounced on day 7 post inoculation with up to 90% suppression of mitogen-induced lymphocyte response compared to control dogs. in the present study, the use of modified live polyvalent canine vaccine combined with stress associated with training, competition and prolonged transportation may have suppressed the innate immune response to s. equi subsp. zooepidemicus, which seldom causes pathology in dogs. the possibility of a co-infection with viral pathogens cannot be excluded and may have contributed to this outbreak. outbreaks of haemorrhagic pneumonia in dogs due to s. equi subsp. zooepidemicus have been described following transportation to a race track associated with sudden change in the weather [8] , in newly arrived densely housed research dogs [4, 6, 7] and in kennelled dogs [3, [10] [11] [12] . in most of these reports the cause of the outbreak of cird is believed to be primary viral agents alone or in combination with environmental factors that induce secondary peracute pneumonia. some of these papers report the use of cdv/cav-2 vaccine prior to infection with s. equi subsp. zooepidemicus [3] or a possible dual infection with cdv [4] , though do not postulate that immunosuppression due to vaccination with modified live polyvalent canine vaccine is significant in this pathogenesis. in a study of experimental parvovirus infection in dogs, potgieter and others [13] observed that dogs vaccinated with modified live cdv and cav-1 five days before figure 6 control radiograph of the lung of case 2 eight weeks after admission to hospital. left lateral radiograph reveals a faint line (white arrow) which is interpreted to represent either mild amount of pleural fluid or mild thickening pleura. challenge with virulent canine parvovirus resulted in disease caused by canine parvovirus whereas unvaccinated dogs remained healthy. this may indicate that these animals had reduced immunity associated with recent vaccination. histopathologically, the pneumonia in case 1 was severe with the presence of inflammatory cells, leakage of blood into the lung tissue, and intralesional gram positive cocci, which were likely to be s. equi subsp. zooepidemicus (figures 2 and 3) . similar histopathological findings were also described in the paper of priestnall and others [14] , who specified that s. equi subsp. zooepidemicus is associated with acute and often fatal clinical disease in dogs. a possible source of s. equi subsp. zooepidemicus in the outbreak described in this paper was neighboring horses. however, there may have been sub-clinical carrier animals within the pack as chalker and others [3] isolated this pathogen from 9.7% of clinically healthy kennel dogs, but where the kennel had a history of endemic cird. s. equi subsp. zooepidemicus is not normally carried by dogs, but it can probably cause disease in a situation where there is high level exposure from diseased dogs or other sources in the environment, an on-going viral infection, temperature stress, transport stress, intense exercise associated with training and competition and vaccine induced immunosuppression. in a retrospective study of 393 cases of streptococcal infections in dogs, four cases (1%) were found to be caused by s. equi subsp. zooepidemicus [15] . in humans, s. equi subsp. zooepidemicus represents a potential virulent zoonotic pathogen, with infection inducing a toxic shock syndrome following contact with horses [16] [17] [18] . one report used a study population from a re-homing shelter with an endemic respiratory disease, including 39 dogs positive for s. equi subsp. zooepidemicus culture on post-mortem lung wash and 16 negative control dogs. an objective scoring system based on histopathological lung examination demonstrated that 26 (67%) of dogs with a positive culture had evidence of pneumonia caused by s. equi subsp. zooepidemicus [14] . the lungs of the dogs affected with severe pneumonia caused by s. equi subsp. zooepidemicus had significantly elevated levels of expression of the genes of pro-inflammatory cytokines indicating a strong innate immune response caused by the invading pathogen. paillot and others [19] demonstrated that three novel genes (szef, n and p) for production of superantigens in s. equi subsp. zooepidemicus are occurring in half of the isolates from cases of disease. the authors indicate that the introduction of these genes to the population of s. equi subsp. zooepidemicus may be the cause of an emerging trend of severe pneumonia caused by this pathogen. however, in the study of priestnall and others [14] there was no indication that the carriage of these superantigen encoding genes is related to the severity of the histopathological changes. the authors emphasise that further knowledge and alertness of early clinical signs will contribute to enhance the treatment success and avoid transmission of the causative strain of s. equi subsp. zooepidemicus to other animals and humans. the rate of resolution of pneumonia in humans has been defined as the resolution of radiographic abnormalities associated with the disease [20] . patients with moderately severe pneumonia have a radiological clearance rate of 70% after 1 month, and more delayed if the pneumonia is severe [21] . a multicenter cohort study that included adult patients classified as at low risk of short-term mortality concluded that full resolution of symptoms from community-acquired pneumonia (cap) may take more than 28 days [22] . this study was not specific to athletes, as they are thought to have a better physical health status compared with the general population. when to recommence training for athletes following pulmonary infection depends upon the underlying microbial aetiology and should be individualised [23] . there is no reason to believe that dog athletes should be advised differently. the long-term rehabilitation of high performance sled dogs may be complicated because of the dual challenge of strenuous exercise and low temperature. as a general rule, caution should be taken to prevent athletes from returning to competition too fast. in human figure 7 control radiograph of the lung of case 2 eight weeks after admission to hospital. ventrodorsal view reveals a mild, mild increased attenuation in the right caudal lung lobe, which was interpreted as likely mild "scar tissue" from the previous severe pneumonia. athletes, potential complications such as pneumothorax, bronchiectasis and hemoptysis has been recorded, as well as acute respiratory failure. furthermore, intense exercise too early in the rehabilitation process can increase susceptibility to viral illness and weaken the muscle performance [24] . we may speculate that the environmental stress related to the transportation, high-end competition, time of vaccination and too early exercise all contributed to the immunosuppression and consequently the susceptibility to virus-and/or bacterial infection, and where potentially more aggressive and opportunistic bacteria like s. equi subsp. zooepidemicus can progress quickly. it may also be advisable to choose a later moment for vaccination than close up to a challenging physical competition, and to avoid any strenuous exercise for at least ten to 14 days after immunisation. return to play is a hot issue within the top-level sports, but without any specific guidelines post pneumonia. these dogs were in a very good physical shape at the start of the outbreak of haemorrhagic pneumonia, and the two surviving individuals recovered relatively fast and returned to their previous performance level by setting time track records in their first race after recovery. environmental stress associated with intense exercise, competition and prolonged transportation combined with canine vaccination may suppress the innate immune response to viral infections and subsequent s. equi subsp. zooepidemicus infection that only rarely cause pathology in dogs. knowledge and alertness of early clinical signs of acute haemorrhagic pneumonia will enhance treatment success and shorten the rehabilitation period. however, caution should be taken to prevent athletes from return to the competition arena too fast. these high performance sled dogs recovered relatively fast and could prove their fitness by competing and winning international sled race championships three months later. canine infectious tracheobronchitis. in infectious diseases of the dog and cat an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs the association of streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease hemorrhagic streptococcal pneumonia in newly procured research dogs sudden deaths in greyhounds due to canine haemorrhagic pneumonia outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus streptococcus zooepidemicus as the cause of septicemia in racing greyhounds effects of vaccines on the canine immune system peracute haemorrhagic pneumonia syndrome in dogs deaths in racing greyhounds haemorrhagic streptococcal pneumonia in a dog experimental parvovirus infection in dogs characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs streptococcal infection in dogs: a retrospective study of 393 cases isolation of streptococcus zooepidemicus from three dogs in close contact with horses fatal case of toxic shock-like syndrome due to group c streptococcus associated with superantigen exotoxin streptococcus zooepidemicus (group c) pneumonia in a human identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep the radiographic resolution of streptococcus pneumonia pneumonia recovery: discrepancies in perspectives of the radiologist, physician and patient measuring symptomatic and functional recovery in patients with community-acquired pneumonia pulmonary infections in the athlete lower respiratory infections and potential complications in athletes submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to thank field veterinarian steinar dagestad for supplement information about the kenneled dogs. we would also like to thank the staff from the norwegian school of veterinary science including radiologists bernadette helmer & lena stenhaug and all the animal nurses for their intensive loving care. a warm thank to john debenham for language consulting and valuable comments. the authors declare that they have no competing interests.authors' contributions gj did the clinical examinations, drafting and revising the manuscript. . hks evaluated the radiographs initially and the controls. øk did the postmortem examination. bb and hs did the microbiology testing. hjsl and hs made substantial contribution to the discussion in the manuscript. all authors read, revised and approved the final version of the manuscript. key: cord-142389-t5swlp04 authors: linden, matthias; dehning, jonas; mohr, sebastian b.; mohring, jan; meyer-hermann, michael; pigeot, iris; schobel, anita; priesemann, viola title: the foreshadow of a second wave: an analysis of current covid-19 fatalities in germany date: 2020-10-12 journal: nan doi: nan sha: doc_id: 142389 cord_uid: t5swlp04 a second wave of sars-cov-2 is unfolding in dozens of countries. however, this second wave manifests itself strongly in new reported cases, but less in death counts compared to the first wave. over the past three months in germany, the reported cases increased by a factor five or more, whereas the death counts hardly grew. this discrepancy fueled speculations that the rise of reported cases would not reflect a second wave but only wider testing. we find that this apparent discrepancy can be explained to a large extent by the age structure of the infected, and predict a pronounced increase of death counts in the near future, as the spread once again expands into older age groups. to re-establish control, and to avoid the tipping point when tti capacity is exceeded, case numbers have to be lowered. otherwise the control of the spread and the protection of vulnerable people will require more restrictive measures latest when the hospital capacity is reached. a second wave of sars-cov-2 is unfolding in dozens of countries. compared to the first wave, this second wave manifests itself strongly in newly reported cases, but less so in death counts. however, already the increasing case numbers put the mitigation strategy at risk, because with case numbers being too high, local tipping points are crossed, which makes the control increasingly difficult, impedes the targeted protection of the vulnerable people, and leads to self-accelerating spread [1, 2] . importantly, a tipping point is reached when case numbers surpass the test-trace-and-isolate (tti) capacity of the local health authority, thus potentially long before hospitals are overwhelmed [1] : when tti breaks down, testing and tracing can only be carried out insufficiently and with considerable delay, which leads to more and more missed chains of infections. thereby increasingly more sars-cov-2 carriers remain unnoticed, and thus transmit the virus inadvertently -also to people at risk. as unaware carriers are strong drivers of the spread of sars-cov-2, the case numbers start to rise more quickly, and the spread spills over to the vulnerable population. in the following, we present evidence for a second wave and the crossing of this tipping point. in germany, as in many other countries, the emergence of the second wave has been questioned, because only the case numbers have been rising since july, while hospitals were not overwhelmed and death numbers stayed almost constant (fig. 1) . we investigated this apparent discrepancy using age-stratified case and death reports [3] , and an age-dependent infection fatality rate (ifr). the ifr is derived from a meta-analysis of seroprevalence studies and extensive pcr tracing programs [4] . approximately, the ifr increases by a factor of 10 for every 20 years of age, being about 10% at age 82. from this age-dependent ifr we predict the temporal evolution of the covid-19associated deaths by delaying each age group's observed weekly cases by two weeks and multiplying by the ifr (see supplementary material). (left) , and the number of predicted and actual covid-19-associated deaths are displayed for each age group (middle). the observed number deaths (black) in each age group matches well the predicted deaths calculated from the case numbers (color) using an age-dependent infection-fatality rate from a metaanalysis [4] . despite a rise in total reported cases since mid july (bottom), cases in the older age groups only started to rise in september (blue arrows). as a consequence, a rise in deaths is expected to unfold soon (right). the forecast scenarios a and b (gray, right column) predict a further increase in case numbers and deaths. the recent rise of infections in the older age groups together with the overall rise in cases clearly indicates a loss of control over the spreading and the start of a second wave. the temporal evolution of the predicted covid-19associated deaths (color) matches the actually observed ones (black) well in each age group (fig. 1, right) . even the absolute numbers match, suggesting a low fraction of unobserved sars-cov-2 infection chains. this agestratified analysis plausibly resolves the apparent discrepancy between the total reported infections and deaths: the rise in reported cases has been mainly driven by younger generations (younger than 60), who contribute only little to the absolute death counts, whereas the elderly (60+ years) managed to maintain low case numbers until recently. however, in september cases among the elderly started to increase steeply. this finding is not confounded by increased testing, because the test numbers in that age group stayed almost constant [3] . this spill-over to the elderly strongly suggests a failure to protect the vulnerable people due to an increased number of unaware sars-cov-2 carriers. moreover, this loss of control over the spread is also marked by the increase of the reproduction number r since begin of october (table i ). the spill-over and the increase of r are clear signatures of crossing the tipping point to uncontrolled spread. during summer 2020, the age groups 60+ apparently were protected well: if the virus had been spreading independently equally among all age groups, an estimated 1.3% to 2.0% of the infected would have died (calculated from the ifr in [4] ). however, the case fatality rate cfr was only 0.39% in august (table i) . now, with the strongly increasing number of infections among the elderly (fig. 1) , the cfr will most likely increase soon. this indicates that protecting the people at risk might be possible whilst case numbers are low, but likely fails under high covid-19 incidence. for the coming two weeks, the already reported cases clearly predict that the number of weekly deaths will almost double. beyond two weeks forecasts are difficult, because the death counts depend on the not-yet-reported cases. to account for this uncertainty, we display two future scenarios, where in (a) we assume a linear increase, and in (b) an exponential increase in cases (see supplementary material). the resulting forecasts range between 500 and 800 weekly deaths in early november, but case numbers might even grow faster in case the second wave fully unfolds. if the test-trace-and-isolate (tti) system is overwhelmed and stops to function as a powerful means of control, mitigation of the spread breaks down and the rise of case numbers accelerates [1] . thus, the current development in germany puts the past successful management of the pandemic seriously at risk. to re-establish control and to avoid crossing the tipping point when the tti capacity is exceeded, case numbers have to be lowered without further delay. otherwise the control of the spread and the protection of vulnerable people will inevitably require much more restrictive measures -at latest when the hospital capacity is reached. a. data sources for germany, the age-stratified data used in this document was retrieved from the national health agency, the robert koch institute (rki): both the number of reported sars-cov-2 cases and the number of deaths are available in age groups of ten years [3] , and the reported cases are also available with 5-year stratification up to the age of 80 from [5] . it was verified that the number of cases in the two sources matches. the time points of the case and death numbers correspond to their reporting dates, and not necessarily to the actual testing or date of death. the population age distribution of germany was retrieved from the united nation's database [6] . b. ifr calculation the overall goal is to estimate death numbers from past reported cases per age group and compare them to the observed number of deaths. we used an age-dependent infection-fatality rate (ifr) equation, following recent meta-studies [4, 7] : let a be the age of an infected person. then the infection fatality rate for this age is estimated as this equation formalizes that at 82 years, the ifr is 10% and decreases by a factor of 10 every 20 years. the age group 100+ is treated separately as 60%, in accordance with the supplementary calculations in [4] . in our analysis, we omit cases below the age of 20, as the weekly reported death numbers in that group are too low for any meaningful analysis. the equation (1) is very close to the published one [4] , but has been rounded to integers for simplicity. for comparison see table ii. as reported cases are available with 5-year stratification, equation (1) was evaluated centered in the respective age group. for instance, the ifr of the age group 40-44 is approximated as: one has to distinguish this ifr calculation from the cfr calculation which we calculate by simply dividing the number of deaths by the observed cases two weeks before, as discussed in more detail in paragraph h. c. estimating the number of deaths from the reported sars-cov-2 cases the number of deaths is estimated by multiplying the published weekly number of reported cases in 5-years-wide age groups by the associated ifr (equation (2)). to obtain the time point of the expected number of deaths, we delayed the number of reported cases by two weeks. this accounts for a one week delay between infection and case reporting, and a three weeks delay between infection and death. thus, the total delay of two weeks between reported case and death amounts to the difference between the infection-case and infectiondeath delay. in addition, two weeks matches a median delay of 10 days, which can be derived from individual case histories supplied by the rki's publicly available database [8] . an alternative approach would have been to take those case histories, infer a delay-distribution between reporting and death with a one-day temporal resolution and predict daily deaths. unfortunately, the age-groups of this dataset are between 20 and 25 years wide, compared to 5 year groups with weekly temporal resolution [5] . due to the strong age-specificity of the ifr we opted for smaller age-groups. the observed number of deaths is only available in 10-years-wide age groups. thus, to match the coarser stratification, we summed the corresponding two 5-yearswide age groups for all the plots. d. uncertainties of predictions our estimations features a number of uncertainties: the ifr probably decreased over the course of the pandemic because of better treatments. this leads to some overestimation of the ifr as reported by levin et al [4] , which uses data from early in the pandemic. a fraction of cases remains unobserved, and people at risk may protect themselves better than earlier in the pandemic. moreover, the assumed two-week delay between case detection and death is an approximation, the observed delay of individual cases features a wider distribution. overall however, the evidence indicates that in germany half or more of all cases are currently detected [9] . in relative numbers across age-groups, uncertainties are smaller, because systematic errors in the ifr and the delay approximations are expected to effect all age groups similarly. e. accounting for non-matching age groups if reported cases are not available with a 5-year stratification, but only coarser , we distribute the reported cases of the coarser age groups in 5-years-wide age groups, proportional to the population-age distribution of the respective country [6] . this approach is necessary for ages above 80 years for germany, where only 10-years-wide age groups are available: 80-89, 90-99 and 100+. f. prediction we consider two distinct scenarios for the future spread of the virus. scenario a is a linear extrapolation fitted to the reported cases in the respective age groups over the last 5 weeks, which we chose since linear growth can reflect the local and heterogeneous spreading of sars-cov-2. furthermore, scenario b is an exponential forecast using the reproduction rate r 0 = 1.2 given by the rki [3] in early october and represents a worser scenario. for the forecast, the corresponding weekly increase of 37.5% is applied to the latest week of age-stratified case numbers. g. effective infection fatality rate the effective infection fatality rate ifr eff is defined as the infection fatality rate given the age distribution of reported cases. it is calculated by weighting the ifr of the age groups (eq. (2)) by the number of current reported cases. this approach to calculate the effective ifr accounts for varying case distributions within the age groups, which therefore leads to a time-dependence of the effective ifr. note that this ifr eff is not a case-fatality rate, because only the age distribution of reported cases is used to weight the ifr(a) in each age group appropriately; this is different from the case-fatality rate, which corresponds to the ratio of observed deaths to reported cases. we used this definition of an effective ifr to calculate the effective ifr for germany assuming eq. (2) and a case distribution proportional to the population's age distribution: ifr de mix = 1.83. with equal spread across all age groups, 65% of deaths would originate from the 80+ age group (tabl. ii). with values from [4] for the age-specific ifr (instead of eq. (2) h. case fatality rate in order to compare the theoretical effective infection fatality rate to the actual reported deaths, we calculated a case fatality rate. the approach was to divide the number reported deaths occurring two weeks later by the reported cases. this doesn't take into account the wide distribution of delays between infections and deaths. we mitigate the errors introduced by this simple assumption by using at least 2 weeks of data (tab. i). i. testing the steep increase in the number of cases throughout all age-groups starting in october is not driven by increased testing activity: between the end of june and the end of september, the rate of positive tests stayed below 1% except for the age group 15-34, which stayed between 1-1.5% [10] . until mid-september, it even remained below 0.5% in the age groups 60-79 and 80+. in conjunction with a consistently high number of tests of 600-700 per 100000 in the age group 80+, well above the 300-400 tests per 100000 in the other groups, we assume adequate coverage of that age group, and as a result a constant low number of undetected cases. the twofold increase in overall testing from beginning to end of august, 500000 to 1 million tests per week [3] , was driven by tests in the age groups < 60, temporarily lowering the fraction of positive tests in those groups until mid-september. from then on the number of tests continues to be around 1 million tests per week, leading to our conclusion that the increase of cases since september is not driven by increased test activity. [4] evaluated at the center of the age group alongside 95% confidence interval bounds. the second column list the ifr values used in the analysis, which are calculated using eq. (2). the right half displays the predicted and observed percentage of deaths in each age group, cumulatively summing over all preceding age groups. for the estimated percentage for germany, the age-specific ifr from the 3rd column is multiplied with the fraction of population in the age group. the observation refers to deaths in weeks 31-40 [3] . the observed fraction of deaths was almost twice as large as the estimated one in the age group up to 60 years (last two columns), reflecting that the virus spread more among the younger people than expected. these columns also show that 5.23 % (10.1%) of all deaths are expected among the age group up to 60, thus almost 95 % (90 %) of all deaths are expected to originate from the age groups 60+ assuming age-independent (or age-dependent) spreading. eine zweite welle von sars-cov-2 breitet sich in dutzenden von ländern aus. die zweite welle zeigt sich zwar stark in neu gemeldeten fällen, aber im vergleich zur ersten welle weniger in der zahl der todesfälle. jedoch gefährden bereits diese steigenden fallzahlen die eindämmung von covid-19, denn bei zu hohen fallzahlen werden lokal kipppunkteüberschritten, was die kontrolle erschwert, den gezielten schutz von risikogruppen behindert und zu einer selbstbeschleunigenden ausbreitung führt [1, 2] . ein kipppunkt wird erreicht, wenn die täglichen neuinfektionen die kapazität einer lokalen gesundheitsbehördeübersteigen, so dass sie nicht mehr effektiv testen und kontakte nachverfolgen können (tti, test-traceisolate). diese situation kann eintreten lange bevor die krankenhäuserüberlastet sind, und ist daher besonders kritisch [1] : wenn das tti zusammenbricht, können tests und rückverfolgung nur unzureichend oder mit großen verzögerungen durchgeführt werden, wodurch infektionsketten zunehmend nicht erkannt werden. dadurch bleiben mehr sars-cov-2-träger unentdeckt und können das virus unbeabsichtigterweiseübertragen -auch auf risikogruppen. da die unentdeckten träger die ausbreitung entscheidend vorantreiben, steigen die fallzahlen immer schneller und ansteckungen breiten sich auch in vorher effektiv geschützten risikogruppen aus. im folgenden präsentieren wir hinweise für eine zweite welle und dië uberschreitung dieses kipppunktes. in deutschland, wie in vielen anderen ländern, ist die entstehung einer zweiten welle in frage gestellt worden, da nur die fallzahlen seit juli gestiegen sind, während die krankenhäuser nichtüberlastet waren und die todesfälle nahezu konstant blieben (abb. 2). wir untersuchten diese offensichtliche diskrepanz anhand von altersstratifiziertn fall-und sterbezahlen [3] und einer altersabhängigen infektionssterblichkeitsrate (ifr, infection fatality rate). die ifr basiert auf einer metaanalyse von seroprävalenzstudien und umfangreichen pcr-tracing-programmen [4] . die ifr steigt für jedes 20. lebensjahr etwa um den faktor und beträgt im alter von 82 jahren rund 10%. aus dieser altersabhängigen ifr prognostizieren wir die zeitliche entwicklung der covid-19-assoziierten todesfälle, indem wir die wöchentlich beobachteten fälle jeder altersgruppe um zwei wochen verzögern und mit der entsprechenden ifr multiplizieren. weitere informationen und ergebnisse finden sich in den "supplementary material". der zeitliche verlauf der vorhergesagten covid-19assoziierten todesfälle (farbig) stimmt mit den tatsächlich beobachteten (schwarz) in jeder altersgruppe gutüberein (abb. 2, rechts). selbst die absoluten zahlen stimmen uberein, was auf einen niedrigen anteil unbeobachteter sars-cov-2-infektionsketten hindeutet. unsere altersstratifizierte analyse löst die scheinbare diskrepanz zwischen den insgesamt gemeldeten infektionen und todesfällen plausibel auf: der anstieg der fig. 2. die anzahl der wöchentlichen covid-19-fälle (links) sowie die anzahl der prognostizierten und beobachteten covid-19-assoziierten todesfälle werden für jede altersgruppe (mitte) angezeigt. die vorhersage der anzahl der todesfälle anhand der fallzahlen (farbig) stimmt mit den gemeldeten todesfällen (schwarz) in jeder altersgruppe guẗ uberein, wobei eine altersabhängige infektionsfatalitätsrate basierend auf einer meta-analyse [4] zugrunde gelegt wurde. trotz eines anstiegs der gesamtzahl der gemeldeten fälle seit mitte juli (unten) stiegen die fälle in denälteren altersgruppen erst seit september an (blaue pfeile). infolgedessen erwarten wir, dass bald ein deutlicher anstieg der todesfälle beobachtet werden kann. die prognoseszenarien a und b (grau, rechte spalte) sagen einen weiteren anstieg der fallzahlen und todesfälle voraus. der jüngste anstieg der infektionen in denälteren altersgruppen zusammen mit dem gesamtanstieg der fälle weist eindeutig auf einen kontrollverlustüber die ausbreitung und den beginn einer zweiten welle hin gemeldeten fälle wurde hauptsächlich von den jüngeren generationen (jünger als 60 jahre) verursacht, die nur wenig zu den absoluten todesfällen beitragen, während es bei denälteren menschen (60+ jahre) bis vor kurzem gelang, die fallzahlen niedrig zu halten. im september stiegen die fälle beiälteren menschen jedoch steil an. diese beobachtung lässt sich nicht auf die zunahme der durchgeführten tests zurückführen, da die anzahl der tests in dieser altersgruppe nahezu konstant blieb [3] . diesesüberschwappen aufältere menschen legt nahe, dass es aufgrund der wachsenden zahl unbemerkter sars-cov-2 infizierter nicht gelungen ist, die risikogruppen zu schützen. darüber hinaus ist dieser kontrollverlust seit anfang oktober auch durch einen anstieg der reproduktionszahl r gekennzeichnet (tabelle iii). das uberschwappen auf dieältere generation und die zunahme in r sind beides klare zeichen dafür, dass der kipppunkt zur unkontrollierten ausbreitungüberschritten wurde. im sommer 2020 waren die altersgruppen 60+ offenbar gut geschützt: hätte sich das virus unabhängig und gleichmäßigüber alle altersgruppen verbreitet, wären schätzungsweise 1,3% bis 2,0% der infizierten gestorben, wenn man von der in [4] berichteten ifr ausgeht. die todesfallrate (cfr, case fatality rate) lag im august jedoch nur bei 0,39% (tabelle iii) . jetzt, mit der stark zunehmenden anzahl der infektionen unterälteren menschen (abb. 2), wird die cfr höchstwahrscheinlich bald ansteigen. dies weist darauf hin, dass ein schutz der risikopersonen möglich ist, solange die fallzahlen niedrig sind, aber wahrscheinlich bei hoher covid-19-inzidenz scheitert. für die kommenden zwei wochen kann alleine aufgrund der bereits gemeldeten fälle der letzten zwei wochen vorhergesagt werden, dass sich die zahl der wöchentlichen todesfälle fast verdoppeln wird. prognosenüber mehr als zwei wochen hinaus gestalten sich als schwierig, da die zahl der todesfälle von den noch nicht gemeldeten fällen abhängt. um diese unsicherheit einzugrenzen, setzen wir zwei zukunftsszenarien an, wobei wir in (a) einen linearen anstieg und in (b) einen exponentiellen anstieg der fälle annehmen (methoden in den "supplementary material"). die sich daraus ergebenden vorhersagen bewegen sich zwischen 500 und 800 wöchentlichen todesfällen anfang november. die fallzahlen könnten jedoch noch schneller ansteigen, falls sich die zweite welle voll entfaltet. wenn die kapazitätsgrenze für testung und kontaktnachverfolgung (tti)überschritten ist, brechen die eindämmungsmaßnahmen dieses kontrollsystems zusammen und der anstieg der fallzahlen beschleunigt sich [1] . die aktuelle entwicklung in deutschland bringt somit die in der vergangenheit erfolgreiche bewältigung der pandemie ernsthaft in gefahr. um die kontrolle wieder zu erlangen und um einüberschreiten des kipppunktes beiüberlastung der tti-kapazität zu vermeiden, müssen die fallzahlen unverzüglich gesenkt werden. andernfalls werden die eindämmung der ausbreitung und der schutz der risikogruppen zwangsläufig sehr viel restriktivere maßnahmen erfordern -spätestens wenn die krankenhauskapazität erreicht ist. the challenges of containing sars-cov-2 via test-trace-andisolate together we can do it: each individual contribution protects health, society, and the economy rki tuesdays' situation reports assessing the age specificity of infection fatality rates for covid-19: systematic review, meta-analysis, and public policy implications. medrxiv survstat@rki 2.0 api united nations world population database assessing the age-and gender-dependence of the severity and case fatality rates of covid-19 disease in spain rki) dl-de/by 2-0. arcgis database of the rki's covid-19 dashboard reconstructing the early global dynamics of under-ascertained covid-19 cases and infections rki laborbasierte surveillance sars-cov-2 report of 2020 key: cord-272909-n6wfe1of authors: peng, shu; huang, liu; zhao, bo; zhou, shuchang; braithwaite, irene; zhang, ni; fu, xiangning title: clinical course of coronavirus disease 2019 in 11 patients after thoracic surgery and challenges in diagnosis date: 2020-04-10 journal: j thorac cardiovasc surg doi: 10.1016/j.jtcvs.2020.04.005 sha: doc_id: 272909 cord_uid: n6wfe1of objectives: to illustrate the clinical course and difficulties in early diagnosis of coronavirus disease 2019 (covid-19) in patients after thoracic surgery. methods: we retrospectively analyzed the clinical course of the first 11 patients diagnosed with covid-19 after thoracic surgery in early january 2020. postoperative clinical, laboratory, and radiologic records and the time line of clinical course were summarized. potential prognostic factors were evaluated. results: in the 11 confirmed cases (3 female, 8 male), median days from symptom onset to case detection was 8. insidious symptom onset and misinterpreted postoperative changes on chest computed tomography (ct) resulted in delay in diagnosis. there were 3 fatalities due to respiratory failure, whereas 4 severe and 4 mild cases recovered and were discharged. all patients had once experienced leukocytosis and eosinopenia. remittent fever and resected lung segments ≥5 were associated with fatality. conclusions: the case fatality rate of postsurgical patients subsequently diagnosed with covid-19 was 27.3%. insidious symptom onset, postoperative leukocytosis with lymphopenia, and postsurgical ct changes overshadowed the early signs of viral pneumonia. dynamic symptom monitoring, serial chest cts, and tests for viral rna and serum antibody improve the chance for prompt detection of covid-19. consideration should be given to preadmission and preoperative screening and strict contact isolation during the postoperative period. the early signs of covid-19 were disguised in 11 postoperative thoracic patients, resulting in a 27.3% fatality rate. the pandemic of coronavirus disease 2019 (covid-19) caused the cancelation of elective surgeries and clinics in many regions in china. however, emergency surgeries continue to be performed, and urgent surgeries in cancer treatment cannot be postponed indefinitely. since severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is infectious in asymptomatic carriers with a variable incubation period (0-24 days), 1 the recognition of infection in clinics and wards is important for physicians of all specialties. although 80% of patients present with apparently mild respiratory infection, covid-19 has a case fatality rate of approximately 2%. [2] [3] [4] thoracic malignancy may also present with mild respiratory symptoms. in addition, after thoracic surgery patients may also have cough, dyspnea, reactive fever, and postoperative radiologic changes. these postoperative changes may overshadow the symptoms of covid-19. without sufficient experience and precaution, prompt diagnosis of covid-19 may be delayed and nosocomial transmission may occur. this study describes the clinical course of 11 patients who underwent thoracic surgery for malignancy in the early phase of the pandemic before the formal declaration of outbreak and were consequently diagnosed with covid-19. we illustrate the risk of thoracic surgery during this pandemic. to be considered as a possible case, a patient needed to have been admitted for and undergone thoracic surgery at the department of thoracic surgery at the central campus of tongji hospital, tongji medical college, huazhong university of science and technology between january 1 and january 24, 2020. all postsurgical patients who tested positive for covid-19 by real-time reverse transcriptase-polymerase chain reaction (rt-pcr) were eligible for inclusion in the case series. cases were classified as nonsevere, severe, or critical according to the clinical guideline of the china national health commission. 5 patients were defined as "critical" if they ever met any of the following criteria: respiratory failure requiring mechanical ventilation, septic shock, or multiple organ dysfunction in the intensive care unit. patients were defined as "severe" when suffering from either dyspnea with respiratory rate !30/minute, or partial pressure of arterial oxygen/percentage of inspired oxygen ratio <300 mm hg or blood oxygen saturation 93%, or lung infiltrates >50% within 24 to 48 hours. the remaining cases were defined as "nonsevere." regardless of consequent recovery, cases were classified by the most severe criteria they ever met during the postsurgical course. positive exposure history was defined as a history of close contact with family members suspicious for covid-19 infection, or if a patient sharing their ward had confirmed covid-19. fever was defined as an axillary temperature more than 37.3 c. a putative case was defined as clinical suspicion of covid-19 resulting isolation of patient before rt-pcr detection of viral rna. delay in diagnosis means covid-19 was not initially suspected due to insidious onset of symptoms that mimicked the usual postoperative course, or due to the first computed tomography (ct) scan of the chest after surgery being interpreted as postoperative changes rather than as being suspicious for viral pneumonia. the case id number (1-11) represents a specific patient and is used to identify that same patient consistently in all figures, tables, and text. demographics, comorbidities, and clinical documents were collected from the hospital information system. for accuracy, 2 researchers independently recorded data using a standardized data-collection form. first symptom and onset of disease was derived from medical records, temperature charts, patient recall, and recall of their primary care surgeons. possible time of exposure was determined through contact tracing, which included evaluation of patient recall, duration of patient placement within beds and wards, co-location with other patients ultimately diagnosed with covid-19, and information about families and visitors during their hospitalization. ct images of the chest were analyzed and recorded by 1 radiologist and 1 thoracic surgeon independently. researchers were not involved in collection of biological samples for rt-pcr, and setting of ct parameters in this retrospective analysis, protocol for rt-pcr 6 and chest ct 7 were same to as that in the reference article from tongji hospital. all patients had perioperative education, including breathing exercises and "intention to cough after operation" once every 2 hours during the day to improve lung expansion. as we experienced rapidly changing patient outcomes in the midst of a pandemic, our treatment modalities also changed rapidly, including an initial period in which patients received prophylactic antibiotic and ambroxol after the case id 1 being diagnosed as covid-19, a practice that was later stopped. as our experience with and our understanding of covid-19 increased, we standardized our approach to initiate oral oseltamivir and moxifloxacin and a ct of the chest immediately after the first onset of fever. rt-pcr tests were requested if a ct of the chest showed characteristics consistent with viral pneumonia. rectal diclofenac (25 mg, every 6 hours) was used to control pain and fever. patients were transferred to isolation wards as soon as covid-19 was suspected. all patients received supportive care with oxygen supplementation regardless of their oxygen saturations. information regarding nonspecific antivirals, corticosteroid and mechanical ventilation are shown in table e1 . ethical application was made to and approved by the institutional review board of tongji hospital, tongji medical college, huazhong university of science and technology (tj-irb20200307; date of approval: march 3, 2020). consent from the covid-19 cases analyzed in this report was waived due to its retrospective nature and that data would be deidentified at publication. median and range or mean ae standard deviation was used to describe continuous data. single comparisons were performed by independent samples t test. normality was tested by the shapiro-wilk test. categorical variables were assessed by fisher exact test to evaluate associations between case fatality and surgical, laboratory, and clinical features. all analysis was computed by ibm spss statistics (version 24; ibm corp, armonk, ny). in total, 205 patients were admitted to the surgical unit between january 1 and january 24, 2020. on january 24, all nonemergent surgeries were cancelled as the pandemic nature of covid-19 was realized. therefore, 84 patients were consequently discharged home without surgical intervention. of the 121 patients who had already been operated covid-19 ¼ coronavirus disease 2019 ct ¼ computed tomography ggo ¼ ground-glass opacity rt-pcr ¼ real-time reverse transcriptasepolymerase chain reaction sars-cov-2 ¼ severe acute respiratory syndrome coronavirus 2 on, 13 developed postoperative fever and were considered putative cases of covid-19, of whom 11 had covid-19 confirmed by rt-pcr and were included in this case series. there was 1 patient who developed fever and died at home without autopsy who was not included in this case series due to uncertainty of cause of death. unknown to the admitting staff, and before the pandemic nature of covid-19 being recognized, 10 patients had postoperative positive exposure history to covid-19 ( figure 1 ). evidence of nosocomial transmission is apparent in the majority. the date of first exposure in case id 9 is uncertain, since her husband had covid-19 diagnosed before her onset of symptoms. demographics, comorbidities, and pathology of neoplasm are shown in table 1 . in general, no one had impaired liver and renal function preoperatively, and 9 patients had thoracic malignancies. all had undergone radical tumor resection within 1 month of developing symptoms consistent with covid-19 infection. duration from possible postoperative exposure to onset of symptom ranges from 1 to 15 days ( figure 1 ). events from surgery to diagnosis of covid-19 are shown in figure 1 . median days from surgery to putative covid-19 infection was 13 (range 1-31 days), to positive rt-pcr test was 17 (range 4-40 days), to death was 35 (range 5-42 days, n ¼ 3), and to discharge upon recovery was 50 days (38-72 days, n ¼ 8). of the 11 confirmed cases, symptom onset included dry cough (1/11), dyspnea (2/ 11), and fever (8/11). there were 3 cases in which infection was not detected at their insidious onset of symptoms but only after fever developed and ct of the chest was done. case id 9 had dry chough after resuscitation with tracheal intubation on the day of operation due to hypokalemia with sudden cardiac arrest. she was discharged home 12 days postoperatively, and her infection was detected after another 12 days by chest ct after her husband had confirmed covid-19 infection. case id 7 had a history of bronchial asthma and became dyspneic 4 days after right upper lobectomy. a ct was taken 7 days later after onset of fever at which time covid-19 was putative. − 24 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 all patients had ct of the chest before and after surgery. of the 11 cases, case id 1 and id 11 had preoperative ground-glass opacifications (ggos). case 11 had multiple ggos proven as synchronous multiple primary lung adenocarcinoma on pathology (stage ia and stage ib, tables 1 and e2). case id 1 (figure 2 ) was asymptomatic during the 3 weeks prior to operation with a normal leucocyte and lymphocyte count; he had abrupt fever within 12 hours after operation, followed by rapid deterioration and death within 5 days. on retrospective analyzes of his chest ct 23 days prior to operation, we revealed subpleural illdefined ggo in the right lower lobe with increased extent and intensity 9 day later ( figure 2 ). of the 10 cases deemed to have had postoperative exposure (except for case id 1), 6 (case ids 4, 6, 7, 8, 9, 11) were diagnosed putatively after the first postoperative chest cts in which the following features were noted: peripheral or subpleural distribution, multifocal located ill-defined ggo, sign of "crazy-paving," bronchial wall thickening, and irregular consolidations that were rapidly increasing in extent and intensity ( figure 2 ). the first postoperative cts of the chest of another 4 patients (id 2, 3, 5, 10) were initially interpreted as having postoperative changes but were later realized to be indicative of covid-19. these ct scans contained the following features: cord-like consolidation or opacity, irregular patchy consolidation, irregular ggo with diameter less than 10 mm, subpleural consolidations, and reticular changes ( figure 3) . at the last follow-up on march 27 2020, 3 critical cases had died (figure 1, ids 1, 2, 3) , and 4 severe (ids 4, 5, 6, 7) and 4 mild cases (ids 8, 9, 10, 11) were discharged upon recovery. clinical and laboratory features are shown in table 2 , and detailed information of each patient is listed in table e1 . the median of symptom-onset days after operation was 3 (range 1-30 days). in general, all patients had fever and dyspnea. the 3 critical cases had remittent fever until death. all patents had normal complete blood count and clinical biochemistry preoperatively, with leukocytosis or lymphopenia at day 1 or day 3 postoperatively followed by eosinopenia. the 3 critical cases had an absolute eosinophil count approximating 0 (3 10 9 /l) when arterial oxygen saturation dropped below 93%. in summary, leukopenia occurred in 2 cases (18.2%), 8 patients had elevated liver enzymes (72.7%), and all patients had hypoproteinemia, hypoalbuminemia, and elevated lactate dehydrogenase. factors potentially associated with fatal cases (n ¼ 3) are shown in table 3 . none of the preoperative factors was associated with death. however, resected lung volume (!5 segments), persistent fever, and postoperative reduction of total protein and albumin were significantly associated with fatality (p < .05). increase in lactate dehydrogenase and liver enzymes also showed marginal association with death (p ¼ .05, p ¼ .055, respectively). across the covid-19 cohort in china, the proportion of severe and critical disease was 2% to 6.1%, and 13.8%, respectively, 2-4 and was 27.3% and 36.4%, respectively, after thoracic surgery in this case series. it appears that sars-cov-2 may significantly increase the risk of death during the thoracic postoperative period. in addition, the virus is contagious during the incubation period, which may lead to nosocomial transmission even within well-organized hospitals with strict infection control procedures. the overlap between symptoms of covid-19 and the usual thoracic postoperative clinical course may result in delayed diagnosis: fever and cough from reactive pleural effusion and atelectasis are common after thoracic surgery 8 ; dyspnea may be secondary to lung resection and chronic obstructive lung disease; many thoracic postoperative patients suffer chest tightness and fatigue, loss of appetite, and nausea due to postoperative analgesics; and antibiotics are usually self-limiting. therefore, in this pandemic, thoracic surgeons must make their patient rounds with greater caution to distinguish infection from noninfectious symptoms, perhaps to assume infection by default. as a result of our experiences, we have provided hospitalized patients with a daily self-checking form to record possible symptoms with onset, duration, severity, and dynamic changes to help early detection of infection. case fatality in this study was significantly associated with resected lung segments (!5), severity of postoperative hypoproteinemia or hypoalbuminemia, and peak value of lactate dehydrogenase. we cannot determine causality, nor control for other factors, including comorbidities and surgery. case id 1 with a smoking index of 1600 had an asymptomatic period for 3 weeks and had onset of fever within 12 hours after operation. whether this was due to a long period of incubation (known to be up to 24 days) or exacerbated by the stress of the surgical procedure cannot be assessed. case id 2 had history of colon cancer, marginal pulmonary function test (forced expiratory volume in 1 second 1.39 l/s, forced expiratory volume in 1 second/forced vital capacity 70.1%), and a smoking index of 1600. case id 3 had arterial hypertension (stage i, high risk), left ventricular dilatation (ejection fraction: 63%), and coronary artery atherosclerosis (coronary artery disease reporting and data system 1). of the 3 fatal cases, matched analysis over a much larger sample size is required to answer whether surgery or types of surgery increase morbidity and mortality in patients with covid-19. we observed leukocytosis with lymphopenia in 10 cases and leukopenia in 2 cases (18.2%), which differs from previously reported covid-19 cases with an incidence of 5.9%, 33.7%, and 70%, respectively. 9 leukocytosis after major surgeries is physiological in the early postoperative period, and in other postoperative settings, lymphopenia can occur in 22% to 35.6% patients within 7 days. 10, 11 thoracic surgery is also associated with lymphocyte reduction. 12 these nonspecific postoperative laboratory changes make detection of infection on these parameters alone extremely challenging. nonetheless, persisting abnormal white cell counts for more than 1 week is infrequent postoperatively but is common in this case series of covid-19. in addition, we observed an impressive reduction of lymphocyte and eosinophil counts in all cases, suggesting their prognostic value should be explored in future studies with larger sample sizes. in this case series, the average time from symptom onset to rt-pcr-confirmed diagnosis was about 2 weeks, a time frame unsuitable for hospital practice, which is highly dependent on rapid diagnosis and isolation of cases in hospitalized patients for infection control. serum antibody detection has better reported sensitivity (88.66%) and specificity (90.63%) 13 ; however, the time to their detection remains uncertain. we know that immunoglobulin m became detectable 3 to 6 days after severe acute respiratory syndrome infection, 14 and if this is similar in the case of covid-19 such delays will remain challenging in the hospital setting. ct scan shows small irregular ggo in the right upper lobe and subpleural cord-like consolidation (c), which were not rare as postoperative reactive change. after 8 days, ct shows typical signs of viral pneumonia (g): diffuse ground glass opacifications with "paving stone" signs and, irregular subpleural cord-like consolidation. d, a 68-year-old male patient (case id 2) 12 days after right lower lobectomy. chest ct (d) shows pneumothorax, subcutaneous emphysema, postoperative changes, and inflammation around chest drain. after 18 days, he had sudden onset of fever and ct scan revealed (h) multiple diffuse ggos in the lung peripheries with reticular consolidation. postoperative changes from thoracic surgery may overshadow early ct signs of covid-19 infection. through retrospective analysis, we found 5 cases of misinterpreted ct of the chest (1 preoperatively, and 4 postoperatively), which had rapid progression of ggos on repeated ct scans indicative of active covid-19 infection. one had ill-defined peripheral ggo with interstitial pneumonia before operation (figure 2 ). the remaining 4 had irregular patchy or cord-like consolidation, small irregular ggo and reticular areas of increased opacity and were misinterpreted as postoperative reactive changes, which later on turned became clearly consistent with active viral pneumonia ( figure 3 ). hence, a ct scan at a single time point cannot exclude suspicious sars-cov-2 infection; rather, repeated ct scans during the postoperative course can assist in diagnosis. during the outbreak, we had to continue emergent surgeries under the risk of covid-19. as a result of our experience we increased our precautions, replacing postoperative chest radiographs with ct scans and ordering covid-19 rt-pcr and serum antibody tests for patients with fever, cough, dyspnea, lymphopenia, eosinopenia, or atypical signs of inflammation on chest ct. although a limited sample size, our experience suggests that physicians should be aware of a possible greater risk of death in patients with covid-19 who have !5 lung segments resected and persistent severe hypoproteinemia, hypoalbuminemia, and elevated liver enzymes after thoracic surgeries. in general, patients infected with covid-19 who underwent recent thoracic surgery were found to have greaterthan-anticipated incidence of severe illness (36.4%) and fatality rate (27.3%). as a result of this, nonemergent surgeries were postponed locally, including stage i thoracic malignancies (except for small cell cancer and low differentiation cancer). case detection in the incubation period remains challenging both pre-and postoperatively. similarities between the usual thoracic postoperative course and symptoms of covid-19 viral pneumonia may cause delays in diagnosis. in our experience, if operating in this pandemic environment for any cause, all patients should undergo the following management regimen: a preadmission ct of the chest, oropharyngeal swab, and serum antibody test for covid-19; if negative, quarantine in a hospital ward for 1 week should be implemented; a second oropharyngeal swab and serum antibody test for covid-19 should then be performed; if both tests are negative, the patient can undergo the planned operation. contact restriction is required for hospitalized patients. postoperative patients with symptoms or known history of exposure to covid-19 should be quarantined and monitored dynamically for symptoms and laboratory abnormalities by repeat cts of the chest postoperatively. there are some methodologic limitations to this case series. due to the retrospective nature of this analysis, the presumed date of exposure and symptom onset from medical records and personal statements may be affected by recall bias. the baseline characteristic of the patient varied with respect to underlying comorbidities and preoperative pulmonary lung function; additionally, the different types of the early signs of covid-19 were disguised in 11 postoperative thoracic patients, resulting in a 27.3% fatality rate. the journal of thoracic and cardiovascular surgery c volume -, number presumed asymptomatic carrier transmission of covid-19 full spectrum of covid-19 severity still being depicted epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention chinese clinical guidance for covid-19 pneumonia diagnosis and treatment clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases does atelectasis cause fever after surgery? putting a damper on dogma clinical features of patients infected with 2019 novel coronavirus in wuhan lymphopenia at 4 days postoperatively is the most significant laboratory marker for early detection of surgical site infection following posterior lumbar instrumentation surgery association of preoperative cell counts with outcomes after operation for congenital heart disease the role of lymphopenia in the development and severity of postoperative pulmonary complications after lung surgery development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis production of specific antibodies against sars-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229e and oc43 key words: covid-19, surgery, lung cancer, esophageal cancer, postoperative, sars-cov-2 authors have nothing to disclose with regard to commercial support. the journal of thoracic and cardiovascular surgery c -2020 key: cord-277563-rc88vn6e authors: merrin, jack title: differences in power-law growth over time and indicators of covid-19 pandemic progression worldwide date: 2020-04-02 journal: nan doi: 10.1101/2020.03.31.20048827 sha: doc_id: 277563 cord_uid: rc88vn6e an automated statistical and error analysis of 45 countries or regions with more than 1000 cases of covid-19 as of march 28, 2020, has been performed. this study reveals differences in the rate of disease spreading rate over time in different countries. this survey observes that most countries undergo a beginning exponential growth phase, which transitions into a power-law phase, as recently suggested by ziff and ziff. tracking indicators of growth, such as the power-law exponent, are a good indication of the relative danger different countries are in and show when social measures are effective towards slowing the spread. the data compiled here are usefully synthesizing a global picture, identifying country to country variation in spreading, and identifying countries most at risk. this analysis may factor into how best to track the effectiveness of social distancing policies and quarantines in real-time as data is updated each day. mathematics is essential to predict and control the course of a pandemic. one approach is mathematical modeling [1] . many epidemiological models derive from the sir (susceptible, infected, recovered) model [2] or some more sophisticated versions of it. if there are susceptible, infected, and recovered (or removed individuals), then there are standard shaped curves and predictions you can make based on differential equations, provided you exactly know the transmission rate and recovery rate. these parameters may only be possible to determine from a retrospective analysis. one parameter is r 0 , the basic reproduction number, which represents the number of people an infected person typically can infect over their progression. clearly, if r 0 is much larger than 1, then there is a significant initial exponential growth of cases that sweep through most of the population. china and south korea have mostly blocked the spread of covid-19 by enacting social policies and testing so r 0 can definitely be reduced or modulated. the sir equations are also entirely deterministic, while in real systems, there is some stochastic element of variation. sir equations do not have an analytical solution so they are more difficult to solve, and determining the fitting parameters requires sophisticated statistics. usually for an epidemic like the flu everything stays open so the parameters are more or less the same over the entire trajectory of the analysis. things start to become very complicated if different countries enact different social policies over time with different degrees of strengths. sir model parameter themselves would then become functions of time complicating the analysis. spreading the virus may also depend on differences in geography, social networks, social dynamics, or individual behaviors. another approach is not to have any mechanistic mathematical model at all, but to use methods of error analysis [5, 6, 7, 8] . one can fit the data to different phenomenological functions observed in the past, such as what happened in china over the last several months to make comparisons. in this study, we attempt to analyze differences in the spread of covid-19 in many countries where growth is rapid to 1000 cases or more as of march 28, 2020. we use the least-squares curve fitting of power-laws and introduce the danger indicator to compare the rate of spread in various countries. both indicators reveal similar information in the current stage of the pandemic. the data analysis follows the proposal of ziff and ziff [3] , where an early stage involves exponential growth, and a middle stage exists where there is power-law growth. the power-law exponents allow us to make reliable short term predictions of a few days of the worst-case scenarios in hot spot countries compared to that of the usual assumption of exponential growth. timescales for mitigation may be three weeks or more and can be tracked by this analysis. these predictions and indicators may be relevant for estimating the burden on medical infrastructure or influence policy decision making before countries enter mitigation or rather the virus completely sweeps through. of all countries so far, only china and south korea have achieved mitigation compared to other countries where there is still significant power-law growth. this analysis identifies some countries which are in a highly critical situation with expectations of more positive cases and deaths than occurred in china, while others have quite some time to achieve a resolution. in this paper, we display the number of measured positive cases (data set who [9]) as a function of time on plots where the x and y variables are both logarithmically scaled. this is referred to as a log-log plot. we will try to distinguish between exponential growth and power law growth. here we show exponential growth on a log-log plot appears like an exponential curve on a linear plot. here we show power law growth on a log-log plot appears linear. increasing power law functions on a log-log plot have positive slope. there can be a high degree of overlap with the power law growth and exponential growth in the middle region, but the long term behaviours are different as shown in figure 1. in this paper, we use error analysis [5, 6, 7, 8] and curve fitting to analyse the progression of positive cases. in weighted least squares curve fitting, one finds the parameters which minimise the chi squared function. as we saw before, a power-law function can be transformed into a linear function on a log-log graph. there is always an analytical solution to the curve fit of a straight line and thus we can determine α, β, and their uncertainties quite objectively in a standard and simple way. the transformation is with a = ln α and b = β. then, all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. if we want to fit data to a straight line, then we need the coordinates of the statistics x i , the measured values y i , and the uncertainties in the values σ i . then we have for chi-squared. in our cases, x i is the number of days of the progression of the disease since 100 cases. y i is the reported positive tested cases at x i . you cannot repeat the measurement, so we have to estimate the typical uncertainty. we have to use the square root n rule. for example, if there are 100 cases, then the uncertainty would be expected to be about 10 cases with a relative uncertainty of 10 percent. if there are 10,000 cases, then the uncertainty would be expected to be about 100 cases with a relative uncertainty of 1 percent. the relative uncertainty is given by more recent points should be weighted more strongly because their relative error is less. a more exact curve fit incorporates the use of error bars. to solve for a and b you calculate ∂χ 2 ∂a = 0 and ∂χ 2 ∂b = 0 and solve the resulting system of linear equations. we use the notation, the uncertainties in the parameters can also be directly computed using propagation of error. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint if a statistic and its uncertainty are known as x ± σ x then we can transform x into f (x) and the new uncertainty is given by propagation of error. of course, β = b and the uncertainty is obtained directly from the linear curve fit. we also used the fact that y = ln y i so it was also attempted to do an unweighted fit with the following formulas [10], but there is not much difference in beta for the two methods. see the table at the end of the results. the weighted fit is preferable to compute the error bar in beta. in the future, it may also be attempted to perform a maximum likelihood estimation of the power law exponent since there are some caveats to the weighted method presented here, such as assuming no uncertainty in the x coordinate [11] . another alternative is to use a software like kaleidagraph, which does a nonlinear least squares analysis based on the levenberg-marquardt method. using a software package like that is cumbersome to deal with all the datasets for each time window as described in the next subsection. also since you didn't write the program, it is not clear that you understand what was computed is right. the weighted linear least squares method is considered sufficient for the time being and works very well automatically in matlab scripts and produces the uncertainty in beta. one last detail is that the data must be linear on a log-log plot, or the points that don't match are outlying. outliers skew the values of α and β and hinder any attempts to make accurate predictions. the initial phase of the data is not power-law, so we restrict ourselves to measuring the last 7 days as a measure of progress in a country. a moving window has the added benefit that you can have a moving window of seven days to see the progress over time as we move the windows backward from now. if the curve stays in a power-law regime, the plot of the exponent is flat versus time. that would be the latest overall exponent. it will also reduce in magnitude as the rate of infections decreases. the data from china and south korea follow this pattern and do not fit a power law for all times because an end-stage of no new infections occurred. basically, a moving window is similar to quantifying the magnitude of slope on a log-log curve over time as an indicator of disease progression. only countries that had reached 1000 cases or more were considered as hot spots for analysis. there was a lower cutoff of 100 cases where the data is often irregular. times before 100 cases are less relevant, and you can't even plot time zero on a log-log plot because log 0 is undefined. more cases bring more predictability. the moving window procedure also has the benefit that you don't have to manually and arbitrarily pick when the power law regime starts in the analysis. you can just track it and see if it locks in for the most recent data. some countries go power law after 1000 cases or more but the plots presented in the results start at 100 cases to get a better picture. in principle, if you limit the spread of the virus as in south korea and china, then the power-law growth is reduced to a zero exponent. a measure of danger is the time it would take to infect the remaining population of a country versus the time required for a useful quarantine to limit growth. the indicator d we introduce here does just that. the d indicator depends on several variables. • p is the population of the country. • n c is ideally the current total number of infections, but we only have measured positive cases. • α, β are the fit parameters to the number of positive cases at the current time, n c = αt β c . • τ q = 14 days is the quarantine time. • δt is defined as the time it takes for the power-law to extrapolate to the total population of that particular country. the d indicator is a positive function with the following properties. when δt >> τ q then d → 0. the country would have much time to implement a social distancing strategy. when δt << τ q then d >> 1. if d is large, and more rapid social distancing is more crucial to reduce future infections. since most countries will not exhaust their populations for some time from march 28, 2020, then d as a function of time looks very similar to just β as a function of time, so i choose not to plot it or report the values. if significant infection occurs relative to population size, we should probably use measures of spread more in lines with d. people are interested to know when the number of new cases saturates. and if they do saturate, is this substantially lower than the total population, or does it sweep through. south korea and china datasets are most interesting because they show it can stop before sweeping through everyone (67400 cases hubei china). the hubei province data is the most interesting because it shows an entire trajectory of the infection. there is one problem with china hubei province data. the criteria for a positive test was extended to include lung imaging on february 13. discontinuities from differences in testing recording are bad for a moving window analysis that we use. instead, as an example, we make an interpolating function, which is a logistic curve. we can see what the moving window method predicts as a basic model on ideal data where testing would have been perfect. for all other analysis in this paper, we assume no interpolating function. also the other china datasets with better measurements for different provinces look like the same progression of beta as we show in figure 3 for this toy model. in summary, we should keep in mind the dropping of beta to zero is what we want to happen as the outbreak comes under control in different countries. if beta is increasing that indicates things are getting worse. 5 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint figure 2 : hubei china data [9] as a basic model fit to logistic curve with kaleiagraph. using the logistic curve fit we can get some idea of how beta progresses over the course of the entire model sequence of events using the analysis in this paper. this is done because the actual data is slightly corrupted. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint 4 results the black dots represent the actual data. the curve fit for the last seven days is extrapolated as the black line. the top edge of the graphs is set to the population of the various countries. the x-axis spans 300 days. one indication of the severity of the spread is the relative slope of the power-law on the log-log plot. a higher slope means things are increasing faster soon. notice for the case of the us that the projected time scale is less than 100 days if the curve does not flatten out. the extrapolation has relevance for setting social policy as a worst case scenario. [ht] 7 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint figure 4 : beta correlates with how drastic the situation is with different countries currently. beta less than 0.5 can be considered as under control, beta greater than 3 indicates a potentially dangerous situation. 23 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint the countries with beta in the 1-2 range with one star, 2-3 ranges with two stars, 3-4 range with three stars, and 4-5 range with four stars. beta picks out the most hard-hit countries such as the us, uk, france, belgium, germany, spain, and israel. surprisingly italy is only 2 stars now. the reason why italy is doing so 24 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.20048827 doi: medrxiv preprint bad could be that they spent many more days at a middle range beta than some of the other countries who started later above 1000 cases. the us is the only country with four stars. it even crossed 5 at one point which is definitely significant. many look closely to follow what happens in the us as this is the epicenter with the largest population. different software and algorithms would computer beta differently. there is not much difference if you try to find beta from a weighted curve fitting scheme where uncertainties are estimated from the square root of the the number of positive tested cases versus an unweighted curve fitting scheme. doing an unweighted fit does not provide a direct way to find the measured error bar in beta versus the weighted method derived in the methods. the difference between the two methods is consistent with the actual computed error bars from the weighted analysis. the weighted curve fitting method from the methods is preferable. a plot of the 7-day window of power-law growth β or d is a good measure of the spread of infections in different populations. the dependence of α does not appear to indicate anything useful for the time being. the powers seem to vary between zero and just over five for the us, which is currently the epicenter. perhaps this has to do with general behavior and connectedness of society rather when the disease first hit the country. the extrapolation of the power-law should enable us to make short term predictions of the number of cases in the future; however, social distancing measures can drop the power as indicated by measurements in south korea and china. in those situations, the crossover occurs when β starts around 2 and takes three weeks or more from the point it starts to reduce. for the case of the us, this might take longer since the exponent is now near 5. log-log plots with the top scaled to the population of the country give some indication of how long a worst-case scenario could last before more or less everyone is infected. in the case, of the us this could be 100 days or less without mitigation. as we know however a power-law does not continue forever. this time scale should be compared to those for finding vaccines, supplying equipment, or finding medicine to treat covid-19. there are several factors to consider in disease progression according to the number of positive cases and if this type of analysis relevant. only people who are sick receive a test. some people who are sick stay at home, and the disease resolves itself. some people are positive but exhibit mild or no symptoms, so never receive a test. the data is only as reliable as countries continue to use the same set of testing standards throughout the pandemic. at some point, there could be so many cases that testing cannot keep up daily with the number of reagents required or available kits. after some point, it would be wise to implement serological testing or random testing of the general population to see a more accurate estimation of the total positive cases. there are several factors to consider in disease progression according to the number of deaths. the numbers of deaths are time delayed from the date of the first infection. the time scale for death is longer than to become symptomatic. the number of deaths may increase in the coming days as hospitals become more overburdened, as is currently seen in spain and italy. there are fewer deaths than positive cases, so the sample sizes are smaller to determine the statistics. whether by using positive cases or death rates, scientists should keep all their options open in tracking the course of covid-19. many countries are mobilizing their manufacturing infrastructure to make additional personal protective equipment (ppe), respirators, and ventilators. one technique is to split the tubing of a ventilator to multiple patients. the timescale to produce this equipment should clearly be tens of days and not a hundred. the diy community is also mobilizing to make 3d printed medical equipment rapidly. companies like dyson have created a ventilator in less than 10 days and plan to deliver 15,000 units. it is not clear if these types of numbers are enough to meet the actual future demand or if hundreds of thousands of ventilators or more are necessary. presumably any person who died could have needed a ventilator. spreading out the disease over time would allow more access to successful treatment. initiating social policy decisions in the initial stages of a pandemic is difficult, but we already saw how things unfolded in china with time to prepare. unfortunately, most countries did little and now suffer the consequences. if the question is when is the best time to enact social distancing policies, the answer is now. the best time to plant a tree to get some shade is now if you did not do it twenty years ago. it is too soon to predict the final outcome in terms of infections and deaths by this method. tracking a decrease in a beta down to 0-0.5 allows one to see the spread being mitigated. it is prudent to follow the examples of south korea and making optimistic predictions depends on what we do now. countries besides south korea and china are all in the middle phase. let's hope we can look towards lowering the beta in the affected area. this study is a step towards trying to visualize the progression of the disease over time to see if social policies are working or not jack merrin would especially like to thank michael sixt for encouraging me to think about these problems while working at home due to restrictions in place. i would like to thank nick barton, simon rella maybe it is time to stop shaking hands. elbow bumps are not good either. perhaps, we should just mathematical epidemiology: past, present, and future the coronavirus curve fractal kinetics of covid-19 pandemic trajectory of covid-19 confirmed cases introduction to error analysis. the science of measurements, uncertaintites, and data analysis introduction to error analysis, the study of uncertainties in physical measurements data reduction and error analysis for the physical sciences measurements and their uncertainties: a practical guide to modern error analysis parameter estimation for power-law distributions by maximum likelihood methods key: cord-103711-tnw82hbm authors: einian, majid; tabarraei, hamid reza title: modeling of covid-19 pandemic and scenarios for containment date: 2020-03-30 journal: nan doi: 10.1101/2020.03.27.20045849 sha: doc_id: 103711 cord_uid: tnw82hbm the impact of covid-19 pandemic on the global health and world's economy have been profound and unseen since the spanish flu of 1918-19. as of now, many countries have been severely affected, partly because of slow responses to the crisis, ill-preparedness of their health system, and the fragile health infrastructure and the shortage of protective equipment. this note evaluates various scenarios, based on an estimation of number of identified and unidentified infected cases, and examines the effectiveness of different policy responses to contain this pandemic. our result, based on an estimation of the model for iran, show that in many instances the number of unidentified cases, including asymptomatic individuals, could be much bigger than the reported numbers. the results confirm that in such circumstances, social distancing alone cannot be an effective policy unless a large portion of the population confine themselves for an extended period of time, which is not only difficult to implement, but it could also prove extremely costly and damaging to the economy. an alternative policy, this note argues, is to couple effective social distancing with extensive testing, even to those who are asymptomatic, and isolate the identified cases actively. otherwise, many lives will be lost, and the health system will collapse, adding to the ongoing economic troubles that many countries have started to encounter. as the impacts of the coronavirus-covid-19-rattle the world on its path to more than 199 countries and territories (as of march 27), countries show astonishingly similar behavior, at least in the first phase, of this pandemic. similar to some deadly diseases that affect individuals, it seems that the society and/or the governing authorities of many countries, go through the similar stages as individuals, namely denial, anger, blame, depression, and acceptance. we understand form scientists, that the sooner a patient accepts her sickness, the sooner she can receive treatment and care. a prolonged process to reach acceptance, makes it more difficult to effectively tackle the disease, and in many instances, if left untreated with unfortunate deadly outcomes. for instance, iran is a prime example of a country that has prolonged the denial phase, and in doing so, has created a situation that might lead to a catastrophic outcome and could result in a human tragedy. the authorities in iran broke the news by announcing the death of two covid-19 victims on february 19, 2020, just a day before nationwide parliamentary elections. although it is almost impossible to know the exact date and the number of infected people prior to the official announcement, the number of new cases suddenly jumped from zero on february 18 to around 150 cases a week later. iran initially refused to quarantine the holy city of qom, the source city of covid-19 in iran, and let the virus spread all over the country, and in a matter of a few weeks, all cities reported new cases of infected patients. today, the pattern of pandemic in iran indicates that, even after one month, the country is still grappling with the early stages of this terrible diseases and is unable to move on to the stage which it accepts the reality and takes extra-ordinary but necessary measures to confront the spread of the virus. the data pattern in iran shows a high death rate at the beginning of the pandemic, which then declined and reached the global average after a couple of weeks. similarly, the pattern of identified cases has been very different across countries. for instance, while the crisis started sooner in iran than many other countries, infected cases rose quickly in europe and the us, and surpassed those of iran. this is in despite of complete shutdown in many european countries and many states in the us. similar to many other countries, this might have been due to the lack of resources for testing and identifying infected individuals. this is why this note tries to account for identified as well as unidentified cases and simulate a model-based number of infected population. we use a modified susceptible, exposed, infected, and recovered (seir) model to simulate the spread of covid-19. although the model is fairly sophisticated, it is easily understood and it is estimated with available public data. the results for iran show that active cases might be significantly higher than officially reported by 3 to 6 times in an optimistic scenario of shelter-inplace policy of 75 percent of the population. in a scenario where half of the individuals confine themselves, still around three million individuals get infected of which 50 thousand will die. therefore, the under-reporting of infected cases, although unintentional, not only undermines the integrity of responses, but ultimately puts more people at risk as they go unrecognized and untreated and continue to spread the virus with no end in sight. the note examines multiple policy scenarios and their effectiveness. although the model is estimated for iran, many countries are in similar stages of the pandemic or soon will be in the same stage and therefore, based on the results, extreme measures need to be adopted to save lives and stop further spread of the virus. social distancing alone cannot stop this disease except if more than 80 percent of the population confine themselves for quite an extended period of time. however, if the number of testing increases massively, and countries' authorities successfully isolate infected individuals, the pandemic can be controlled before the summertime, even with a less degree of confinement. while the paper simulates various scenarios of containment, it does not address the economic cost of each scenario. in line with recent findings, the note argues that a fast and aggressive multi-layer intervention is needed to have a substantial dent of the transmission. what the paper calls for is a clear and transparent strategy by governments to implement social distancing and aggressive testing. the testing, should be targeted to geographic locations that the virus is wide spread, to airports and cruise ships, to road travellers, and anyone who has been in areas that are hot spot or has been in any contact with someone in affected locations over the past two weeks. the strategy should not "just" focus on those that show symptoms but should include "all" who might have been exposed. in this exceptional environment, governments can use temporarily a combination of location data and credit card information to track covid-19 in their countries and use surveillance tools to monitor quarantined individuals. deterministic compartmental models are simple yet powerful models in describing this kind of epidemic outbreaks. we developed a variant of the seir model for the epidemic dynamic and control of covid-19, using seven state variables and a minimal number of parameters to avoid over-fitting. the model is estimated for iran, but it can be estimated for other country examples. it is different from a standard seir model by distinguishing hidden and identified infected cases. indeed, standard seir models are estimated assuming that all infected people are reported. such an assumption for the novel coronavirus pandemic is largely unreasonable, as many infected people show no or mild symptoms and as the testing procedure is not available in mass, many remain undetected. thus, we divide infected compartment into infected but not reported (i) and infected, detected and reported (q) groups. we assume that identified cases are less infectious by taking self-precautionary measures and/or due to hospitalization. while unidentified cases can largely continue to infect the rest of the population. the model, therefore, 3 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . distinguishes between the confirmed infected individuals, which are observable in data, and the total infected individuals. in reality, the number of confirmed cases is a function of medical tests and the preparedness of the healthcare system. covid-19 has an infection capability during the incubation (pre-symptomatic) period. in addition, based on the detection method, there exists a delay between the time that an individual is infected and the time this person is detected. state variables and parameters of the model in equations (2.1)-(2.7) capture these characteristics. figure 1 shows the schematic diagrams illustrating these states (compartments). we denote the share of susceptible individuals with no resistance to the disease in the population by s t . e t is the share of people who are exposed to the virus but are not yet infected nor infectious. i t is the infected population share who are undetected and hence un-quarantined. these individuals can be either symptomatic or pre-symptomatic. a share of these individuals, q, are detected and move to somewhat quarantined population q t . among both infected populations, r t = r i t + r q t recover and for some time become resistant to new infections. finally, we assume those who are detected are the individuals who need medical assistance and those that their general health condition might have deteriorated quickly. a share of these individuals, δ, pass away (d t ). finally, due to the high pace of the current outbreak, the model is a closed model in the sense that there are no new birth and death, except for the disease itself. in a simple sier model, people are removed (hence the letter r in seir) from the analyzed population due to either immunity/ resistance to the disease or death. dividing the recovered into "recovered from i" and "recovered from q" groups, and keeping in mind that we only have data on recovered from q and number of deaths (d), we can fit the model compartments to these series. the values of β i and β q depend on the contact rate between individuals and the probability of transmission in an encounter. 1/σ is the mean latent period and 1/γ i and 1/γ q are the mean infectious periods for each group of patients. finally, γ i and γ q are the recovery rates and δ is the death rate among the q group. the model is simulated by solving a deterministic ordinary differential equations (ode), given the parameters (β 1 , β 2 , σ, γ i , q, γ q , δ) and the initial conditions to obtain a realistic simulation of the baseline model and possible scenarios, we needed to estimate or calibrate these parameters and initial conditions. since we are at the beginning of the epidemic cycle and the full distribution is not yet revealed, the model cannot be properly fit to a probability distribution function. as a result, the model is estimated by minimizing the differences between actual and predicted share of cases for observable data series which are q, r q , and d based on an extension of drake (2011) . the model is estimated using iran's official data for the number of confirmed, recovered and death cases 1 . as there are limited observable state variables, we fix or restrict a few parameters based on the reports in the literature to avoid over-fitting. first, based on the genomic studies on the phylogeny of sars-cov-2, two genetic mutations of the virus has happened in iran (the nextstrain team, 2020) 2 , first between january 10 th and january 27 th , and the second between january 24 th and january 30 th . thus, the virus was present in iran before january 27 th , long before the first official record of the disease in iran (february 19 th ), but not before january 1 st as the parent mutation has occurred in hubei, china between january 1 st and january 14 th . so, the starting time of the simulations should be between january 1 st and january 27 th . initial values for observable states are obviously zero. second, we pin down the relationship between β i and γ i , and between β q and γ q , such that β q γ q = 1.4, i.e., the minimum basic reproduction value and β i γ i to be equal to maximum basic reproduction number of 6.6, based on various reported values 3 . third, the relationship between γ q and δ is fixed at 5.7 percent to match the case fatality rate with the one estimated by baud et al. (2020) . without these restrictions, the parameters and initial states can be estimated, but the issue of over-fitting resides and leads to misspecification. based on the optimization, the epidemic started on jan 15 th with 1 infected and 290 exposed people. in the absence of any control measures and/or changes in individual behaviors, we would expect a peak of infection (some of identified and unidentified patients) to occur around may 23, 2020 (figure 2). as discussed in the previous section, we fix r i 0 = 6.6 and r q 0 = 1.4 in this scenario for the whole epidemic period. under this assumption, 70 percent of the population will be infected by around may 11 th . as the epidemic progresses, at the peak, around 9 million identified individuals will be sick and will need medical assistance. in total, in an uncontrolled epidemic, around 900 thousand iranians will die from this disease, not accounting for all other patients who will not get proper medical assistance during this period due to an overwhelmed medical system being. in a cabinet meeting, iran's minister of health mentioned that the maximum capacity of intensive care of all hospitals in iran stands around 14,000 beds. under the baseline scenario and without any control measures, at a 10 percent hospitalization rate of identified cases, all icu beds in iran will be filled by april 26 and even all hospital beds will be filled on may 26 (figure 3). some 13,000 individuals will die per day around mid-june (figure 4). . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 7 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.27.20045849 doi: medrxiv preprint although iran initially refused to fully quarantine the holy city of qom and later the other cities, the government started to react to the epidemic by cancelling all cultural, religious and sportive events, and closing of universities and schools in several cities. the authorities refused to quarantine areas affected by the outbreak and instead asked people to remain inside their houses on a voluntary basis, without any enforcing measures. some religious sites remained open until mid-march. unlike every year in late march (persian new year) the authorities urged the public to avoid travelling for the two-weeks holidays. however, according the official statistics, roads were still busy, and millions of people travelled, making the self-quarantine policy ineffective. in the absence of firm suppression policies 4 , we simulate the model based on the changes in the behaviors of the public by comparing i) the baseline or uncontrolled scenario, ii) with complete isolation of identified infected cases, iii) case isolation and self-quarantine, iv) intensive testing, identification of cases and quarantine, v) a combination of previous scenarios, vi) abandoning shelter-in-place policy one month after the peak of the epidemic under scenario v. scenario (ii) is implemented by assuming β q = 0, meaning that as soon as an individual is reported as infected, all necessary measures are taken to stop contagion from this person to other people. china forced hospitalization of all "confirmed cases" and not just those who required hospital care, to reduce onward transmission in the household and potentially in other social settings. results show that this scenario is not effective and the epidemic progress almost at the same rate with and without complete isolation of identified cases. scenario (iii) is implemented by choosing smaller values for β i of the base scenario, as it depends on the contact rate between individuals, in addition to β q = 0. adding social distancing saves many lives (figure 6), but at this stage of the epidemic at least 80 to 90 percent of the population should practice shelter-in-place. sadly, if only one quarter of the population continue regular contacts with each other, the number of death cases can easily surpass a quarter of a million, and clearly, this policy will not be enough to overrun hospitals' maximum capacity (figure 5). we implement scenario (iv) by assuming that the health authorities will increase number of testing by 10 folds. this helps isolate infected cases, provides patients with necessary medical assistance and decelerates spread of the disease to the rest of population. this policy would shift individuals from i group to q group, enforcing better implementation of precautionary measures and control of contagion. the results are shown in figure 7. even in this case, more than 75 percent of the population will contract the disease and the death toll will remain very heavy. only by combining this scenario with previous scenarios, one can be hopeful to control the epidemic and save lives. figure 6 : total death count in case isolation and partial self-quarantine in different levels. 9 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https: //doi.org/10.1101 //doi.org/10. /2020 scenario (v) shows the result for the combined policies, assuming that only 50 percent of the population respect the shelter-in-place policy, while testing and identification process intensify by 10 folds. figure 8 shows the results for the combined policy. if implemented immediately, the total number of deaths by end-june will remain below 5 thousand individuals. the maximum number of infected cases (i + q) will reach its maximum at 175,000 on may 9 th and the majority of the population will not contract the virus. finally, in the last scenario, we assume that last set of policies were effective enough to reach the peak of the epidemic on may 9 and the shelter-in-place policy continues by an additional 30-days period. as a result, businesses and government will open on june 9, while the intensive testing continues. we assume that as the shelter-in-place policy ends, all individuals remain vigilant and take hygiene measures. under this scenario, social interaction reduces by 25 percent compared to normal times. as a result, the number of patients in need of intensive care surpasses the total icu beds capacity before the end of june, while it remains below the total number of hospital beds in the country until the end of year. . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint figure 10 : ending the shelter-in-place policy 30-days after the peak of scenario (v), assuming that intensive testing continues and social interaction diminishes by 25 percent. table 1 reports the number of death and recoveries at end-2020. obviously, the combined set of policies, described in scenario v yields the best outcome with the lowest fatality and infected cases. comparing scenario iii and iv, we conclude that a combined intensive testing, identifying infected individuals, and quarantine is a more effective policy than just a shelter-in-place policy. as discussed, scenario vi is the same as scenario v, assuming that the authorities end the shelterin-place policy 30-days after the peak of the pandemic. unfortunately, under such scenario the death toll can be still multiplied by 4 times compared to the scenario where social interactions are just reduced and kept low by 50 percent. if the social interaction is increased to normal times, the death toll can be 15 times larger than in the scenario v. many governments are impatient to open their countries businesses. they are rightly worried about the economic cost of a prolonged shutdown of economic activity. there are already outcry across countries by those who have lost their jobs. many in low income and developing countries cannot afford to stay in home for long, since there are not enough safety nets to protect the most vulnerable. on the other hand, even in advanced economies, the ripple effect of a lengthy and uncertain shutdown of the economy is too painful. this is the reason that countries' authorities have to start implementing a broad range of decisive actions. such policies should be put in place to save lives of thousands of people, alleviate pressures on the health system, and provide an opportunity for the people to go back to a normal living setting. these comprehensive set of measures include isolation of infected cases, shelter-inplace, and intensive and extensive testing. good examples are germany, south korea and not surprisingly china, where it not only hospitalized all cases, but also enforced a population-wide 12 . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10. 1101 /2020 scenario d r i 891000 81772000 ii 890000 81670000 iii 805000 74137000 iv 246000 60772000 v 12000 2816000 vi 53000 13680000 table 1 : end of 2020 results social distancing to curb the contagion. unlike the current approach in some countries, where only the symptomatic cases are tested, the authorities should provide intensive and extensive testing and try to identify almost-all infected cases. this process would identify asymptomatic but infected cases and stop them of spreading the virus. this is key for a successful containment policy. given the geographic distribution of the pandemic, the testing should focus on the most affected locations first and then expand to those people who have visited the hot spots or have been in contact with anyone in those areas, while everyone practice social distancing. a combination policy, as discussed above, not only reduces the number of infected, and ultimately the death toll, but it shorten the shelter-in-place period and consequently lowers the economic cost. correspondence real estimates of mortality following covid-19 infection. the lancet infectious diseases fitting epidemics in r how scientists quantify the intensity of an outbreak like covid-19 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) time-varying transmission dynamics of novel coronavirus pneumonia in china novel coronavirus 2019-ncov: early estimation of epidemiological parameters and epidemic predictions pattern of early human-to-human transmission of wuhan the novel coronavirus, 2019-ncov, is highly contagious and more infectious than initially estimated genomic epidemiology of novel coronavirus estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china key: cord-022147-istz1iql authors: nan title: procedures to investigate waterborne illness date: 2016-07-13 journal: procedures to investigate waterborne illness doi: 10.1007/978-3-319-26027-3_1 sha: doc_id: 22147 cord_uid: istz1iql humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early 1900s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. not only safe water is limited to drinking water, but recreational water can also be a source for waterborne illness—both from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early 1900s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. safe water is not limited to drinking water, since recreational water and aerosolized water can also be sources for waterborne illness, from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. often when an outbreak is first suspected, the source is not clear, i.e., food, water, animal contact. investigation is usually needed to find the common source. in some outbreaks the food may first be identified as the source, such as with produce, but the ultimate source could be contaminated irrigation water. investigators have to keep an open mind until laboratory and/or epidemiologic evidence links cases to the primary source. although we frequently think of waterborne illness originating from a microbiological agent, we should be aware that water may also be contaminated by pesticides, fertilizers, and other chemicals which may enter through industrial discharge, agriculture runoff, or deliberate contamination. waterborne illness acquired from microorganisms may be classified as: • toxin-mediated infections caused by bacteria that produce enterotoxins or emetic toxins that affect water, glucose, and electrolyte transfer during their colonization and growth in the intestinal tract; • infections caused when microorganisms invade and multiply in the intestinal mucosa, eyes, ears, or respiratory tract, or contact the skin; • intoxications caused by ingestion of water containing poisonous chemicals or toxins produced by other microorganisms manifestations range from slight discomfort to acute illness to severe reactions that may terminate in death or chronic sequelae, depending on the nature of the causative agent, number of pathogenic microorganism or concentration of poisonous substances ingested, and host susceptibility and reaction. the public relies on public health regulators to investigate and mitigate waterborne illness. mitigation depends upon rapid detection of outbreaks and a thorough knowledge of the agents and factors responsible for waterborne illness. public health and law enforcement agency officials should always be alert to the rare possibility of an intentional contamination of water supplies by disgruntled employees or terrorists. the purposes of a waterborne illness investigation are to stop the outbreak or prevent further exposure by: • identifying illness associated with an exposure and verifying that the causative agent is waterborne • detecting all cases, the causative agent, and the place of exposure • determining the water source, mode of contamination, processes, or practices by which proliferation and/or survival of the etiological agent occurred • implementing emergency measures to control the spread of the outbreak • gathering information on the epidemiology of waterborne diseases and the etiology of the causative agents that can be used for education, training, and program planning, thereby impacting on the prevention of waterborne illness • determining if the outbreak under investigation is part of a larger outbreak by immediately reporting to state/provincial/national epidemiologists in the instance of a bottled water outbreak, halting of distribution and sale of product and recall of product, some of which may already be in consumers' homes, are necessary to prevent further illness. as epidemiologic data accumulate, information will indicate the source of the problem, whether a municipal water treatment plant, bottled water manufacturing plant, or recreational water exposure, and suggest methods for controlling and preventing waterborne illness. this information will guide administrators in making informed decisions to provide the highest degree of waterborne safety. a flowchart, sequence of events in investigating a typical outbreak of waterborne illness (fig. 1) shows the sequential steps, as presented in this manual, in investigating a typical outbreak of waterborne illness and illustrates their relationships. a description of each step is presented in this manual. can be recognized at any point during the outbreak investigation. if intentional contamination is suspected follow your notification scheme in emergency response plans (this could include law enforcement, emergency management and other government agencies) the primary purpose of a waterborne disease surveillance system is to systematically gather accurate information on the occurrence of water-related illnesses in a community, thus allowing development of a rational approach for the detection, control and prevention of waterborne illness. other purposes are to (a) determine trends in the incidence of waterborne diseases, (b) characterize the epidemiology of waterborne diseases, (c) gather and disseminate information on waterborne diseases, and (d) develop a basis for evaluating control efforts. it may be useful to coordinate this system with, or integrate it into a foodborne disease surveillance system. however, while the procedures are quite similar from an epidemiologic viewpoint, they may differ with respect to personnel or agencies involved. an effective disease surveillance system is essential for detection of disease caused by either unintentional or intentional contamination of food. an effective waterborne disease surveillance system consists of: • early reports of enteric and other illnesses that may be related to water exposure or consumption • coordinated effort between local and state public health partners, water utility and water recreation staff • systematic organization and interpretation of data • timely investigation of identified outbreaks or clusters of illness • dissemination of outbreak reports and surveillance summaries to all appropriate stakeholders many types of reporting systems may already exist at the local or state/provincial level, and these should be incorporated into a waterborne disease surveillance program. these include (a) mandatory (or voluntary) laboratory-or physician-based reporting of specific infectious diseases, (b) national-based surveillance systems such as calicinet (cdc 2009 ) or nors (cdc 2009 in the us, (c) physician office, hospital emergency, and urgent-care clinic medical records, (d) public complaints made to health agencies and/or local water utilities, (e) school illness and absentee records, (f) absentee records of major employers, (g) water treatment records kept by water utilities (e.g., turbidity, disinfection levels, occurrence of coliforms), (h) increased sales of antidiarrheal drugs and anti-nausea medications, and (i) source water quality data kept by environmental agencies (e.g., departments of natural resources and geological survey agencies). another type of surveillance mechanism that may supplement or enhance existing reporting systems is a daily log of illness and water quality complaints. agency contemplates initiation or development of a waterborne illness surveillance program, give top priority to identification of appropriate financial, political, strategic, and administrative support. then, identify a key person to create, implement, and manage the system. this person takes responsibility for: • reviewing the types of reporting systems that already exist in your agency or in other agencies that could be incorporated into a waterborne illness surveillance system • identifying the types of information that cannot be obtained from existing reporting systems but that need to be collected or addressed by the waterborne illness surveillance system • identifying ways to merge or integrate the data collected by existing systems with data gathered in the waterborne illness surveillance system • identifying collaborating agencies and staff • develop a mechanism to communicate and update all stakeholders (may be by blast e-mail or periodic conference calls) • providing training in surveillance methods for agency staff and other partners to enhance cooperation • assembling materials that will be required during an outbreak investigation • evaluating the effectiveness of the system. develop procedures to seek and record complaints about waterborne illnesses, water supplies, and water recreational sites. for example, list the telephone number of the waterborne illness investigation unit prominently on local and state public health and water utility websites. to be most effective, have this number monitored 24/7 by staff or an answering service. if possible, the utilization of social media such as facebook or twitter should be considered and monitored as many large municipalities (including drinking water utilities) and recreational facilities have an internet presence. if your agency has social media accounts, consider using this vehicle to further disseminate information regarding waterborne illness clusters or outbreaks. identify medical care facilities and practitioners and seek their participation. direct educational activities, such as newsletters and talks at meetings, to stimulate participation in the program. encourage water treatment utilities and operators of recreational water sites to report suspected complaints of waterborne illness to the appropriate local agencies. also, encourage private and hospital laboratories to report isolations of parasitic agents (e.g., giardia, cryptosporidium), viruses (e.g., norovirus and hepatitis a virus), bacteria (e.g., e. coli (pathogenic), salmonella, shigella, vibrio cholerae), and other agents that may be waterborne. develop a protocol for notification and coordination with agencies that might cooperate in investigational activities, including 24-h-a-day, 7-days-a-week contacts. a comprehensive contact list should be constructed and updated at least twice a year as individuals may change. notify and coordinate with state/provincial or district agencies, national agencies that have surveillance and water regulatory responsibilities, and other national and international health agencies, as appropriate. for example, it may be useful to find out the level of participation within a certain jurisdiction in national-level outbreak surveillance programs such as nors (cdc, 2015) or other national surveillance system. delegate responsibility to a professionally trained person who is familiar with epidemiologic methods and with the principles of water treatment and recreational water protection. this person will (a) direct the surveillance program, (b) take charge if waterborne and enteric outbreaks are suspected, and (c) handle publicity during outbreaks. delegate responsibility to others who will carry out specific epidemiologic, laboratory and on-site investigations. if an intentional contamination event is suspected, local and national law enforcement agencies will likely become the lead agency responsible for the investigation. with this in mind, it is critical to identify appropriate individuals and include them in communication and any practice drills that may occur. if a relationship has been established prior to any event, the investigation may run more smoothly. enlist help from a team of epidemiologists, microbiologists, sanitarians/environmental health officers/public health inspectors, engineers, chemists, nurses, physicians, public information specialists, and other (e.g., toxicologists) as needed. free flow of information and coordination among those participating in waterborne disease surveillance and investigation are essential, particularly when several different agencies are involved. water-related complaints are equally likely to be directed at health departments or water utilities but also perhaps to different jurisdictions. therefore, it is essential that these complaints be registered by an agency and that the information is rapidly shared within and perhaps outside of a particular jurisdiction as part of an integrated surveillance system. whenever possible, share the information with participating parties by rapid means such as e-mail and by calling 24/7 contact phone numbers. if an intentional contamination event is suspected, contact emergency response and law enforcement for their early involvement. select staff members who will participate in the waterborne disease surveillance program on the basis of interest and ability. inform them of the objectives and protocol of the program. emphasize not only the value of disease surveillance, but also the value of monitoring water quality and treatment performance. if possible, provide printed learning material that can be referenced later. encourage the use of epidemiologic information and approaches in routine disease surveillance and prevention activities. develop their skills so that they can carry out their role effectively during an investigation, and teach them how to interpret data collected during investigations. conduct seminars routinely and during or after investigations to update staff and keep agency personnel informed. train office workers who will receive calls concerning waterborne illnesses to give appropriate instructions. those who participate in the investigation will learn from the experience and often are in a position to implement improvements after completion of the investigation. assemble and have readily available kits with forms and equipment as specified in table a (equipment useful for investigations). restock and maintain kits on a schedule recommended by, and in cooperation with, laboratory staff to ensure their stability and sterility. verify expiration dates, and use kits before this date or discard and reorder. assemble a reference library on waterborne illnesses, investigation techniques, and control measures from reference books, scientific journal articles, manuals, and reputable internet sources (e.g., www.cdc.gov, www.who.int/en/, www.hc-sc.gc.ca/ index-eng.php, www.gov.uk/topic/health-protection/infectious-diseases); make it available to the staff in an easy-to-access format. (see further reading for suggestions). organize a multiagency team with representatives from public health agencies, regulatory agencies, and water utilities and with local political officials to review and exercise existing emergency response plans in the event of a large scale waterborne disease outbreak or other disaster likely to result in waterborne illnesses. local public health agencies have the primary responsibility for the restoration and protection of health of a community following an outbreak event or other emergency. emergency operational procedures should include the following: • an emergency notification list that includes phone numbers and e-mail addresses of key persons/agencies that need to be informed about possible outbreaks and that should receive emergency press releases. every state/province has an emergency management agency and depending upon the scale of the event, it may be useful to coordinate efforts. • clear guidelines for household water consumption following an event. for example, boil-water advisories or instructions to drink only bottled water. statements should be reviewed to ensure current relevance and updated to reflect the most current knowledge. • a plan for dissemination of information to the public; select a coordination point to which all news media and outside agencies will be directed, and designate one person or one telephone number as the contact. (more than one contact person can create confusion). • alternative drinking water sources to be used in cases of emergency and plans for the distribution of this water, if necessary. these include alternative munici-pal systems, bottled water supplies, portable filtration and/or disinfection devices and home treatment units. (special attention should be given to backup supplies for hospitals, nursing homes and other places where lack of safe water would be immediately life-threatening). • identification of specialty laboratories at the state/provincial and national level that are capable of performing (and willing to perform) procedures not routinely done at local laboratories (e.g., large volume water sampling and processing for pathogenic parasites and viruses, serotyping, electron microscopic examination of stool samples, molecular and immunodiagnostic tests for pathogens in environmental and clinical samples). one or more of these tests may be necessary to identify the causative agents in an outbreak and confirm their transmission route. • a plan to exercise procedure with tabletop exercises involving all pertinent partners on a regular basis and implement any necessary adjustments based upon review of after-action findings. notification of a suspected outbreak is often received by health authorities from a laboratory report or a family physician and can be documented on a log such as form a (foodborne, waterborne, enteric illness complaint report). public health investigators will then interview cases and persons at risk who are well (controls) to make epidemiologic associations to find a common source. from here a hypothesis can be formed. further investigation will involve: • collecting clinical samples and water samples • conducting an on-site investigation at the facility alleged to be responsible to determine the mode of contamination or process failure, e.g., low disinfectant level • characterizing the etiologic agents by laboratory analysis using various typing schemes. dna profiling or pulsed-field gel electrophoresis (pfge), of isolates from clinical and water samples to "fingerprint" and link strains of the etiologic agent among cases and to the source prompt handling and referral of water-related complaints, rapid recognition of the problem, and prevention of further illnesses are the foundations of a successful investigation. complaints of water problems are as likely to be reported to a water utility as to a health department. communication is essential between these agencies. this first contact with the public is a vital aspect of an investigation of a potential outbreak and needs to begin by public health professionals as quickly as possible, usually within 24 hours, sometimes by putting less urgent activities aside. as indicated earlier, any action respecting a potential deliberate contamination of water will generate a specific approach to further action. upon receiving a complaint or an alert about a water supply or water exposure or illness potentially attributed to these sources, record the information on form a. alerts may be initiated by reports from physicians, laboratories, or from hospital emergency rooms. alerts may also include an increase in a particular pfge pattern from clinical isolates. an investigation may be initiated to determine if there is a common water exposure among patients with the pfge pattern. the pattern may be compared with similar pfge patterns in pulsenet databases to determine if there are similar occurrences of the pattern in water and clinical isolates nationwide or internationally, e.g., for food that might have been contaminated with water, bottled water. the form provides information upon which to decide whether an incident should be investigated. form a is not difficult to fill out and can be completed by a public health professional, a trained water utility staff member, or trained office worker. assign a sequential number to each complaint. if additional space is needed to record information, use the reverse side or attach additional sheets. always ask the complainant to provide names of other persons at the event, or using the water supply or recreational water under suspicion, whether or not ill, and names of any other persons who are known to be ill with the same symptoms. follow up by contacting these additional persons. emphasize to the persons making alerts or complaints the need to retain a sample of the suspect water and to save clinical specimens (vomitus and stool) from ill persons using a clean utensil in a clean jar or plastic bag and to seal tightly and label clearly with the name of the person and date, and store in a refrigerator (do not freeze). also consider family members not ill for case-control studies. advise complainants to collect a liter (quart) of water immediately, preferably in sterile containers but otherwise in jars that have been boiled or in plastic bags, or if this is not feasible, in other clean containers. tell the complainant to save any ice cubes or refrigerated water, either in their present containers or in unused plastic bags, in the refrigerator or freezer (if already frozen) where they are normally kept. instruct the ill person to hold all clinical specimens and water samples until the health agency evaluates the epidemiological evidence and arranges, if necessary, to collect them. if it is determined that the specimen or sample is not necessary, notify the complainant and advise on proper disposal of the material. unfortunately, the specific etiologic agent cannot be identified in a large proportion of waterborne outbreaks because water samples and clinical specimens (a) were not collected in an appropriate time-frame (not early enough during illness), investigate outbreaks (b) are too old, (c) are too small in volume, especially for giardia and viruses which require liters, (d) have not been examined for the appropriate agent. contaminants may be in the water system for only a short time, and concentrations of toxic substances and numbers of microorganisms may decrease significantly because of dilution or disinfection. if there is a cluster of cases, monitor reports from physicians, complaints about water, or records of laboratory isolation of enteric pathogens that may suggest outbreaks of disease or contributory situations. collect clinical specimens and water as soon as practicable according to the section obtain clinical specimens in this book. extract key information (see* and † entries) from form a and enter it onto form b (foodborne, waterborne, enteric illness and complaint log). record time of onset of the first symptom or sign of illness, number of persons who became ill, predominant symptoms and signs, whether ice or water was ingested, and the name of the water supply or recreational water alleged to have caused the illness, and whether a physician had been consulted, and/or had taken feces or emesis samples, and/or prescribed antibiotics. also, enter on form b names of the places or common gatherings (other than home) at which the stricken persons ingested water during the 2 weeks before onset of illness (see table 1 for an example). enter a code for the water source (e.g., community, non-community, individual well, bottled, stream/ lake, vended, or untreated) . under "history of exposures" column indicate whether the afflicted person had recent domestic or international travel, attended a child care facility, or had recent contact with ill persons or animals. under "comment" column, enter notations of type of agent isolated, results of specimen tests, places where water was consumed during travel, names and locations of restaurants or other foodservice facilities, and other pertinent information including hospitalization, occupation, or place of employment. at this phase of the investigation it will probably not be known whether the illness is waterborne, foodborne, or person-toperson spread. this log can be kept either in hardcopy or in electronic format. see table 1 (below) as an example of a log. interpretation of table 1 . entry 101-get further details on the patient's symptoms and seek other cases. the report of foreign travel suggests an infection that may have been acquired outside the country. follow-up of such cases may identify an outbreak of international scope. if so, inform state/provincial and national authorities concerned with surveillance of waterborne disease about the situation. entry 102-possibly food associated; alert food safety officials. entry 103-initiate investigation; the two cases of conjunctivitis suggest the possibility of a common-source outbreak associated with the motel pool. entry 104-initiate investigation; 12 cases indicate an outbreak that has a common time-place association. entry 105-this could be related to entry 103, because this person reported swimming in the same pool. exposure history: dt domestic travel (out of town, within country); it international travel; cc child care; ci contact with ill person outside household or visitor to household; a an exposure to ill animal; c contact with ill person within household responsible for the investigation, if this was not done when the surveillance protocol was established. delegate sufficient authority and provide resources to the head investigator so that the investigation tasks can be accomplished effectively and efficiently. inform all outbreak investigative team members that any findings are to be reported to this delegated authority. a list of all team members and additional contacts such as administrative contacts, sanitarians/environmental health officers/ public health inspectors, local and regional contacts, physicians, clinical laboratories, or other persons who may become involved in outbreak investigations should be assembled. before beginning the investigation, check the supply of forms and the availability of equipment suggested in table a (equipment useful for investigations) and obtain any needed materials or additional equipment. general resource materials describing signs and symptoms, incubation times, and specifics regarding specimen collection and appropriate kits to be used should be maintained and readily available to those processing the initial calls, which may help to formulate the initial hypothesis. if the alert or complaint suggests a possible outbreak, inform laboratory personnel of the type of outbreak and estimated quantity and arrival time of clinical specimens and water samples collected. this information will give laboratory managers time to prepare laboratory culture media, prepare reagents, and allocate personnel. at a minimum, the laboratory should have six to eight stool culture kits on hand or readily available, since in many cases, stool specimens must be collected within 72 h of onset of illness to isolate and identify certain pathogens (e.g., campylobacter spp., and salmonella spp.). consult laboratory personnel about proper methods for collecting, preserving, and shipping environmental samples and clinical specimens if such information is needed. obtain appropriate specimen containers and sample submission (chain of custody forms) from them. once the investigation is underway, the proper clinical specimens should be collected as soon as possible before patients recover and become less likely to submit specimens. all suspected waterborne outbreaks should be examined and a determination made regarding the feasibility of conducting a thorough investigation even if the time to collect proper clinical specimens has passed. an ill person or family member, physician, hospital staff member, or operator of a water utility or recreational site may report suspected cases of waterborne illness. whatever the source of the report, verify the diagnosis by taking a thorough case history and, if possible, by reviewing clinical information and laboratory findings. (this analysis can be further substantiated by detecting suspected etiologic agents in water). verification of the diagnosis is done in consultation with medical professionals. when a complaint involves illness, complete forms c1-2 (case history: clinical data and case history: food/water history and common sources) either at the time of initial notification, during a personal visit, or during a telephone call to the person reported to be ill. use this same detailed interview approach with every person who has been identified in the initial complaint or alert, even though some may not have been ill. be aware that potential cultural and language barriers can make interviews difficult. a different interviewer may be needed to accommodate these barriers. continue this until sufficient information is obtained to decide whether there is, indeed, an outbreak of waterborne illness. from persons who are at risk of illness but who remained well, also obtain water and 72-hour food histories, inquire about recreational water exposure in past 2 weeks, and information about their activities in common with the ill persons. information from these persons is as important to make epidemiologic associations as it is from the cases. when it is apparent that an outbreak has occurred and a specific event has come under suspicion, substitute forms d1-2 (case history summaries: clinical data and case history summaries: water/laboratory data) for form c. form d1 can be used initially in many routine waterborne illness outbreak investigations where it is obvious that a common-source outbreak has occurred or when all of the ill persons consumed water together (e.g., drank from the same public system, consumed ice at an event) or recreated at the same place (e.g., swam in the same lake or used the same hot tubs). this will simplify recording, because most affected persons will give similar information. at this time, notify the district, state, or provincial epidemiologist about the outbreak. if a specific pathogen (e.g., norovirus, e. coli o157:h7, cryptosporidium spp.) has been identified as the etiologic agent, consider developing a form for recording relevant information. many state/provincial or national public health agencies have standard forms tailored to specific pathogens. include signs and symptoms of the illness and other clinical information, the etiology of the agent, and usual methods of transmission. computer programs (e.g., epi info™) can aid in the design of such standard forms. upon contact with the affected person, identify yourself and your agency and explain the purpose of the visit or call. a professional attitude, appropriate attire, friendly manner, and confidence in discussing epidemiology and control of waterborne illnesses are essential for developing rapport with affected persons or their families and in projecting a good image of the investigating agency. keep in mind that you are not interviewing someone you inspect or regulate, but that you are providing a service to the affected person. exhibit genuine concern for persons affected and be sincere when requesting personal and confidential information. communicate a sense of the urgency of the investigation, and emphasize that their participation will make a positive contribution for the control and prevention of waterborne illness. parental consent must be obtained before interviewing children under 18 years of age. in some locations, consent from the affected person's physician may also be required. after asking open-ended questions about the person's food exposures and illness history, follow up with more specific questions to fill in the details and better ensure a thorough recall. base your level of communication on a general impression of the person being interviewed, considering information about age, occupation, education, or socioeconomic status. tact is essential. use either form c or form d, as appropriate, as a guide. state questions so that the persons who are being interviewed will describe their illnesses and associated events in their own words. try not to suggest answers by the way you phrase questions. fill in form c1-2 (if appropriate) and take additional notes during the interview. ask specific questions to clarify the patient's comments. think questions through before conducting the interview. realize that people are sometimes sensitive to questions about age, sex, special dietary habits, ethnic group, excreta disposal, and housing conditions. nevertheless, any or all information of this type can be relevant. word questions thoughtfully when discussing these characteristics and habits. such information can often be deduced from observations. if doubt remains, confirm your guesses by asking indirect questions. information on recent travel, gatherings, or visitors may provide a clue to common sources or events that would otherwise be difficult to pinpoint. review known allergies, recent immunizations, recent changes in the patient's medical status, and similar information. remember that the agents associated with waterborne disease can also be spread by other means such as consuming food, person-to-person, visiting child care centers, animal-to-person in petting zoos, through walk-in-spray fountains, and pools for young children. as persons describe their illnesses, check boxes next to appropriate symptoms or signs on form c1. do not ask about all symptoms or signs listed; however, ask about those marked with an asterisk if the ill person does not mention them. if there are questions, explain symptoms to the patient in understandable terms. the symptoms and signs in the first two columns of form c1 are usually associated with poisoning or intoxication, although some occur during infections. those in the third, fourth, and fifth columns are usually associated with enteric infections, generalized infections, and localized infections, respectively. those in the last column are usually associated with disturbance of the central nervous system. diseases in any category will sometimes be characterized by a few symptoms and signs listed in the other columns, and not all signs and symptoms occur for any one ailment or for all persons reporting illness. if an illness seems to fall into one of these categories, mention other symptoms in the category and record the patient's response. whenever possible, use physician and hospital records to verify signs and symptoms reported by patients. clinical data may strengthen or dismiss the possibility of waterborne illness. before contacting a physician or a hospital, become familiar with laws and codes relating to medical records to ensure that you have legal access to these records. legal release forms may be necessary to obtain some records. do not distribute names of patients, their other personal identities (e.g., address, phone number), or their clinical information to unauthorized persons. the entries begin with the day of illness, followed by the previous 2 days. if the illness, however, began early in the day or before any of the listed meals, modify the entries on the form so that the 72-hour history can be completed in the space pro-vided on the form. if the incubation period is 3 days to a week in duration, use additional copies of form c2 and modify day or day before subtitles. signs and symptoms will sometimes give clues to the transmission route by indicating the organ systems affected. if the early and predominant symptoms are nausea and vomiting, ask about the most recently ingested water or beverage within the past 6 h. in these situations, suspect high-acid water supplies, carbonated beverages and fruit drinks, because these tend to leach metallic ions from water pipes and containers. if diarrhea, chills, and fever predominate, be suspicious of water and beverages ingested 12-72 hours before onset of illness for salmonellosis, shigellosis, and norovirus related gastroenteritis. if the incubation period averages 1-2 weeks, consider typhoid fever, cryptosporidiosis or giardiasis. diseases with incubation periods exceeding 2 weeks (e.g., hepatitis a and e, amebic dysentery, or schistosomiasis) can be handled as special cases for which longer histories would be sought. others, such as chronic lead and arsenic poisoning, have incubation periods of variable durations and onsets so gradual as to be indeterminable. see table b (illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents) for details on specific pathogens, table c (illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents), and table d (illnesses acquired by inhalation of microorganisms aerosolized from water. a classification by symptoms, incubation period, and type of agent). other microorganisms not listed in tables b, c, and d that can be potentially spread by water include the bacteria klebsiella pneumoniae, mycobacterium avium complex, acinetobacter calcoaceticus, elizabethkingia meningoseptica, stenotrophomonas maltophilia, pseudomonas putida, serratia marcescens, protozoa isospora, microsporidium, algae schizothrix calcicola. these microorganisms are less frequently identified with waterborne illness, but they may become opportunistic pathogens, particularly for highly susceptible and immunosuppressed persons. further investigation is needed to confirm their role in the spread of waterborne diseases. gather information about all sources of water to which the patient(s) may have been exposed 2 weeks before onset of illness. the water supply and the event that precipitated the illness might not be obvious. persons often have difficulty recalling exposure to all water sources including; ice or water ingested; aerosols and recreational water contact. therefore, if the person does not remember specific exposures to water, ask about the water consumed in usual or routine daily habits and the amounts ingested; exposure to recreational waters; and unusual exposures or events attended during this interval. this may stimulate recall of away-from-home water consumption or contact that was unusual. ask about other risk factors for enteric illness, such as contact with young children and child care centers, animal contact, ingestion of raw foods of animal origin, and usual food preference habits. for persons who have been traveling, ask them where (both cities and rural areas) they have traveled during the incubation period of suspected agents. determine if they drank water from any taps or pumps in rural areas they visited. ask whether unheated (or untreated) tap water or beverages containing unheated (or untreated) water or ice was ingested at restaurants, in hotels or at events in the places they visited. also, ask whether they ingested bottled water and, if so, the brand name. find out whether they drank water from streams, ponds, springs, or other natural water sources. if they did, ask if they observed any abnormal condition of the water such as algal growth, high turbidity or discoloration. ask if domestic or wild animals had access to the water. if they have skin or eye infections or generalized infections, ask them to name all swimming pools, water slides, beaches, lakes, ponds, or other chlorinated and nonchlorinated water courses where they swam, waded or bathed during their trip. also ask them whether they used any hot tubs, spas, whirlpools, or similar devices. this information sometimes provides clues to common sources or to events that otherwise would be difficult to discover. record the information on form c1. in a protracted outbreak, or when investigating an outbreak of a disease with a long incubation period, expect recall to be poor. in this situation, obtain from ill persons and others at risk a listing of their water, ice, and beverage preferences and amounts usually ingested, or their purchases of these items within the range of the incubation period of the suspected disease. as a guide, draw up a list of either water, ice, and beverages that are commonly consumed by the affected group or those waters, ice, and beverages previously identified as vehicles of the suspected disease under investigation. summarize data from all copies of forms c1-2 on form d. form d allows rapid review of all exposed persons (ill or not ill) and serves as a basis for analyzing the data. diagnosis of most diseases can be confirmed only if etiologic agents are isolated and identified from specimens obtained from ill persons. get specimens from the ill persons to confirm an etiologic agent. • in large outbreaks, obtain fecal specimens from at least ten persons who manifest illness typical of the outbreak • in smaller outbreaks, obtain specimens from as many of those ill and those at risk as practicable, but from at least two, and preferably ten, ill persons • try to collect specimens before the patient takes any medication. if medication has already been taken, collect specimens anyway, and find out the kinds and amounts of medicine taken and the time that each dose was taken • also get control specimens from persons with similar exposure histories that did not become ill obtain clinical specimens at the time of the initial interview during acute illness or as soon as practicable thereafter. even though this is not always possible, take specimens even after recovery because etiologic agents may remain in low populations or concentrations. if a disease has already been diagnosed, collect specimens as listed in table b . if a disease has not yet been diagnosed, choose kinds of speci-mens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in the treatment of cases detected later. apart from the fact that people are more likely to cooperate while they are ill, some pathogens or poisonous substances remain in the intestinal tract for only a day or so after onset of illness. if the patient is reluctant to provide a fecal specimen explain that the specimen will be tested to identify the causative agent and compare it to any agent recovered from the water. if a disease has not yet been diagnosed, choose specimens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in treating cases detected later. some pathogens (e.g., salmonella, parasites) may be recovered for weeks after symptoms have abated. if applicable for the disease under investigation, take specimens even after recovery because some etiologic agents may remain in low numbers, and changes in serologic titers can be detected. before collecting specimens, review table e (guidelines for specimen collection) and, if necessary, get additional instructions from laboratory personnel and seek their advice on how to preserve the stool specimens if you cannot deliver them to the laboratory immediately. many public health agencies have special fecal specimen kits. demonstrate to the patient how to use the materials in the kit, how to complete the form in the kit and how to mail it if you are not going to pick it up. if mailing specimens, make sure that you are aware of the regulatory requirements that may apply to the transport of infectious material. stool specimen containers for intestinal parasite examination are not suitable for bacterial or viral examinations because they ordinarily contain a preservative, such as formalin or polyvinyl alcohol. if an inappropriate transport medium is used, a specimen can be rendered unsuitable for laboratory examination. feces. if the patient has diarrhea or is suspected of having had an enteric disease, obtain a stool specimen (preferred specimen) or a rectal swab. instruct patients to provide you with their own specimens by one of the following means. 1. if practicable, give the patient a stool specimen container with a wooden or plastic spoon or a tongue depressor. a clean container available in the home (e.g., a jar, or disposable container that can be sealed) and a clean plastic spoon or similar utensil can be used if laboratory containers are not available. 2. label the specimen container with the patient's name age/date of birth and date of collection. 3. collect the stool specimen by one of the following methods: (a) put sheets of plastic wrap or aluminum foil under the toilet seat and push them down slightly in the center, but not so far as to touch the water in the bowl. sheets of paper can be tacked on the rise of a latrine and pushed down to form a depression in which to catch feces. take care to ensure that toilet cleaning chemicals and other microorganisms in the toilet bowl do not contaminate the fecal specimen. after defecating, use a clean spoon or other utensil to transfer about 10 g of feces into a specimen container or other clean container. (b) defecate directly into a large clean dry container or bedpan. use a clean spoon or other utensil to transfer about 10 g or the size of a walnut of feces into a specimen container or other clean container. (c) scrape feces off a diaper with a clean spoon or other utensil to transfer about 10 g of feces into a specimen container or other clean container. 4. collect fecal swabs by twisting the cotton-wrapped end of the swab into the stool obtained in one of the ways described above. follow instructions given in table e . if necessary, use fecal-soiled toilet paper or cloth diaper and twist a swab into the top of feces. take care to ensure that there is no carryover of toilet paper as they are impregnated with barium salts which are inhibitory to some fecal pathogens. dispose of excess fecal material into the toilet and carefully wrap all soiled articles (e.g., by placing them inside two plastic bags) and dispose of in domestic waste. check that the specimen container is tightly sealed and properly labeled and place into a clean outer plastic bag (special zip lock bags for clinical specimens, if available). store the specimen in a cool place, preferably at 4°c to await pick-up or despatch. do not freeze. feces from rectal swabs. collect rectal swabs by carefully inserting the swab approximately 2.5 cm (1 in) beyond the anal sphincter. gently rotate the swab. fecal matter should be evident on the swab. vomitus. if the person is vomiting or subsequently does so, arrange to collect vomitus. tell the patient to vomit directly into a sterile specimen container or a plastic bag. otherwise, transfer some vomitus from a clean receptacle into the container with a clean spoon. refrigerate, but do not freeze, this specimen until it can be picked up or delivered to the laboratory. blood. take blood if a patient has a febrile infection or when infectious agents are suspected (see tables b, c, and d) . blood specimens are collected for: • bacterial culture • detection of antibodies to specific agents • detection of certain toxins before collecting specimens, get additional instructions from laboratory personnel and seek their advice. blood should be obtained by an appropriately trained and accredited person (check appropriate laws). collect blood during the acute phase of illness, as soon as the febrile patient is seen (within a week after onset of illness) and, if comparing of serologic titers, again within 6 weeks (usually 2-4 weeks later) during the convalescent phase. draw 15 ml of blood (from an adult) or 3 ml (from a child) or 1-2 ml (from an infant). if possible, collect the blood from the same patients from which stool specimens were obtained if both specimens are to be examined. label tubes and vials at every step of serum transfer. do not freeze whole blood because the resultant hemolysis interferes with serologic reactions. collect into a sterile syringe or evacuated sterile tube that does not contain anticoagulants. if practicable, centrifuge the blood at 1,000 rpm for 10 min; pour off the serum into small screw-cap vials and store at approximately −18°c. if the serum cannot be separated immediately, rim the clot with a sterile applicator stick and refrigerate approximately 4°c to get maximum clot retraction if the specimen is to be stored unfrozen overnight. if centrifugation cannot be done, store the blood specimens in a refrigerator until a clot has formed, then remove the serum and transfer it with a pasteur pipette into an empty sterile tube. send only the serum for analysis urine. instruct patients to collect urine in the following manner. clean the area immediately around the urethral orifice with a paper pad that has been pre-moistened with 4% tincture of iodine or other appropriate antiseptic. then begin to urinate into a toilet and collect 30 ml (about 1 oz) of midstream urine into a sterile bottle. use either a second antiseptic-moistened pad or an alcohol-moistened cotton ball or tissue to clean any drops from the top or side of the bottle. other instructions. follow applicable instructions given in table e . before or immediately after collecting clinical specimens, use waterproof permanent markers to label each container with the patients name, complaint number, case identification number, specimen number, date and time of collection, tests requested, and other appropriate information. tightly seal all containers. clinical specimen collection report for each specimen. complete form e (clinical specimen collection report). the complaint number, case identification (id) number, and specimen number must be entered on each report so that laboratory results can later be correlated with other data. on form c1 record the type of specimen collected, and submit both the specimen and a copy of form e to the laboratory. send a copy of the laboratory report to the patient's physician or call if urgent. if the patient/case or other household member collected any water, ice, or beverage as instructed during initial contact, label containers with the complaint/outbreak and sample numbers. proceed as instructed in table f (general instructions for collecting water samples for microbiological analysis) and complete form f (water/ice collection report) and/or forms g3-g8 as applicable. record conditions of collection as called for on the forms. if a hypothesis associates the illness with water, caution these persons not to use the water source unless the water is first boiled and to discard all previously prepared ice and water-containing beverages until notified otherwise. develop a working case definition to classify exposed persons as either cases or non-cases. start with the most specific symptoms (such as diarrhea and vomiting) rather than broader symptoms such as nausea or malaise. for example in an outbreak of gastroenteritis, a case might be defined as a person from whose stool a specific pathogen was isolated. it may be a person who was at risk and developed diarrhea within a specified period of time. diarrhea will have to be defined, perhaps as three or more loose, watery stools during a 24-hour period. in some cases, a particular pathogen responsible for the outbreak might have been identified from clinical specimens. a case definition, which is developed later in the investigation, might include either a person having specific signs and/or symptoms within a period of time or a person from whom a specific pathogen was isolated. the ultimate case definition has a tremendous impact on the investigator's ability to make illness and exposure associations and to calculate probability of these associations. sometimes the first symptom or sign provides a clue to developing a case definition. information in tables b, c, d, g, and h can be useful in making case definitions. compare newly identified cases with the definition to see whether each is part of the outbreak. classify cases into categories: • a confirmed case is a person with signs and symptoms that are clinically compatible with the disease under consideration and for which there is either (a) isolation of an etiologic agent from (or otherwise identified in) an appropriate specimen from the patient, or (b) serologic evidence of a fourfold or greater rise in convalescent antibody titer. a confirmed case must also have possible exposure to the etiologic agent within the incubation period of disease. see table e . criteria for confirmation of etiologic agent responsible for outbreaks of waterborne illnesses for definitions of confirmed cases for specific waterborne diseases: • a presumptive case is a person with signs and symptoms that are clinically compatible with the disease under consideration, and for which there is laboratory evidence of infection (e.g., an elevated antibody titer but less than a fourfold increase), but the etiologic agent was not found in specimens from patients or no specimens were collected. a presumptive case must also have possible exposure to the etiologic agent within the incubation period of disease. • a suspected case is a person with signs and symptoms that are clinically compatible with the disease under consideration and history of possible exposure, but laboratory evidence is absent, inconclusive or incomplete. • a secondary case is a person who became infected from contact with a primary (outbreak-associated) case or from a vehicle contaminated by a primary case. onset of illness for secondary cases typically is one or more incubation periods after the outbreak-associated cases. it is not essential, however, to classify cases into these categories. do so only if it aids in developing a final case definition or in making comparative analyses of data. consider doing analyses using case definitions of both confirmed and combined confirmed, presumptive, and highly suspect cases, and compare the results. make a preliminary evaluation of the data collected as soon as possible. if you decide that there is an outbreak, use the information you have to develop a hypothesis about the causal factors. an outbreak is an incident in which two or more persons have the same disease, have similar clinical features, or have the same pathogen (thus meeting the case definition), and there is a time, place, or person association among these persons. a waterborne outbreak is traceable to ingestion of contaminated water or ice or to contact with contaminated water. a single case of either chemical poisoning or a disease that can be definitely related to ingestion of drinking water or contact with water can be considered an incident of waterborne illness and warrants further investigation. waterborne methemoglobinemia in an infant who resides in a rural area having a high concentration of nitrates in well water is an example of a single case of waterborne illness due to ingestion. a rare diagnosis such as primary amebic meningoencephalitis in a person who swam in a body of freshwater and inadvertently ingested the ameba, naegleria fowleri, through the nose is an example of a single incident related to water contact. sometimes it will be obvious from an initial report that an outbreak of waterborne disease has occurred simply because of the number of persons displaying certain signs and symptoms at or near the same time. many complaints, however, involve illness in only one or a few persons. it is often difficult to decide whether ingestion or contact with a particular water source and onset of illness was associated or coincidental. certain diseases that are highly communicable (e.g., shigellosis and epidemic viral gastroenteritis) may result in secondary infections from person-to-person spread or from subsequently contaminated food or water. however, if complaints are received from several persons who are associated with ingesting water or contact with water at the same place, water is likely to be involved. routine review of the log pertaining to potential waterborne illnesses for similar complaints can often be useful in detecting time, place or person associations. an investigation may also proceed based upon the suspicion of an intentional contamination of a water source. a time association exists if the time of onset of similar illnesses is within a few hours or days of each other. place associations exist when persons have ingested water from a particular single source, have swum in, worked in or otherwise been exposed to the same water, have attended the same event, or reside in an area common to all. person associations indicate a shared personal characteristic, such as being of the same age group, sex, ethnic group, occupation, social group, or religion. waterborne illnesses transmitted by a community water supply usually afflict persons of both sexes and all ages throughout the community. non-community water sources, such as bottled water, ice, water from individual wells, or water from areas of recreation should also be considered when making associations. keep in mind that water can contaminate foods during washing or freshening, and it can contaminate utensils and vessels that are used to handle or store foods. water may therefore be a source of contamination of another vehicle. also, water can be ingested as aerosols generated by shower heads, whirlpools, hot tubs, fountains, cooling towers, and irrigation devices. once some of these associations become obvious, question other persons who could be at risk because of their time, place, or person associations with the ill persons. from time, place, or person associations that have been established or suggested by the investigation, formulate hypotheses to explain (a) the most likely type of illness, (b) the most likely vehicle involved, (c) where and the manner by which the vehicle became contaminated, and (d) other possible causal relationships. the section "collection and analysis of data" describes calculations that can aid in the formation of these hypotheses. test hypotheses by obtaining additional information to support or reject them. if the hypothesis includes food contamination, the instructions given in the manual, procedures to investigate foodborne illness, might be useful. guidelines for confirmation of waterborne outbreaks are presented in table g and guidelines for confirmation that water is responsible for illness are presented in table h . if there is strong evidence to support a hypothesis that the outbreak is waterborne, take precautionary actions. the choice of action is dictated by the (a) suspected causal agent, (b) size of the water source, (c) availability of alternate water sources, and (d) expected use of the water. on the basis of available information, estimate the population at risk and engage any public relations staff with your organization to help inform all persons potentially impacted. when dealing with a microbiological contaminant or agent, consider issuing a boil-water advisory with water treatment guidelines (e.g., heating water in a covered container to a rolling boil for at least 1 min and keeping it covered until use). other options that can be explored include chlorinators that can be installed in individual and non-community systems. for community and non-community supplies in which chlorine is already used, increasing the chlorine dosage and opening hydrants and taps to draw the super-chlorinated water through the whole system might be an option. increasing chlorine is sometimes not effective because the chlorine contact time is too short or super-chlorinated water does not reach some parts of the system. furthermore, chlorination is ineffective against cryptosporidium oocysts and requires a long contact time to kill other human pathogens like hepatitis a virus and giardia. for suspected chemical contamination contact a specialist for further assessment and remedial strategies, such as activated charcoal filters. as a last resort, shut off the contaminated system until the source of contamination is found and controlled. be cautious when you take this drastic measure, because it may do greater harm than good by causing lack of water for hospitals, nursing homes, or for firefighters to extinguish fires. if the water is shut off or the treatment facility or distribution system disrupted (as in the case of floods or other disasters), consider means to distribute water from an alternative source to healthcare facilities and homes. if an illness could have resulted from water contact, close the offending water source, post warning signs around it, and patrol the area. where there is a swimming pool, hot tub, spa, fountain, or whirlpool, evaluate the recirculation system and its operation. it may be that increasing disinfectant concentration by super-disinfection could resolve the problem. where there may be chronic operational problems, evaluate ph, disinfectant concentration, and bacteriological laboratory records. choose your course of action, including consultation with appropriate professional experts, depending on the contributing factors existing at the time of investigation. verify the effectiveness of these actions (e.g., boil-water advisory, superchlorination, provision of alternate water source) to protect public health by monitoring illness levels in the population to determine if the outbreak terminates. if the outbreak continues unabated, consider the possibility of other transmission routes. also, verify the effectiveness of repairs to the water system, super-disinfection, and other actions by closely monitoring the quality of the water supply or recreational water to determine if laboratory reports indicate that the water is now safe for consumption or contact. if there is a public health threat, work with any available public relations staff to announce the outbreak in the mass media so that the public who consumed or was otherwise exposed to the implicated water can be alerted to take appropriate action including seeking medical consultation or treatment. provide only objective factual information about the outbreak. coordinate among the investigating agencies to assure that a consistent and accurate message is delivered. it is easy for agencies to miscommunicate before and during a water crisis (see box 1, false alarm; box 2, the walkerton outbreak; box 3, the flint water crisis). it is often preferable to have one spokesperson for all agencies. do not release preliminary information that has not been confirmed. the person giving information about an outbreak should be well informed about the etiologic agent being investigated and prepared to deal with questions. if the health hazard warrants a public warning at the hypothesis stage, tell the public why emergency measures are being invoked and that subsequent information may be cause to modify the action. as the investigation proceeds and the etiologic agent is confirmed and contributory factors are identified, consider terminating emergency measures, and give advice on specific control and preventive measures. attempt to reach all segments of the population at risk; this may require communication in multiple languages. route all news releases or statements to all persons involved in the investigation. in situations involving large outbreaks or highly virulent or toxigenic etiologic agents, set up an emergency hotline for the public to call to ask questions. this is likely to occur if there is an intentional contamination event where there is high publicity and public concern. train staff to handle these calls in a consistent manner so that the advice is the same who gives it. faulty information derived from poorly tested hypotheses can lead to severe political, legal or economic consequences. an example of this occurred in sydney, australia, in 1998 when an apparent water contamination event was publicized for the public to take precautionary actions. the false alarm was costly because of rebates to water customers, additional water testing, and for hiring extra staff, as well as a loss of confidence in the facility (see box 1, false alarm). they may then be disseminated by the mass media with inappropriate interpretations of the public health significance. furthermore, this information may be used as an unrealistic base for water programs or water regulations because of either misinterpretations or pressure from misinformed consumer-advocate groups. all involved parties should follow a written protocol for cross-agency communication and release of information to the public. unreasonable delays are unacceptable. test hypotheses by obtaining additional information to either confirm or refute their validity. do this by case-control or cohort studies, additional laboratory investigations, and on-site investigations (e.g., laboratory reports of water testing). sydney water (a new south wales state-owned corporation) supplies 1600 million liters of water each day to 1.5 million properties in sydney and its outlying areas. the city has a large and complex catchment with nine major dams and several storage reservoirs. about 21,000 km of water main, almost 200 pumping stations and many tunnels deliver water from four main river systems. the water is filtered through eleven treatment plants. seven are owned by sydney water and four are privately owned. these plants provide 90% of sydney's drinking water and one plant, prospect, provides up to 80%. in 1998, the quality of sydney's drinking water came under acute review when giardia and cryptosporidium were found in the city's main water supply at the warragamba dam. initially, low levels of these parasites were first detected in the water supply on 21 july, but these were within the acceptable health limits. in days following, much higher levels were recorded, and on july 27 the first "boil water" alert (in which residents were instructed to boil their tap water before use) was declared for the eastern central business district of sydney. however, by late on july 29 high readings were found in samples at the prospect filtration plant, in a reservoir and at a location further down the system, and a "boil water" alert was issued for the south of sydney harbour, and on july 30 a sydney-wide "boil water" alert was issued affecting most of sydney's residents. on august 4 the warning was discontinued. however, high levels were again found on august 13 (the second event), although it was believed that most organisms would likely be dead. more positive readings were found on august 14, although at lower levels. further contamination was identified on august 24 and an extended boil water alert was again declared. this was progressively lifted suburb by suburb until further contamination was reported on september 5 (the third event). a 2-week alert was then instituted, which was finally lifted on september 19. it was determined that the parasitic contamination was caused by low-quality surface water entering the dam. this contaminated source was attributed to moderate rainfall in july, followed by heavy rainfall in august and september which caused intermittent supplies of the raw water to enter the dam. despite high levels of cryptosporidium (up to >12,000 oocysts) and giardia (up to >7600 cysts) being recorded in july and august, 1998, no increase in human cryptosporidiosis or giardiasis was detected in the exposed population. the incident was highly publicized and caused major a public alarm because the number of people affected, the on and off boil water alerts, and the fact that the filtration plant had been advertised as one of the best in the world. the economic and political repercussions were extensive. the cost of the crisis to sydney water was estimated at a$33 million which included $20 million paid in rebates to customers, $13 million in lost revenue, water testing and staff costs and at least $2.5 million for damages claims. these costs exclude those relating to improvements to the system and infrastructure. the lack of cases of cryptosporidiosis, giardiasis or other water-related health problems led to suggestions that the parasites were either not an infectious type, or not as extensively distributed. an inquiry after the event revealed the publicity as an exaggeration of fact, with australian water technologies, part of sydney water, severely overestimating levels of cryptosporidium and giardia present in the water, with the recorded levels exposed to consumers as not harmful to human health. the handling of the crisis by state-owned sydney water was heavily criticized, causing the resignation of both the chairman and the managing director, and bringing up issues of private vs. public ownership and scientific uncertainty. the eventual consequence of the state inquiry was the establishment of the sydney catchment authority in 1999 to assume control of sydney's catchments and dams, while sydney water maintained responsibility for water treatment and distribution and for sewage collection, treatment and disposal. if an outbreak investigation requires resources beyond your agency's capacity, request assistance from other health professionals. it is desirable to have a team including, if feasible, an epidemiologist, an engineer, a microbiologist, a sanitarian/ environmental health office/public health inspector, a chemist, a physician and others, to undertake a detailed waterborne illness investigation. such personnel can usually be provided by local, state/provincial, or national agencies concerned with health, environment, or agriculture, depending on the expertise needed. for events suspected to arise from intentionally contaminated food, contact emergency response or law enforcement agencies. continue to search for and interview both ill persons who have had time, place, or person associations with the identified cases (see the section on "make time, place, and/or person associations"). review recently received complaints in the water-related complaint log (form b). contact other nearby health agencies, hospital emergency rooms, elderly care centers, and local physicians to discover other epidemiologically related cases. call previously contacted persons to see whether they know anyone else who has become ill or had a common association suggested by data in the log. the illness you are investigating may be part of a larger multijurisdictional outbreak, and therefore communicate with adjoining local and state agencies to learn if they are seeing similar illnesses. state or provincial public health agencies can check reportable disease records and state/provincial public health laboratories can start looking for clusters in isolates that they are characterizing. for outbreaks where intentional contamination of water is suspected or confirmed, public health and law enforcement agency officials may conduct the investigation jointly. if it becomes apparent that an outbreak is associated with a specific water supply (source) or recreational water or event, use form d1 for recording information. at this stage of the investigation, interviews can be expedited by reviewing the event itself to stimulate each person's recall. ask about specific symptoms and signs that are known to be common to the syndrome, as well as, time of ingestion or contact with water and onset of illness. mention each source of water to which the person may have been exposed, and ask each person (whether a case and well persons at risk) which of the water sources had been ingested or contacted. the number of persons to be interviewed depends on the number exposed and the proportion of them who are probably affected; if fewer than 100 persons were at risk, try to interview all of them; if several hundred are involved, interview a representative sample. be sure to obtain clinical specimens from these cases and well persons at risk (controls). it is more difficult to obtain positive results if symptoms from persons have ceased. there may be situations where self-administrated questionnaires are sent to cases and persons at risk. use either form c or form d or modified versions for this purpose. after questionnaires have been completed, summarize the data on form d. also, identify and interview secondary cases if they become apparent. because no two waterborne disease outbreaks are identical, the order of the expanded investigation may not always follow the outlined sequence of procedures. some investigative steps can usually be done simultaneously by different investigators. additional procedures may also be required. the principles and techniques described will suffice for most investigations. modify forms, if necessary, to accommodate the type and amount of information to be collected. make on-site observations. prove or refute hypotheses developed during the epidemiological portion of the investigation. focus on sources and modes of contamination and ways contaminants could survive and pass through water treatment. as applicable, conduct an on-site investigation of source (lakes, streams, areas around groundwater, etc.), treatment facilities, distribution lines, cross connections, water reservoirs, places of recreational water contact and/or sites at which aerosols were generated. such an epidemiologically focused investigation is quite different from sanitary surveys done during routine evaluations of water source sites, treatment plants or recreational water facilities. not all drinking water (even municipal and bottled water) is disinfected; so, it is important to identify whether the water source is treated and if so, how. some treatments (filtration, reverse osmosis, membrane treatments, riverbank filtration, and others) may not be complemented with a disinfection step. sanitary survey information can provide information about potential sources of contamination in the area of a usually pristine water source. microbiological records of a water supplier, particularly if any total coliform positive samples were found by the system in the last 6 months, may help identify a contamination pathway. if significant matters relating to water quality are observed or otherwise identified during the investigation, note them and communicate them to those responsible for the water system and to the proper authorities. do not lose the focus and objectivity of the investigation by confusing matters of quality and aesthetics with factors related to contamination by, and survival of, infectious and toxic agents. use the haccp-system, also known as systems analysis, way of thinking in your investigation. contact the person with the highest responsibility for the operation and maintenance for the implicated water source, water treatment facility, and/or distribution lines. identify the types of records that ought to be reviewed during the investigation and their likely source. do not forget that the responsible authorities also can have records (about water quality, if there has been a change of municipal water supply, industrial water pollution, wastewater pollution). they can be good sources of information about recent pipe breaks and other water system issues that could be related. in many cases they will be aware of the potential for contamination upstream of source water intakes. if applicable, obtain water distribution maps and recent water quality reports from appropriate departments. if you are not familiar with the community in which the investigation is to be done, obtain maps of the area to locate streams, lakes, water treatment facilities, and other community features that might have a bearing on the investigation. check if there are water protection areas and their rules. get plans and specifications on design of treatment facilities from consulting engineers or state agencies that approve these facilities. contact weather bureaus, airports, radio/television stations, or newspapers for information on heavy rainfall, flooding, extremely low temperatures, droughts, or other unusual weather conditions that preceded the outbreak, if this information is unknown to investigators. contact police or fire departments about traffic accidents, which can be the source of the outbreak. review all background data pertaining to the suspect water. as information is gathered, record it on applicable parts of form g. discuss with laboratory personnel that a field investigation will be made, and get their suggestions regarding samples and specimens that should be collected (see tables e and f) . confer with them about special analyses, media, and sampling procedures; make arrangements for rapid transport of samples to the laboratory. the samples must maybe be transported at the right temperature. pick up appropriate forms and sample collection equipment (preferably preassembled in a kit-see table a ). the laboratory can probably help assemble this kit. during the investigation, identify factors that contributed to contamination and survival of the etiologic agents and perhaps also to their growth or amplification or another cause of the outbreak. identified factors and situations that have contributed to waterborne disease outbreaks include those listed in table 2 . focus the investigation on the potential situations listed in table 2 , as applicable. remember that other possibilities can occur. describe circumstances that contributed to contamination and that permitted the etiologic agent to survive so that it reached drinking, agricultural, industrial, or recreational water. also describe circumstances that allowed pathogenic bacteria or algae to multiply in the water. write your findings down on the back of form g1 (illustration of contamination flow) or on a separate sheet. continually update the listing in table 2 with newly available data. introduce yourself (who you are, where you come from, who ordered you there) to the owner, resident, or persons in charge and state your purpose, when you arrive at the place of the suspected contaminated water source. emphasize that your visit is to confirm or eliminate suspicion that this water was a source of illness. tell him or her that a complete epidemiologic study is in progress and that other possible sources (such as food) will be investigated as well as operations of this site. explain that your investigation is not to fix blame but to identify the cause of the outbreak. emphasize that the findings can yield benefits related to the ability to identify needed improvements, to educate staff and to provide public support. try to create an atmosphere of cooperation. maintain an open mind and try to answer all questions. if you can not answer a question, tell the person that you will come back with an answer. come back to the person within 1 or 2 weeks even if you do not have any new information. give the person your phone number and e-mail address and tell the person to contact you if the person has more information later. privately interview key persons responsible for operating or repairing water facilities. do not forget to interview persons from other work shifts. identify persons who were working there at the likely time of contamination and have since left and interview them as well. ask questions to determine the flow of water and operations from intake through distribution through plumbing systems. ask about any changes in operation, unusual events in the watershed or repairs to the water facilities. ask if you can check records, both in paper form and on the computer (monitoring system), analyses of results, and/or incident reports. plant personnel may not describe water treatment or installations exactly as they existed at the time that a mishap occurred. they may fear criticism or punitive action as a result of their possible role in the causation of the outbreak. their descriptions should be plausible and should account for possible sources and modes of contamination and indicate possibilities for survival of pathogens. if a description does not contain all the information desired, reword questions and continue the inquiry. confirm accounts by private interviews with others knowledgeable of water treatment or operation of the facility. be alert for inconsistencies among the accounts told by different persons. seek resolution of discrepancies in accounts by watching actual procedures as they are being carried out, by taking appropriate samples, or by conducting experiments. a communicative working relationship between the plant management and the investigator influences plant workers' attitudes toward the investigative team. consider the position, feelings, and concerns of the manager and staff; defensive reactions are normal on their part. diagram each phase of the water system or situation under study on form g1 (illustration of contamination flow). insert special symbols and notes for all sites that might be involved in introducing contamination to the water or where contaminants might have survived treatment. record other information gathered on the appropriate parts of forms g2-8. review and collate appropriate information on quality control and operational records from the water utility and from responsible agencies. as applicable, obtain information on quality of untreated surface or groundwater from a local, state/ provincial, or national pollution control or geological survey agency. also, seek water distribution maps, well logs, descriptions of geological conditions and indices of groundwater quality from them. for surface water supplies, obtain information on upstream discharges and unusual events that may have affected raw water quality. get data on finished water quality in the distribution system from a local, state/ provincial water surveillance or regulatory agency. water suppliers also frequently have records of raw and finished water quality. review data on quality control tests (e.g., ph, chlorine residual, chlorine demand, bacteriological and chemical tests, turbidity, jar test data, incident reports) that are available. obtain data on cross connection control programs and sewer repairs from the water supplier or other local agencies (e.g., building inspectors, sewage departments). review files for data concerning potential sources of contamination for individual or semipublic water supplies (e.g., diagrams of septic tank systems, sewer line locations, well logs, small individual wastewater plants, accidental industrial pollution of water, traffic accidents involving chemicals, salting of roads or sawmills). check if they have any haccp-systems or water safety plan and, if so, how they monitor their ccps (critical control points) and if they are implementing control measures. ask them about haccp, to see if they understand the system and if results are documented. check if the haccp-system is validated (should be documented) and that they are conducting internal audits. get information on all aspects of normal operations as well as unusual events or conditions to determine whether such events were coincidental with the time of suspected contamination as determined from the epidemic curve. also, consider the time it takes for a contaminant in the raw water or treatment plant to reach households in the affected community. ask responsible persons for this information. compare data on heterotrophic plate and total coliform counts of raw and finished water leaving the treatment facility and of water in distribution lines. also, compare data on chlorine residuals within the plant with that in distribution and check, if they have, that the uv-light is functioning. review other test data (e.g., turbidity and chemical analyses) that may indicate a problem situation. identify locations and dates of sample collection. take photos, if it is allowed, of things you suspect are not right. go back more than one incubation period of the disease under investigation. record this information on form g2 (record review of on-site investigations, and test results prior to and during outbreak). photocopy appropriate records for confirmation and subsequent review and attach them to the record review form. be alert for evidence of falsification of records. while reviewing records, watch for evidence of the following: • potential contamination of groundwater systems because of proximity to septic tank systems, latrines, animals manure or landfills, industrial contamination of the water supply, small sewage plants, especially old and nearly forgotten ones, and recent heavy rain • high heterotrophic plate or coliform counts, or counts that exceeded the average (median) or typical count • sudden changes in water quality or operating practices that suggest the possibility of contamination or treatment failure • high turbidity, unusual odor, color, or taste, or high coliform counts in raw water, which can indicate potential overloading of the normal treatment process • high levels of ammonium, nitrate and nitrite, which can indicate organic and inorganic contaminants • low chlorine residuals in treated water or higher-than-normal amounts of chlorine used, which can indicate either a high chlorine demand or a sudden high level of contamination • a sudden drop in amount of disinfectant used, possibly indicating failure or interruption of a disinfection process. no functioning alarm system • a sudden change in the amount of a chemical (e.g., alum or ferric sulfate) used, suggesting equipment disfunction or inadequate coagulation or flocculation and thus poor filtration • lack of treatment chemicals if a more corrosive water supply is used (see box 3, the flint water crisis) • pump failures, draining of distribution lines or reservoirs, or massive pumping to fight fires, which can produce low pressures that can cause contamination through cross connections or back siphonage • repairs to water mains, wells or pumps where contamination could have been introduced record this information on form g2 or other appropriate form in the g series. as applicable, investigate the water source, treatment facility, distribution and plumbing systems, sites where water was contacted, and sites at which microorganisms amplified and aerosols disseminated. use forms in the g series as guides while observing facilities, gathering data, making measurements and collecting samples. google or bing maps or other similar resources' views of the watershed can be very helpful in identifying potential sources of contamination that you will need to investigate further. these maps can also facilitate your own map and diagram making on form g1. the water source may be surface or ground or in some cases a combination of the two. verify this by observations at the site and by talking to the property owner or persons responsible for operation or maintenance of water supply or recreational facilities, as applicable. examination of "weather events" such as heavy rainfall may indicate a potential for surface water contamination (see box 2, the walkerton outbreak). when a surface water is either suspected or implicated as the source of a contaminated supply, get information about the watershed concerning possible sites of contamination of the suspected etiologic agent. this includes, but is not limited to (a) land use, (b) sewage effluent from treatment plants and septic tanks, (c) industrial plants that may be discharging toxic waste, (d) mining wastes, (e) landfill leachates, (f) slaughterhouse discharge wastes, (g) animal feed lots, (h) both domestic and wild animals that use the source water for drinking, (i) sludge disposal from sewage treatment plants or septic tanks (e.g., land spreading or lagoons), (j) storm water discharge. if this information is not available from records or persons familiar with the site, visit the site and observe possible sources of contamination and pollution (e.g., while traveling by foot, vehicle, boat, or helicopter, as applicable). record this information on form g3 (source and mode of contamination of surface water). diagram the surface water and sites of contamination on form g1. note type and location of sources of contamination and their distances from the water. visit groundwater sources. using form g4 (source and mode of contamination of groundwaters) as a guide, question owner or operator and inspect groundwater installations to ascertain character of the land and surface and subsurface soil and water. when a well or improved spring is under consideration as the source of the contaminated supply, observe its location relative to possible sites of contamination and to whether its construction allows contaminants to reach the water. determine locations of all sewage outflows or disposal sites (e.g., septic tanks and absorption lines, cesspools, privies, and other sewage disposal facilities), gradients, and distances from the well or spring. determine the type of soil at the site. if the soil is limestone or fissured rock or if there is a high ground or perched water table, pollution may travel many miles. in this case, the search for sources of contamination may have to be expanded for a considerable distance from the well or spring. ascertain whether there were heavy rains, heavy snow melts, or sudden discharge in may, 2000, many people in walkerton, a small ontario, canada, community of about 5,000 people, began to simultaneously experience bloody diarrhea and other gastrointestinal infections. on may 8-12, torrential rain had unknowingly contaminated the town's water system, but operators failed to check residual levels for a period of several days, allowing unchlorinated water to enter the distribution system. however, the privatelyowned walkerton public utilities commission insisted there was no problem with the water despite other laboratory tests showing evidence of e. coli contamination. illnesses began about may 18, with the first death occurring on may 22 and the seventh and last on may 30. by may 21, however, many more cases had been diagnosed, the infectious agent determined to be e. coli o157:h7, and contaminated well water was confirmed as the source of the e. coli; all this allowed the region's medical officer of health to issue a boil water advisory, warning residents not to drink the tap water. two days later, laboratory results identified the presence of campylobacter and e. coli o151:h7 and dna testing showed that the contaminating source was a cattle farm a short distance from a well used for the water supply. by the time the outbreak was over, >2300 were ill and 7 had died. the people who died directly from drinking the e. coli-contaminated water might have been saved if the walkerton public utilities commission had admitted to contaminated water sooner. those in charge of the water utilities at the commission had no formal training in their positions, retaining their jobs through three decades of on-the-job experience. they were later found to fail to use adequate doses of chlorine, fail to monitor chlorine residuals daily, make false entries about residuals in daily operating records, and misstate the locations at which microbiological samples were taken. regulations state that water suppliers are required to treat groundwater with chlorine to sufficiently neutralize contaminants and sustain a chlorine residual of 0.5 mg/l of water after 15 min of contact. had utility operators adhered to the protocol, the disaster most likely would have been averted. the operators knew that these practices were unacceptable and contrary to ministry of environment guidelines and directives; they eventually admitted falsifying reports and were sentenced to short jail terms. the ontario government was also blamed for not regulating water quality and not enforcing the guidelines that had been in place. the water testing had been privatized in october 1996. an enquiry found that the water supply, drawn from groundwater, became contaminated with the e. coli o157:h7 strain from manure from cattle on a farm washing into a shallow water supply well after heavy rainfall. the risk of contamination from farm runoff into the adjacent water well had been known since 1978. key recommendations from the enquiry included source water protection as part of a comprehensive multibarrier approach, the training and certification of operators, a quality management system for water suppliers, and more competent enforcement, which were incorporated into ontario new legislation. the bottom line of the enquiry was that officials and municipal water facilities operators and managers across north america need to recognize public waters are a most valued but vulnerable public resource. investment in keeping them safe and secure needs to be a community top priority. from dams that could have resulted in flooding within the duration of the incubation period of the disease under investigation. obtain information on the depth of the well in reference to the ground water table from the owner or by referring to any available well logs on public file or from local drillers. observe well construction and get information about depth of casing, depth and method of grouting, and whether there is an underground discharge. observe whether there is an impervious well platform and whether the pump or casing seal was subjected to flooding. illustrate the situation by showing location of the well in this event is considered a disaster, still unfolding, initiated from a political decision to save money, and ending up with acute and chronic illnesses and deaths to residents of a michigan city, as well as high system remediation and health-related costs to the taxpayer. on april 24, 2014, flint, genesee county, michigan, switched its water supply from detroit's system to the flint river as a cost-saving measure for the struggling, majority-black city on the recommendation of the state-appointed emergency manager. flint agreed to separate from the detroit water and sewerage department and go with the karegnondi water authority, including the decision to pump flint river water. this was to be an interim measure until a new pipeline from lake huron was constructed in 2016 to serve the region. soon after the switch, residents begin to complain about the water's color, taste and odor, and to report rashes and concerns about bacteria. in august and september 2014 city officials issued boil-water advisories after coliform bacteria were detected in tap water. in october 2014, the michigan department of environmental quality (mdeq) blamed the cold weather, aging pipes and a population decline. in the same month a general motors plant in flint (continued) reference to possible sites of contamination on form g1. note distances between the well and contamination sites and elevations. determine whether any pumps were out of order or had been repaired during the interval of concern. if priming of the pump was done, find out the source of the water used. record this information on form g4. test hypotheses of modes of contamination by conducting a dye test and/or sampling the water. (see appropriate sections of this manual for directions.) collect samples of water from these sites and submit them for analysis of the suspected etiologic agent or for any physical, bacterial, or chemical tests that will provide evidence of contamination or movement of the contaminants. (see procedures for collecting water samples) record these results on forms g3 or g4 and i (laboratory results summary). when appropriate, confirm hypotheses by a dye or other tracer test. (see section on this subject). determine the means by which the etiologic agent survived treatment or was otherwise not eliminated or inactivated. consider the treatment process as a series of barriers placed between contaminants and consumption of the treated water. the operation of each barrier should be optimized. review available data for each step in the treatment process. records of well-maintained and properly calibrated continuous monitoring equipment will be especially valuable. look for failures in the barriers, which could include (a) lack of disinfection, (b) inadequate concentration of disinfectant or contact time, (c) interruption of disinfection, (d) inadequate filtration, (e) lack of corrosion inhibitors, which may follow inadequate pre-filtration treatments. in 2015 in flint, michigan excessive levels of lead were found in drinking water from corrosion of water distribution pipes (see box 3, the flint water crisis). corrosion inhibitor had not been added. also, look for possible introduction of contaminants within the treatment process, such as in treatment chemicals. stopped using municipal water, saying it was rusting car parts. on january 4, 2015, the city announced that flint's water contained a high level of trihalomethanes, a byproduct from increased disinfection by the city. though this is in violation of the safe drinking water act, officials told residents with normal immune systems that they have nothing to worry about. in january 2015, detroit's water system offered to reconnect to flint, waiving a $4 million connection fee but the offer was declined by the emergency manager. by february, state officials continued to play down any water problems saying that the water was not an imminent "threat to public health." on february 18, 104 parts per billion (ppb) of lead were detected in drinking water at a flint home and the federal environmental protection agency (epa) was notified. the epa does not require action until levels reach 15 parts per billion, but science indicates that there is no safe level for lead in potable water. officials from epa and mdeq discussed the lead level in the sample, and epa found that the state was testing the water in a way that could profoundly understate the lead levels. on march 3, 2015, a second testing detected 397 ppb of lead in flint drinking water. a consultant group hired by flint, reported that the city's water met state and federal standards, and it did not specifically report on any lead levels. in may, tests revealed high lead levels in two more homes in flint. in july, an epa administrator told flint's mayor that "it would be premature to draw any conclusions" based on a leaked internal epa memo regarding lead. however, in september, flint was asked to stop using the flint water supply or consider corrosion control for it, because it was causing lead to leach from the water pipes and children had high levels in their blood. state regulators insisted the water was safe. nevertheless, on october 1, the governor of michigan ordered the distribution of filters, the testing of water in schools, and the expansion of water and blood testing after a briefing on the lead problems with the mdeq and federal officials. at the same time, flint city officials urged residents to stop drinking water. on october 16, flint reconnected to detroit's water system, and residents were advised not to use unfiltered tap water for drinking, cooking or bathing. on october 19, the director of mdeq reported that his staff had used inappropriate federal protocol for corrosion control, and soon after, the governor announced that an independent advisory task force would review water use and testing in flint. on december 9 flint added additional corrosion controls, and soon after an emergency was declared. at the end of december, the task force found that the mdeq was accountable for its lack of appropriate action, and the director resigned. on january 16, 2016, the governor asked the national guard to distribute bottled water and filters in flint, and president obama declared a state of emergency in the city and surrounding county, allowing the federal emergency management agency to provide up to $5 million in aid. three days earlier the crisis expanded to include legionnaires' disease, because of a spike in cases, including ten deaths, after the city started using river water. on january 21, the michigan department of health and human services stated it did not have enough information to conclude that the increase in cases was related to the ongoing flint water crisis, although the. head of michigan's communicable disease division had said three months earlier that the number of legionella cases at that time "likely represents the tip of the iceberg." as of february 2016, the number of reported cases was close to 100. a flint hospital official was surprised that michigan and local health agencies did not inform the public about the legionnaires' outbreak in genesee county in 2014-15 until january 13; the hospital earlier had spent more than $300,000 on a water treatment system and bought bottled water for patients. the source of legionella is not known but it was likely in the flint river, and possibly extensive flushing of flint's colored water, which had undesirable odors and tastes, by residents may have caused chlorine residual in the pipes to be washed away, leaving the pipes susceptible to growth of the legionella; in addition, aerosols from the extensive flushing from turned-on faucets might have led to close contact between the residents and the pathogen. the investigation of the cause of the illnesses continues with criminal charges laid against michigan departmental employees. observe treatment processes from the water inlet to the finished water discharge. diagram on form g1 the treatment process; insert notations of hazardous situations that were observed. collect samples of water at the inlet, after each phase of treatment that may have functioned suboptimally or failed, and at the outlet. test the samples for pathogens that cause a syndrome characteristic of that being investigated, for indicator organisms and for physical and chemical characteristics of the water, as appropriate to the situation. evaluate effectiveness of the disinfection process and resulting residuals. determine the type of disinfectant (e.g., gaseous chlorine, hypochlorite, chlorine dioxide, chloramine, ozone, ultraviolet irradiation) used and whether the disinfection treatment was adequate for the volume of water treated. determine, by talking to water treatment plant employees and reviewing records of the plant or regulatory agency, whether there were any interruptions of disinfection during the two weeks prior to the first onset date. determine contact time between the point of addition of the disinfectant and the first point of use. measure the chlorine residual, ph and temperature of the water just before it leaves the plant. observe the condition, operation, and maintenance of disinfectant dispensing equipment. review plant records to identify any sudden changes in disinfectant demand that causes temporary depletion of disinfectant residuals and allows survival of pathogens. review maintenance records for disinfectant dispensing equipment and quality assurance records for online analyzers. record this information on form g5a (disinfection failures that allowed survival of pathogens or toxic substances). calculate disinfectant rate applied and usage (see formulae in form 5a). for example, to calculate disinfectant rate, if the flow rate is 1,000,000 gal/day and the dosage is 15 lb/day: the destruction of pathogens is dependent on (a) type and condition of microorganisms present, (b) type of disinfectant used, (c) concentration of available chlorine or other disinfectant, (d) contact time, (e) water temperature, (f) ph, (g) degree of mixing, (h) presence of interfering substances (which may be related to turbidity). utilize treatment records that provide small scale time resolution, such as online monitoring data, to determine whether the process was stable during the time period in question. daily averages may provide evidence of massive failures, but will not provide information about whether consistent treatment was being provided. in general, the relative effectiveness of microorganisms' resistance to free chlorine, from high resistance to low resistance, is as follows: • protozoan oocysts (i.e., cryptosporidium) • protozoan cysts (i.e., giardia, entamoeba histolytica) • viruses (hepatitis a virus, poliovirus) • vegetative bacterial cells (shigella, escherichia coli) protozoan oocysts are highly resistant to chemical disinfectants, but not to physical means such as uv light or ozone (gas). microorganisms within each group and strains among the same species differ somewhat in resistance. the state of injury induced by environmental impacts and selection of resistant strains influence survival. aggregation of microorganisms and/or close association with debris shield them to various degrees from lethal effects of disinfectants and attachment to surfaces such as pipe walls to form biofilms that protect organisms from inactivation by disinfectants. a measurement of microbiological inactivation by disinfectants is the ct value (ct calc ), which is the product of the free residual disinfectant concentration (c) in mg/l that is determined before or at the first user (customer) and the corresponding disinfectant contact time (t) in minutes (i.e., c × t). refer to table i (ct 99.9 values for inactivation of giardia cysts at different concentrations of disinfectants, temperatures, and ph values) and table i (ct values for inactivation of viruses at ph 6-9, at different temperatures with different disinfectants for comparing disinfectant efficiencies). make residual measurements during peak hourly flow. for comparisons of ct values between the indicated ph, temperature, and concentration values, use linear interpolation. (for example, for free chlorine, 10°c, concentration 1 mg/l, ph 7.5 = [166 − 112 = 54; 54/2 = 27; 112 + 27] = 139). if no interpolation is done, use the ct 99.9 value at the higher temperature, at the higher ph and higher concentration. a simple ct calculation, for example, using a disinfectant concentration (c) at the basin effluent of 1.3 mg/l and a detention time (t) of 22 min, is as follows: use this calculation for comparing to values in table i or j. the calculated ct value should be higher than the value stated in the table for specific conditions of disinfection, temperature, ph, and concentration (residual). in this situation, if the temperature of the water was 15°c, the ph 7 and the concentration of chlorine 1 mg/l, a ct value of 75 would be needed for a 99.9 reduction of giardia cysts. the ct value of 28.6 would have been inadequate to meet the criteria and could explain the survival of the pathogen under investigation. microbial inactivation efficiencies vary considerably among different disinfectants and are influenced by the characteristics of the water and water temperature. tables i and j show that, in general, ozone is more effective than chlorine dioxide, which is more effective than free chlorine, which is more effective than chloramines. also, in general, longer contact time increases the degree of inactivation, and higher water temperatures as well as lower ph values increase rates of inactivation. rapid mixing of the disinfectant with water increases disinfection efficiencies, whereas dissolved organic matter reacts with and consumes the disinfectant and forms products that have weak or no disinfection activity. certain inorganic compounds and particulate matter also react with disinfectants. the ct value must be determined sequentially whenever a disinfectant is added to water. contact time (t) is the duration in minutes for water to move from the point of application of the disinfectant or the previous point of residual disinfectant measurement to the point where residual disinfectant concentration (c) is measured. it is measured from the first point of disinfectant application and from all subsequent applications until or before the water reaches the first user. determine contact time in pipelines by dividing internal volume of the pipe by the maximal hourly flow rate through the pipe. determine the flow rate from (a) plant records, (b) continuous monitoring device readings, (c) measurements at hourly intervals, or (d) if this sort of information is unavailable, measurements at expected high flow periods. use tracer studies to determine contact time within mixing basins and storage reservoirs. these values represent only 90% effectiveness because of short circuiting. chlorine, fluoride, and rhodamine wt (but not b) are commonly used tracer chemicals. contact time is usually measured by a step-dose method, but a slug-dose method is used where chemical feed equipment is not available at the designated point of addition or where such equipment does not have the capacity to provide the necessary concentration. (see appropriate epa literature for procedures, and consider getting engineering expertise if these matters are too complex.) estimate whether pathogens had been inactivated. to do this, divide the ct calc value by a value (ct x% ) resulting in a certain percentage inactivation (e.g., 99.9% [3-log] or ct 99.9 for giardia cysts and 99.99% [4-log] or ct 99.99 for viruses). this gives an inactivation ratio. see table i for ct 99.9 values for giardia and table j for ct 99.99 values for viruses. following is a sample calculation for data in table i when water temperature is 20°c, ph in a clearwell (reservoir for storing filtered water) is 7.0, time (t) (either calculated or measured by dye test) is 38 min, and the disinfectant used is chlorine: the desired ct value for 99.9% inactivation of giardia for ph 7 at 20°c is between 52 and 68 depending on concentration of disinfectant. in this case, the disinfectant measured at the clearwell outlet is 2.0 mg/l. therefore, the result, 76, is larger than the value, 62, required in the table; hence, these disinfection concentration (c) and time (t) conditions should result in a 99.9% or greater inactivation of giardia cysts. for free chlorine, a 3-log inactivation of giardia cysts provides greater than a 4-log inactivation of viruses. the following example, using the data in table i , demonstrates a means to calculate the increased disinfectant dosage needed for a plant during the transition from summer to winter, when the water temperature fell from 15 to 5°c, chlorine dioxide was the disinfectant used and the t value (calculated or measured) is 12 min. using table i in this situation, the plant should have increased the chlorine dioxide concentration from 1.58 mg/l to 2.17 mg/l to maintain the same efficiency of disinfection. if this had not been done, it may explain the survival of pathogens in the water supply. the sum of these ratios gives the total inactivation ratio, which should equal 1 or more to provide effective disinfection. make calculations and record information on form g5a. the following example shows the way this is done. chlorine is added to three basins. chlorine concentration, contact time, temperature and ph are measured at these locations and recorded as shown in table 3 . data from table i is combined to do the calculation. the resulting sum exceeds 1.0. this ensures that the plant met the recommended or required ct. regulations may require that community and non-community public water systems that use surface water or water under direct influence of surface water meet a criterion (e.g., minimum of 99.9% [3-log] removal and/or inactivation of giardia cysts and a minimum of 99.99% [4-log] removal and/or inactivation of viruses of fecal origin that are infectious to humans). removal and/or inactivation of microorganisms may be accomplished by either filtration plus disinfection or disinfection alone, depending on the water source. water systems using chlorine with ct values that attain minimal level or inactivation of giardia cysts will result in inactivation of 99.99% (4-log) of viruses. evaluate the prefiltration processes (e.g., coagulation, flocculation and sedimentation). coagulation is a process that uses coagulant chemicals and mixing, by which colloidal and suspended materials are destabilized and aggregated into flocs. flocculation is the process that enhances agglutination of smaller floc particles into larger ones by stirring. sedimentation is the process by which solids are removed by gravity separation before filtration. observe whether these processes reduce turbidity. calculate detention (transit) time within the settling tank and seek information about frequency and method of cleaning the tank. for example, if an 8-ft-deep sedimentation basin has a volume of 1 million gal, and the plant flow rate is 20 million gal/day, detention time in the basin is: (in your country you may want to calculate rates based on metric measurements) several different types of filtration may be used in water treatment facilities. these are conventional, direct (both conventional and direct are referred to as "rapid" filtration), slow sand filtration, and diatomaceous earth filtration. conventional filtration consists of a series of processes including coagulation, flocculation, sedimentation, and filtration. direct filtration consists of a series of processes including coagulation and filtration but excluding sedimentation. slow sand filtration is a process involving passage of raw water through a bed of sand at low velocity (usually less than 0.4 m/h), utilizing both physical and biological means to remove particles and microorganisms. in diatomaceous earth filtration, water is passed through a precoat cake of diatomaceous earth filter medium while additional filter medium is continuously added to the feed water to maintain the permeability of the filter cake. if done properly, each filtration method results in substantial particulate removal. when rapid sand filters have a head loss of about 7-10 ft, they require back washing. filters are backwashed by reversing the flow of the filtered water back through the filter at a rate between 15 and 30 gal/min/ft 2 of sand-bed area. sometimes water jets at the surface aid in loosening and removing deposited material on the sand. observe an actual backwash and look for indications of short-circuiting or areas of the filter material that seem agglomerated or resist being cleaned by the flowing water. if backwash water is not discharged to waste, evaluate where it is released. slow sand filters eventually become clogged. when this occurs, a scraper or flat shovel is used to remove the top layer of clogged sand, and new sand (equivalent to the depth removed by scraping) replaces the old. test the effectiveness of filtration for each filter unit by observing capacity and filtering area relative to volume and turbidity of the filtered water. also, review turbidity, headloss, and filter rate record. look for anomalies, especially in the few hours after a filter is returned to service, and before the filter is backwashed. review criteria that cause a backwash to be initiated, and establish if these criteria were followed during the time preceeding the outbreak. determine the source of backwash water and the frequency of back washing of filters from records and head gauge readings. check whether the water used to back wash or clean filters came from an untreated source and determine the fate of the backwash water. in the case of illness due to chemical substances, evaluate types of chemicals used and condition, operation and maintenance of chemical feeding equipment. consider sampling backwash water for pathogens under investigation. review plant records for results of monitoring and be alert for changes that suggest treatment failure. record this information on form g5b (source of contamination and treatment failures that allowed survival of pathogens or toxic substances.) data in table k (estimated removal of giardia and viruses by various methods of filtration), give a summary of expected minimal removal of giardia and viruses in well operated filtration systems. values can be subtracted from ct values required for disinfection. although contamination is likely to be associated with raw incoming surface water, look for bypass connections where raw or partly treated water can get into treated water. also look for common walls that separate treated and untreated water. consider the possibility that a contaminant was introduced in any of the treatment chemicals themselves, or as an act of sabotage. determine whether any flooding has occurred during the interval of concern. check absentee records for possible enteric illness of the water treatment plant staff. such illness may reflect either sources of contamination or victims. record this information on form g5b. at domestic locations, evaluate treatment devices (such as chlorinators, filtration units, softening equipment) as described above, but modified to fit the situation under investigation. record observations and measurements on forms g5a and g5b, as applicable. the water distribution system can be complex, with multiple entrance points for treated water and different pressure zones in which water can enter but not leave. water flows in the direction in which it is being "requested," so can flow in different directions in the same pipes from one hour to the next. contaminated water can enter a potable water supply from a non-potable water supply when the two are directly connected. such interconnections are referred to as cross connections. to evaluate such situations, trace lines of the treated supply from the point of treatment or entrance into a building to points of use and associated plumbing. look for any interconnections of other water supplies, such as wells, waste lines or holding tanks for water intended for fire control. if cross connections are found, look to see whether backflow prevention devices are inserted between the lines and, if so, whether they are functioning properly. also, look to see whether there is an air gap between the water inlet and vessel or tank. evaluate the arrangement and operation of check valves on connections between the two water systems. review inspection report for backflow prevention devices. contaminated water can also enter a treated supply by siphonage from a contaminated vessel or sewerage to the potable water line having negative pressure. this is referred to as back siphonage. examine all water vessels to see whether they contain submerged inlets or hoses connected to water faucets, and if so, whether properly functioning vacuum breakers are in place. without proper air gaps or properly functioning vacuum breakers, there is a possibility of siphonage of water from plumbing fixtures in upper stories to lower stories when line pressure is negative. this may occur when faucets on lower floors are opened after the water supply valve has been turned off for repairs or when the supply line has had a sudden loss of pressure, as can happen with nearby heavy use of water (e.g., to fight fires or irrigate) or when pressure lines are broken. measure water pressure on upper stories of buildings to determine whether negative pressure occurs. (pressure losses may be transient and of very short duration.) interview building managers and residents about whether there were (a) any repairs of water service during the past month, (b) fires that occurred nearby, or (c) other situations that could have caused negative pressure in the water line. also, if appropriate, review fire and utility department records for information about these situations. get dates of line repairs to see whether they correlate with the time of incubation periods of early cases. measure chlorine residual (of chlorinated water systems) and take samples for microbiological tests at several strategic locations in the distribution systems. perform calculation on comparison of disinfectant residual. if a toxic chemical poisoning is under investigation, talk to home owner, building manager or maintenance staff about whether pesticides or other toxic compounds were sprayed with equipment connected to a hose or a sprinkler system. furthermore, interview building managers and residents about whether there are persons residing there who either are or recently were ill with diarrhea. they may represent sources of the etiologic agent or may identify victims. interview those identified about the onset of their illness and symptoms and examine their plumbing systems. record information obtained during the investigation of distribution and plumbing systems, and record related calculations on form g6 (source and mode of contamination during distribution and at point-of-use). evaluate implicated waters used for swimming, water skiing, bathing, clothes washing by hand, or agricultural activities, in a manner similar to that described under the section on investigation of surface water source. if the potential site of contact was natural surface water, determine whether the water is likely to be infested by parasites and look for the presence of snails (swimmer's itch). for swimming pools, measure the water's ph, chlorine residual, water temperature, and turbidity, if applicable. also, review pool records for previous information on these characteristics. high turbidity in pools, hot tubs, and spas is a sign of either poor filtration or inadequate disinfection. evaluate whether the resulting water would adequately protect those who swam or waded in it or had any physical contact with it. evaluate filter and chlorination equipment as described for water treatment. backwash filters and collect a sample to get an indication of microorganisms present on the filter (thus obtaining historical information). this approach has been useful for identification of pseudomonas aeruginosa. look for the presence of slime on tub, whirlpool, slide and pool surfaces, and collect some of this material for analysis for p. aeruginosa. if the answer is not obvious, ask ill persons whether they had puncture injuries or wounds or scrapes while immersed in water. record this information on appropriate parts of form g7 (contamination source and survival of pathogens or toxic substances for recreational waters). collect samples of the water (see section on "collect water samples"), and test them for pathogens and/or indicator organisms, as applicable. the agents listed in table d can multiply in water and if such water is aerosolized, they can be transmitted to human beings via the respiratory route. highly susceptible persons (e.g., the elderly, smokers, immunosuppressed individuals) are the usual victims. look for possible sites where water may have been or is being disseminated as aerosols. consider (a) air conditioning cooling towers and evaporative towers, (b) hot water systems (heaters and tanks), (c) shower heads, (d) faucets with aerators, (e) mist machines used to freshen fruits and vegetables in markets, (f) humidifiers, (g) nebulizers/respiratory therapy equipment, (h) whirlpools and spas, (i) dental drills and cleaners, (j) cooling water apparatus for grinders, (k) splash from hoses, (l) water pressure line breaks, (m) decorative water features, (n) outside misters, (o) other aerosol-producing devices. sample water from all suspect sites for legionella or other waterborne agents that may cause illness when inhaled. it is not possible to recognize by visual inspection the potential for water to be contaminated with legionellae. warm temperatures, especially those between 27°c (80°f) and 46°c (115°f), are conducive to growth of legionellae. additionally, stagnant water allows time for legionellae to multiply, especially in dead-end lines, reservoirs and hot water tanks, and in water trapped in shower heads and faucet aerators. if it is deemed appropriate or necessary to sample for detection of legionella in the environment, collect water samples from suspect sources. it is important to use a lab with proven expertise in isolating and characterizing legionella, such as those labs in the u.s. certified under the environmental legionella isolation techniques evaluation (elite) program. the centers for disease control (cdc) have a convenient form for recording case histories (http:// www.cdc.gov/legionella/downloads/case-report-form.pdf). it is not appropriate to sample air for detection of legionella hazards. it may, however, be appropriate to use micromanometers or smoke to trace direction of air flow to determine route of dissemination. micromanometers measure pressure differences, and flow can be assumed to travel from high to low pressure areas. smoke moves from areas of higher pressure to areas of lower pressure and is extremely sensitive to air currents. observe direction and spread of smoke movement. record this information on form g8 (contamination source and sites of amplification and aerosolization of pathogens). prior to the collection of samples, investigators should consult with the testing laboratory that will be used, to receive specific laboratory sampling instructions and sampling kits. sampling protocols for potable and non-potable sources are dependent on the specific etiological agent and the related analytical procedures performed by the testing laboratory. collect samples promptly to test for possible etiologic agents and for microorganisms indicative of fecal contamination. contaminants in water are in a dynamic state; their presence and quantity differ with time and place. see table f (general instructions for collecting drinking water samples) for guidance on collecting and shipping samples for viral, bacterial, and parasitic analyses. samples for bacteriological tests can be collected in one of three ways: (a) by letting a stream of water flow into a container or by submersing a container into a volume of water, (b) by passing a large volume of water through a filter, (c) by putting moore swabs (see table a for description) or similar absorbent materials in surface water or drains for a few days (see table f ). use bottles that have been cleaned, rinsed, and sterilized, or use sterile plastic bags to collect and store samples for bacteriological examination. for a chlorinated water supply, or when in doubt about the presence of residual chlorine, use bottles containing 100 mg/l sodium thiosulfate to combine with any free chlorine in the sample and prevent lethal effects of chlorine on microorganisms in the sample. this compound will not interfere if used for non-chlorinated water. when collecting water samples, first try to get "historical" samples that might give an indication of the condition of the water at the time it was ingested by those who became ill. obtain historical samples from water in bottles in refrigerators, toilet tanks, hot water tanks (for chemical analyses only), fire truck reservoirs, storage tanks, and taps at seldom-used and dead-end locations, and from ice in refrigerators and commercial ice plants. direct the laboratory to test historical samples for pathogenic organisms or toxic chemicals, as well as indicator organisms, because these samples have a chance of still containing the etiologic agent, whereas samples collected during the investigation several days or weeks after the event may be of water that has been flushed free of contamination or has been significantly diluted. take samples from 8 to 10 points throughout the distribution system. sample dead-end locations if they are found. do not neglect to obtain raw water samples even though treatment is provided. this is important, as it suggests possible sources of contamination and reflects the effectiveness of treatment. compare these test results with records of results on previous samples of raw or treated water. before drawing a sample from a water tap, make sure the tap is connected to the supply to be tested. do not collect samples (other than for legionella) from hose connections, sprays, or swivel faucets; uncouple these connections or choose different outlets. it is unnecessary to flame outlets, as this does not improve the quality of the sample. first, ensure your hands have been thoroughly washed then take a line sample by allowing the water to run to waste for 5-10 min. adjust the flow of water so that the thiosulfate will not wash out of the bottle or bag (do not overfill-most laboratory bottles indicate a maximum fill line). keep sample containers closed until the moment they are to be filled. hold the bottle near the base, fill to the "fill line" or within an inch of the top without rinsing, and immediately replace the stopper or cap and secure the hood, if attached. if a whirl-pak™-type plastic bag is used instead of a bottle, hold the base, rip off the perforated top, open the bag by pulling the side tabs apart, grasp the end wires, and place the bag under the flowing water. remove the bag before it is completely filled and squeeze most of the air out; fold over the top of the bag several times and secure by twisting the end wires. take a source or a distribution line sample by opening the tap fully and letting the water run to waste for sufficient time to empty the service line (or if in doubt, for 5 min) and proceed as above. collect samples from open shallow wells and step wells by dropping a clean wide mouth container on a string or rope into the well. allow the container to sink below the water surface and then pull it out of the well. pour contents into a sample jar or bag. collect samples from rivers, streams, lakes, reservoirs, springs, toilet tanks, and non-pressurized storage tanks by holding a 200 ml sample bottle near the bottom and plunging it neck down to a depth of 15 cm (6 in) below the surface; turn it right side up, and allow it to fill. don a plastic disposable glove when small vessels used for drinking are sampled in this manner. when collecting these samples, move the bottle in a sweeping, continuous, arc-shaped motion, counter to stream flow or in a direction away from the hand. collect samples at locations approximately one-quarter, one-half, and three-quarters the width of the stream or water course. special apparatus can be used for sampling at various depths. samples can then be taken by positioning large bottles on a rod or pole at the desired depth and location before pulling their stoppers with a wire, string or thin rod. samples of bottom sediments are sometimes useful for the detection of certain pathogens. collect surface scum or regions containing dense particulate colored material when seeking cyanobacteria (blue-green algae). collect slime, if present, when seeking pseudomonas. if large amounts of water are needed, seek assistance and obtain specialized sampling equipment from agencies responsible for water quality. if possible, avoid wading when sampling bodies of water because wading often stirs up bottom sediments. if this is the only way to get a sample, however, wade against any current (e.g., upstream in creek or river) and keep moving forward until sample taking is completed. piers or similar structures, or the front end of a drifting or slow moving boat, make good sampling stations. concentration of bacteria by the use of swabs, filters, or by absorption, is particularly important when waterborne pathogens are sought. to concentrate bacterial pathogens from flowing water (e.g., streams, lakes, sewer lines, or drains), suspend moore swabs (or non-medicated sanitary napkins or non-medicated tampons if moore swabs are unavailable) for 3-5 days. these can be held in place by wire just below the surface or at other depths. if rodents are about, put moore swabs in wire baskets. after the sampling period, either put swabs or pads into a plastic bag and pack in ice, or put the swabs or pads directly into an enrichment broth for the pathogen sought. take or send these to the laboratory promptly. concentration of microorganisms can be increased by filtration with a variety of filters (e.g., membrane filters, cartridge filters, or other filter media). when membrane filters are used for pathogenic bacteria recovery, pass at least 1 l of water (relatively free of turbidity) through a sterile 0.45 μm membrane filter. for viral analysis, use virus-absorbing electropositive cartridge filter to concentrate 400 l or more water (see table f ). keep filters cool (but not frozen) and ship to a reference laboratory for further processing. for giardia cysts and cryptosporidium detection, collect samples by passing at least 400 l water through a cartridge filter (see table f ). for inorganic chemical analyses, use 1 l polyethylene containers. these should be new, or acid-washed if previously used. collect the water without flushing the lines, preferably in the early morning before water is used. for trace metal analyses, preserve one sample with 2 ml of high-grade nitric acid to a ph of 1 or less. this is particularly important whenever it is suspected that metals may have leached from water pipes or vessels. for organic chemical analysis, use 4 l glass containers with teflon-lined caps. clean and rinse the containers with a good quality laboratory solvent and heat at 400°c for 20 min. rinse the cap thoroughly with distilled water. fill the container so that there is a minimum of air space. for physical analyses, collect at least 2 l, or other amounts requested by the laboratory. collect ice aseptically in sterile plastic bags or jars. use sterile tongs to collect cubes; sterile spoons for collecting chipped or crushed ice; and sterile chisel, hammer, or pick to chip block ice. put block ice or large chips into plastic bags. if legionella is sought, sample water at sites of any source that may have been aerosolized and send to a lab with proven expertise in legionella isolation and characterization, such those in the cdc elite program. this includes cooling towers, evaporative condensers, water heaters and holding tanks, humidifiers, nebulizers, decorative fountains and whirlpool baths (see section on investigating sites where aerosols are disseminated for a more complete listing). turn off fans of condensers before sampling; if this is not possible, wear a respirator. use 250 ml to 1 l polyethylene bottles that have had sodium thiosulfate added if the water to be tested has been chlorinated. for each sample, don disposable plastic gloves and collect the sample by inverting the bottle and moving it in a continuous arc away from the hand. measure and record water temperature. handle samples as described in table f . rub swab over faucet aerators and shower heads if these are considered as sources of aerosols. break stick and allow tip to fall in a tube containing 3-5 ml sterile water (not saline). investigators are often requested to test air to demonstrate the presence of legionella in aerosols. although legionellosis is an airborne disease, legionellae are susceptible to low humidity and become non-viable on drying. therefore, air sampling is an ineffective and inefficient way of determining whether a legionella hazard exists, and it can thus be misleading. label each container with sample number, date, time of collection, and your name or initials. complete the water/ice sample collection report, form f, for the first sample. list additional samples with sample numbers and other pertinent information on the back of the form. in those situations where the laboratory needs additional information, attach the appropriate g series forms. send the original form f and list with samples to the laboratory; retain a copy for your files. inform the laboratory of the type and number of samples and specimens; also, consult with the laboratory on methods to preserve and transport samples, if necessary, and on time of their arrival. if legal proceedings are anticipated, deliver sample personally to the analyst, or seal the sample container in such a way that it cannot be opened without breaking the seal. note on form f the method by which the bottle was sealed. maintain a chain-of-custody log to document the handling of the sample, and have the log signed and dated each time it changes hands. consult with state/provincial regulatory agency on complying with legal requirements for chain-of-custody procedures. recipient should record on the form whether the sample was sealed when the laboratory received it. if analysis cannot be done on the day of collection, chill water samples rapidly and hold them at temperatures at or below 4°c (39°f), but do not freeze, because populations of bacteria such as escherichia coli and of parasites decrease during frozen storage. hold ice samples frozen; if this is not possible, keep the temperature below 4°c h (39°f). investigators should consult with the testing laboratory that will be used to receive specific laboratory sample packaging, labeling, and transportation instructions as protocols are dependent on specific transportation regulations (iafta, tdgr) within each jurisdiction. ensure each sample is uniquely identified and labeled (as per the receiving laboratories requirements). many laboratories include barcode labels along with the sample containers within the sample collection kits. ensure that the correct label is affixed onto the correct sample container and that this information is transferred to the shipping manifest accurately (chain of custody form). specimens should be packed and the packages labeled according to applicable regulations governing transport of hazardous materials. generally, the transport of samples of water and ice intended for laboratory analyses are packed and shipped in a manner to ensure the sample does not change from the time of sampling to the time received by the testing laboratory and shipped using the most expeditious means (e.g., personal delivery or overnight mail). typically samples of water or ice are packed with refrigerant (ice packs, dry ice, etc.) in insulated and sealed containers (see table f ). several measurements are routinely called for during on-site investigations. brief instructions are given for those that are commonly done; nevertheless, follow manufacturer's instructions if these are available. color comparison kits are available for testing for free, combined and total residual chlorine. the diethyl-p-phenylenediamine (dpd) test is an example (see table a ). check instrument calibration regularly. use dry reagents, because the liquid forms are unstable. chlorine comparators can be used to test for bromine by multiplying the result by the factor 2.25 and to test for iodine by multiplying the result by the factor 3.6. measure temperature. measure water temperature by immersing the sensing end of either thermocouples, transistors, or thermometers into the water. sometimes measurements need to be made at various depths; use thermocouples with wire leads of sufficient length for this purpose. calibrate temperature measuring devices periodically. measure ph. calibrate the ph meter as recommended by the manufacturer with at least two standard buffers (e.g., ph 7.0 or 10.0) and compensate for temperature, if the meter does not do it automatically, before each series of tests. remove a sample of water to be tested and immerse the ph electrode into the sample; record the reading. ph can also be measured by color comparators that employ color indicator solutions or discs. (ranges of ph color indicator solutions are bromophenol blue, 3.0-4.6; bromocresol green, 4.0-5.6; methyl red, 4.4-6.0; bromocresol purple, 5.0-6.6; bromothymol blue, 6.0-7.6; phenol red, 6. 8-8.4; cresol red, 7.2-8.8; thymol blue, 8.0-9.6; and phenolphthalein, 8.6-10.2.) in this case, water containing more than 1 mg/l chlorine in any form must be dechlorinated with sodium thiosulfate before the ph indicator solution is added to prevent decolorization of the indicator. always report temperature at which the ph is measured. measure turbidity. nephelometric turbidity unit (ntu) is the usual standard unit, but other turbidity measurements (such as particle counts) are used. the ntu requires a nephelometer, which measures the amount of light scattered predominantly at right angles and absorbed by suspended particles (e.g., clay, silt, finely divided organic matter, inorganic matter, soluble colored organic compounds, and microscopic organisms) in the water sample. calibrate turbidimeters with a standard reference suspension. make turbidity measures on the day samples are taken. vigorously shake samples, wait until all air bubbles have disappeared, and then pour sample into turbidimeter tube. read directly from scale on instrument or from an appropriate calibration scale. pump chemical smoke into the air at the exit of the device suspected of releasing aerosols. observe the direction and spread of the smoke. otherwise, measure pressure differentials with a micromanometer. measure other attributes of water. follow instructions given by manufacturers or in standard reference books (see further reading). use fluorescein dye, lithium or other tracers in appropriate soils to determine the means by which contamination from sewage, industrial wastes, or other sites of pollution reached the water supply. fluorescein dye is particularly helpful in evaluating flow of contamination through fissured rock, limestone, gravel, and certain other soils. this dye is not readily absorbed or discolored by passage through these soils or sand, as are many other dyes, but it is discolored by peaty formations or highly acid (ph < 5.5) soils. make a concentrated fluorescein dye solution by mixing 300 g of fluorescein powder into a liter of water. usually, 2/3 to 3 l of this solution are sufficient for the test for up to 60,000 l of water. fluorescein dye is also available in liquid and tablet form. one tablet will dye approximately 480 l (~120 us gal). pour the calculated amount of fluorescein solution or put a sufficient number of fluorescein tablets into a receptacle at a point of potential pollution. usually this point will be located within 100 yards and at a higher elevation than the water source under study. cesspools, latrines, distribution boxes, sink holes, borings, septic tanks, drains, manholes, toilets and plumbing fixtures are typical places to introduce the dye. if dye is poured into a plumbing fixture or dry hole or boring, add water to wash it down. the amount of dye to use varies with the distance the dye must travel, the expected time of the journey, the size of the aquifer or water channel, and the nature of the soil. take samples of the water when the dye is introduced into the test hole or fixture and then hourly for up to 12 h to detect arrival and departure of fluorescein. if no dye is observed, repeat the test with twice the amount of dye. whenever possible, use a fluorescent light or fluorometer to analyze water samples for evidence of fluorescein. a fluorometer can be set up and calibrated, and a continuous recording can be made. this meter can detect fluorescein in concentrations of μg/l (ppb). fluorescein dye will temporarily color water, which discourages use of the water until the dye is sufficiently degraded or diluted. alternate tracers can be used if specific ion meters are available. the dye stains all it touches. methanol is a good solvent for the dye, and hypochlorite solutions decolorize it; both can aid in removing stains. abrasive soaps are useful for cleaning stained skin; fluorescein-stained clothing should be washed separately. appearance of dye in a water supply is conclusive evidence of seepage from the site where the dye was introduced. failure to detect dye, however, is not conclusive evidence that seepage did not or would not occur if more dye had been added or if weather conditions or subsurface flow had been different at the time of the test than during the outbreak event. illustrate source and direction of contaminated water flow as indicated by the dye test on form g1. take photographs of sources of contamination and evidence of staining of the ground at the site or dye-stained color of the water. in situations where a single source of contamination is obvious or where multiple sources are readily apparent, dye studies serve little purpose. drinking water, however, is not the only source of water that may contribute to outbreaks. other sources of water that can contribute to outbreaks include water not intended for drinking, recreational water and water used in agriculture during harvesting and packaging. legionnaires disease is the pneumonia caused by the inhalation of contaminated water aerosol containing the bacteria legionella, with legionella pneumophila being responsible for 85% of all infections. it is also a common cause of healthcare associated pneumonia. legionella can replicate within free-living amebae in water, allowing it to resist low levels of chlorine used in water distribution systems. risk of infection is more common in warm and humid weather, when water droplets are able to drift further due to higher absolute humidity. fifty percent of all legionella outbreaks have been traced to cooling towers with l. pneumophila serogroup 1 responsible for all cooling tower outbreaks. all aerosol generating devices, however, can be potential sources of legionella. some other sources of aerosolization that may have contributed to or be associated with outbreaks include: whirlpool displays, building's air conditioning systems, water spray fountains, public bath houses, vegetable misting systems in grocery stores, evaporative condensers, showerheads, humidifiers, air scrubbers, car washes, ornamental and decorative fountains, potting soil, respiratory therapy equipment, dental units, road asphalt paving machines, car windshield washer fluid and car air-conditioning systems. in the investigation of a legionella outbreak, (see box 3, the flint water crisis, which describes a likely legionella outbreak from a commercial water source) due to the varied sources, there is a need to use a broad investigative questionnaire and the collection of environmental data. environmental factors such as dry bulb temperature, relative humidity and wind rose data can provide information regarding drift evaporation, deposition (settling) and the size of the affected zone. although aerosol drift can carry legionella up to 6 mi (10 km), the risk of infection is usually highest within 1600 ft (500 m) of the source. there are also air dispersion models that can be used to determine drift zone and the use of human activity mapping in the identification of potential sources. in general, e. coli and norovirus are the most common pathogens responsible for recreational waterborne outbreaks associated with non-treated water such as beaches and lakes. cryptosporidium, which is resistant to chlorination, is the most common pathogen resulting in outbreaks in treated water venues such as swimming pools and water spray parks. it should be noted that e. coli, the indicator of choice of recreational water samples, is not indicative for the presence of norovirus and giardia, cryptosporidium. e. coli can also be "naturalized" and have been found to survive and multiply in beach sand. beach water sampling results therefore may provide false positive or false negative results and may not be the best indicator for the presence or absence of pathogens. recreational waterborne outbreaks are not just traced to the ingestion of contaminated water (table c . illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents). hot tub rash, or pseudomonas dermatitis/folliculitis commonly occurs in public hot tubs or spas such as those found in hotels. the rash is often a result of skin infection from the bacteria pseudomonas aeruginosa colonizing in the hair follicles after exposure to contaminated water. pseudomonas aeruginosa is an opportunistic pathogen that can survive within the biofilm on the tub surface or within the piping system. outbreaks can occur when there is a heavy bather load resulting in an increase in chlorine demand, which in turn reduces the effectiveness of the disinfectant to control the population of pseudomonas. blue-green algae or cyanobacteria bloom can occur in warm, slow-moving or still water. when conditions are favorable, mostly during hot summer weather, cyanobacteria populations may increase dramatically, resulting in a "bloom" as they rise to the surfaces of lakes and ponds. they resemble thick pea soup and are often blue-green in color. although blooms can occur naturally, water bodies which have been enriched with plant nutrients from municipal, industrial, and agricultural sources are particularly susceptible. some cyanobacterial species may contain various toxins, some are known to attack the liver (hepatotoxins) or the nervous system (neurotoxins); others simply irritate the skin. health effects from cyanotoxin exposure may include dermatologic, gastrointestinal, respiratory and neurologic signs and symptoms (table b . illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents). water can also be an indirect cause of foodborne outbreaks by providing a media for the survival, transportation and the introduction of pathogens into food products. water used during production, including irrigation, pesticides and fertilizers application and washing, frost protection, harvesting, has long been recognized by food safety scientists as one of plausible and probable sources of the contamination of fresh fruits and vegetables. there have been many outbreaks from produce traced to pathogens being introduced by contaminated irrigation water. although harvested products are sometimes washed with chlorine solution, pathogens may still survive the process through internalization. e. coli o157:h7 may migrate to internal locations in plant tissue and be protected from the action of sanitizing agents by virtue of its inaccessibility. experiments have also demonstrated that e. coli o157:h7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant. however, this claim has been refuted by others. salmonella and e. coli can also adhere to the surface of plants, and enter through stomata, stem and bud scars and breaks in the plant surface caused by harvesting and processing. water containing bacteria can be drawn into the produce if it is immersed in or sprayed with water that is colder than the produce itself. e. coli o157:h7 may also use its flagella to penetrate the plant cell walls and attached to the inside of the plant. once attached, it may be able to grow and colonize the surface of the plant. the concerns are not just with bacteria. the present of norovirus in the hydroponic water can result in internalization via roots and dissemination to the shoots and leaves of the hydroponically grown lettuce. irrigation water may be contaminated from runoff from nearby domesticated animals and their lagoons, feedlots, ranches into rivers; from feral/domestic animals with direct access to creeks, ditches, rivers, ponds; from sewage flows into waterways and contaminated wells. in some parts of the world sewage contaminated water is preferred for irrigation despite a potential risk of transporting enteric pathogens, since it carries nutrients (n and p) for the plants. there is sufficient information to conclude that the application method of irrigation water to fresh produce can have an effect on the microbiological risks associated with the crop. in general, keeping water away from the edible parts of ready-to-eat crops that are consumed without cooking can result in a lowered risk of a foodborne outbreak. the least to more risky methods for irrigations for microbial contamination are: subsurface irrigation (buried soak hoses) < drip irrigation < indoor flood irrigation (hydroponics) < outdoors flood irrigation (water-filled furrows) < overhead irrigation (sprinklers). an outbreak of illness arising from exposure to water demands immediate epidemiological investigation to assess the situation, gather, evaluate, and analyze all relevant information, with the goal of (1) halting further spread of this illness, and (2) predicting, preventing, and/or attenuating future outbreaks. this twofold mandate of epidemiology is usually described as "surveillance and containment." at the commencement of an investigation, the unknowns usually outnumber the known facts. there is no substitute for prompt, thorough, and careful collection of interview data from ill and well persons who ingested or contacted the suspect water, attended a common event, or who were part of a group of persons where illness occurred. careful analysis of these data, particularly with reference to common patterns of "time," "place," and the characteristics of the persons involved, can often eliminate many vehicles, agents, and pathways quite early in the investigation, and focus on the remaining possible vehicles, routes, and agents. later, laboratory results may confirm the agent, the specific pathology, the route taken by the infection or toxic agent, and indicate what is needed to stop the spread, but early epidemiology can often be invaluable in predicting the outcome and taking preventive steps to contain the problem before the lab results are available. lessons can be learned from most outbreak investigations and are invaluable for increasing our understanding of these pathologies, and preventing their future occurrence. an outbreak is defined as either an unusually large occurrence of an expected illness at that time of year in that place, or the occurrence of a type of illness that does not usually appear at that season and location. the "time" factor should be studied immediately by plotting the onset time of each case on a time-based grid, to create the epidemic curve. although any number of cases can be involved, the minimum number for an "outbreak" to be declared is two associated cases, with special exceptions such as naegleria fowleri where, because of the severity and the possibility that cases may have been missed, a single case constitutes an "outbreak." although the epidemic curve is usually measured in hours or days, protracted exposure to agents in water may mean apparent sporadic cases linked to a common source over months or years. if an "outbreak" is suspected by a sudden increase of cases, determining who is to be categorized as a "case" is not necessarily a simple process. many people notoriously fail to report enteric illness for many reasons: embarrassment, lack of time, no clear idea which agency should be notified, mild self-treatable symptoms, or simply because they prefer not to make a fuss. they may therefore be incorrectly classified at least initially as "nonill." consider also that 4-6% of the general population will have experienced some form of "upset stomach" in the last 24 hours, regardless of exposure to the suspect item, and they may be incorrectly classified, at least initially, as "ill." to reduce the "false negatives" and "false positives" that are expected with self-reporting, the investigator needs to establish a working case-definition. a careful case definition categorizes people as "case" or "control" with the best accuracy possible within the time constraints and resources available. a case definition could be considered "too sensitive" if it classifies as a "case" a person who experiences: "… at least one episode of stomach cramps, nausea, vomiting, or diarrhea in the last 48 hours." this would confuse subsequent analysis, and produce more false positives. similarly, a case definition could be considered "too specific" if it classifies as "not-ill" a person who had experienced only three episodes of diarrhea or vomiting, because they failed to satisfy a case definition requiring "…at least four episodes of vomiting or diarrhea in the last 48 hours." should this last individual, having been declared as not fitting the case definition, be taken into the "notill" group, the error and subsequent analysis is confounded even further. a reasonable case definition therefore attempts to reduce both types of errors, and will depend upon the early indications of what the etiology may be. in the instance of a suspected salmonellosis, a case could be defined as "a person who was in good health before attending the event on monday may 3rd, and who experienced two or more of the following symptoms anytime up to midnight, sunday may 9th.: nausea, vomiting, stomach-cramps, diarrhea, headache, or fever." note that a case definition should include a place of exposure if known, a timeframe during which symptoms may have been experienced (salmonellosis has a range from 6 to 72 h. usually 24-30 h), and the additional footnote that the individual was not already symptomatic before the suspected "exposure." calculate the percentage of ill persons who manifest each symptom by dividing the number of persons reporting the given symptom by the number of cases (205 for the example, table 4 ) and multiplying the quotient by 100. the distribution of symptoms can be used to identify the most likely pathogen, and aids in requests to the laboratory for microbiological assays of samples and specimens. other symptoms (e.g., prostration, lethargy, weakness) may be included if deemed appropriate or helpful, but the six symptoms in table 4 should always be included. headache, for instance, is associated with many viral infections (e.g., norovirus, rotavirus), but much less so with bacterial infections. fever is usually associated with an invasive bacterial infection (such as salmonellosis or campylobacteriosis), and is not usually seen in outbreaks of simple enteritis (such as with cholera). this information helps to determine whether the outbreak was caused by an agent that produced intoxication, an enteric infection, or generalized illness. in the example given, a predominantly diarrheal syndrome without much fever or headache tends to eliminate some of the viral infections (norovirus or rotavirus) or the host-adaptive/invasive serotypes of salmonella (e.g., s. dublin or s. choleraesuis). median onset time calculations may further reduce possible candidate etiologies. in historical investigations, or where no laboratory confirmation is possible, the symptom profile and onset times can sometimes predict the etiology of the outbreak within reasonable certainty. an epidemic curve (also called an onset curve or onset distribution) is a graphic illustration called a histogram that shows the distribution of the time of onset of first symptoms for all cases that are associated with the disease outbreak. paper printed with square "grid" lines will allow the investigator while on site to represent each case as a single "block." the horizontal axis is the sequence of intervals of time and date. the unit of time that defines the width of each interval depends on the characteristics of the illness under investigation. for example, intervals of days or weeks are appropriate for diseases with long incubation periods, such as cryptosporidiosis or hepatitis a. intervals of a day or half-day are appropriate for outbreaks of enterohemorrhagic e. coli strains or shigellosis, while single-hour, 4-h, or 6-h intervals will be more suitable for illnesses with shorter incubation periods, such as chemical poisonings. the vertical axis is always the actual count, or "frequency" of cases (blocks) stacked at each interval. it is often necessary to redraw the onset curve as more accurate information becomes available. if the illness is known, a rule of thumb is that the time interval used for each "block" on the x-axis should be no more than ¼ the incubation period of the disease under investigation. if the illness is not known, select an interval where the data produces a bell-shaped curve; not too flat and not too tall. construct this graph using time-of-onset data from forms c or d, employing an appropriate time scale. once all the onset times for the cases have been plotted on the histogram, determine the range as the interval between the shortest and longest incubation periods. in fig. 2a , the range is the 5 day period from the 12th to 16th march. the median onset time is preferred to the mean because the latter is vulnerable to a few or even a single very small or very large value. the median on the other hand, is the midvalue of a list of all individual onset times, including duplicate entries, that are ordered in a series, from shortest to longest. if the series comprises an even number of values, the median is the mean of the two middle values. most standard reference texts on communicable diseases give onset times as median values. the mode is simply the interval having the largest number of observations. a distribution with a single "peak" is called a uni-modal distribution, while an outbreak with two peaks is called "bi-modal." subsequent modal peaks following the first may indicate either a "secondary wave" of cases or the exposure of other people at a later time. the shape of the epidemic curve helps to determine whether the initial cases originated from a single point-source exposure (such as water or food available for only part of a day), or from repeated exposures for a longer time, or even more gradual person-to-person spread. a point-source epidemic curve is characterized by a sharp rise to a peak, followed by a fall that is almost as steep ( fig. 2a ). an "explosive" outbreak of this type is common where a municipal water supply is the vehicle, affecting large numbers of people in a very short period of time, but without secondary cases occurring, or any evidence of onward spread within the community. propagated outbreaks are those in which the initial victims ("primary cases") manage to spread the agent to other people ("secondary cases") such as family members, patients, clients, or other contacts in crowded places through aerosols, personal contact, or contaminated water/food/utensils/surfaces, etc. propagation following a point-source exposure is demonstrated by a second increase in reported cases following the decline of the first cluster. sometimes this takes the form of a second "modal peak" separated by approximately one incubation period, but this distinction is soon lost. figure 2a shows no evidence of propagation; fig. 2b suggests that propagation may have taken place, although care must be taken to consider other explanations. in addition to (1) true propagation, where the secondary wave can be expected to appear one incubation period after the first, secondary waves may be also explained by (2) exposure to the same point source (e.g., food or water supply) at different, but specific times by other people; this might be a repeated offering of contaminated food or water at two or more mealtimes; (3) a second pathogen (perhaps from the same unhygienic food or water source) which may have a different symptom profile and a different (incubation) time. slow propagation from the beginning of an outbreak with neither an obvious point-source, nor any distinctive "waves" separated by an incubation period as in fig. 2c , usually indicates one-at-a-time person-to-person spread through closecontact, poor personal hygiene, aerosol (e.g., influenza, or sars), or sexual transmission (e.g., hiv/aids). it can also be explained by (non-propagated) continuing exposure, for example drinking of contaminated surface water following a conflict, natural disaster or other breakdown of infrastructure. as such it is commonly associated with waterborne cholera, shigellosis, typhoid fever, or e. coli infection, and characterized by scattered cases which continue until the chain of infection is cut. slow, constant and/or intermittent exposure to persons over time to pathogenic microorganisms can also result from sewage run-off after a series of heavy rainfalls. in addition to revealing whether the outbreak was due to a single point-source, or had been spread steadily through the community by propagation in some way, another important objective in constructing the epidemic curve is to estimate the incubation period of the illness if it is not already known. with waterborne illness especially, the time of exposure may be further obscured because people usually drink water several times a day. hence, the incubation period cannot always be determined for each case, but the actual time of onset is usually available. the incubation period is the interval between exposure to food or water that is contaminated (with enough pathogens or with a sufficient concentration of toxic substances to cause illness), and the appearance of the first sign or symptom of the illness. each etiology is characterized by a typical incubation period (tables b, c, d, and g). individual onset times will vary due to immune factors, co-morbidities, the dose ingested, and other ingested materials, but the investigator can often make a rough estimate of the average incubation time by examining the aggregation of all onset times as an epidemic curve. the modal peak of a single "cluster" or distribution is the time interval in which most cases commence symptoms. in fig. 2a this occurs on march 13, and in fig. 2d that occurs at the double interval feb 10-11th. where two separate modal peaks (a "bi-modal distribution") suggests secondary cases ("propagation"), then the distance between the first two modes is a good estimate of the incubation period. figure 2b shows about 4 days between primary and secondary modal peaks, suggesting that the initial exposure is likely to have been 4 days before the first mode. in fig. 2b this would be sometime on or near the 10th of the month. if the exposure point is known but the agent is not, then that estimation of the median incubation period will allow many etiologic agents to be excluded due to 7 6 5 4 3 2 1 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 date of onset of symptoms: between march 12 and april 2, 2014 count per day onset histogram for 30 cases of shigellosis, march 12 to april 2, illustrating slow spread through a community through either propagation (person-to-person spread via poor hygiene), or exposure by many people to a small well at different times (a non-propagated route) incubation periods that are clearly outside the range of times observed. the list of possible candidates can be further reduced by examining the symptom profile and other characteristics of the illness and suspect food or water vehicle. as time passes, the onset curve also provides an ongoing measure of the potential for propagation, and the incidence rate. all this information can be useful in deciding whether the illness in question is an infection or intoxication and thereby determining which laboratory tests should be requested (tables b, c, d, and g). note that not all water or foodborne illnesses listed in a standard reference such as the "control of communicable diseases manual" (apha 2014), are directly communicable person-to-person; many require a suitable substrate (food or drink) and adequate time/temperature combinations to attain sufficient numbers or the production of enough toxin to induce a pathological condition. an exposure time can sometimes be estimated from a clear, point-source, singleexposure onset distribution (fig. 2d ). it has no solid basis in statistics, but has sometimes been found to be useful in practice. the typical incubation periods for most foodborne and waterborne illnesses are readily available for comparison (e.g., control of communicable diseases manual, apha/cdc, 2014), and in this manual in tables b, c, d, and g. an incidence rate is the number of new cases of a specified disease reported during a given time period in relation to the size of the population being studied, multiplied by a constant, usually 100, to give percentages. thus 14 new cases of e. coli o157:h7 infection among the 140 residents of a children's summer camp in july is an incidence rate of (14/140) × 100 or (0.10) × 100 = 10.0% for that month. if several people have left and their state of wellness is not known, their impact should be expressed in the form of the possible range of values around the known incidence rate within the two extremes whereby they may all be well or they may all be ill. thus, where six children who were at the camp had departed around the time of the outbreak and their health is unknown, the range could be from a possible (14/146) × 100 (or 9.6%) if all of the six had been well, up to (20/146) × 100 (or 13.7%) if all had been ill. note that the "missing" six are added to the denominator only when we speculate that none were ill, whereas they are added to the numerator and denominator if we speculate that they might all have been ill. in this example, the overall incidence rate would be reported as "10%, with a possible range from 9.6 to 13.7%." depending upon the situation, it is often necessary to identify exposures which may be related to the illness, and to calculate an incidence rate for each such exposure. for example, in the summer camp illustration (above) it might be useful to enquire if gender, age, location, or some other attribute or activity increased the risk of becoming ill. this should not be interpreted automatically as implying that a given exposure would be associated with the outcome in any situation. by hypothesizing that gender was linked to the risk of illness, for example, does not imply that males are more vulnerable to the illness (the outcome) than females, but it can indicate that gender may have been related to the exposure, which in turn increased the risk. as an illustration, suppose that boys at the camp had been swimming, while the girls had gone on a nature walk. the boys may subsequently show increased incidence rate for e. coli o157 infection, not because they are more susceptible, but because of their activity. every proportion or percentage statement should be made with clear reference to the appropriate denominator used. incidence rates of waterborne illnesses are usually similar for both sexes at any given age group in the population, but differences in activities or dietary habits or susceptibility due to age or underlying health status can change the risk. the very young, the elderly and the immunocompromised can be at more susceptible, while in some instances, previously exposed populations may have developed a measure of immunity to an infection that may still cause more serious illness among visitors. a further complication arises where the "at-risk" population (perhaps residents at an institution, summer camp, or on a cruise ship) have generally consumed all the food and water for the extended period. careful interviewing of affected persons often uncovers one or more persons who entered the subject community shortly before becoming ill or who visited the community for a short time and became ill after leaving it. example: the south-west part of the county is served by three semi-private water systems. thirty cases of waterborne illness are being investigated in the area. when the numbers of cases are displayed for each water system, no clear grouping or clustering is evident, although the delta supply appears associated with about 50% more cases than the other two (table 5a) . however, when the analysis introduces the total population of persons who depend upon each water system (as denominators), a different scenario emerges. the incidence rates (expressed here as percentages) now allow a meaningful comparison (table 5b ). we can see that persons using the bravo system have roughly five times the risk of illness compared to people who are served by the other two systems. the use of the denominator is vital for most calculations. caution: numerous other factors may also explain the outbreak and these should be carefully examined. for example, the households using the bravo supply may be closer to an unhygienic corner store, drink from a cross-connected public water fountain, or their children may swim in a more polluted pond than the other communities. potential sources such as these should be eliminated before the water supply is announced as the source of the illness. sometimes a spot map may be useful in showing the location of the residence of each case, while on a larger scale, the rates of illness can be shown using city blocks, census tracts, townships, or other subdivisions. different colors or symbols to indicate cases with different time of onset periods (such as weeks) may help to support a hypothesis as to where contamination was introduced, inasmuch as the earliest cases tend to cluster around the point where contamination first occurred. the weakness of this procedure is that if the exposure had been at a restaurant, workplace, or school, plotting the relationship to the location of the home would not be useful. the investigation of waterborne or foodborne disease outbreaks invariably commences after both exposure and illness have happened. this is the classical "casecontrol" study, where a group of ill people ("cases") and a group of non-ill people ("controls") are compared in terms of their exposures. 1 to measure the association between exposure and illness, the data are typically displayed in a 2 × 2 contingency table. table 6 compares 37 cases and 30 controls in terms of their exposure to a suspected factor "x." the table is ready for analysis using odds ratio, as well as the chi-square or the fisher's exact tests where appropriate. one 2 × 2 table will be used for each possible exposure (e.g., each beverage, food, or other material). as many cases as can be identified and contacted, and as many non-ill people (controls) as can be found, should be interviewed as quickly as possible about their exposures to each suspect item. fading memories, the chance of obtaining stillavailable samples of implicated food or water, and the opportunity to obtain fecal specimens before the patient is started on the ubiquitous broad-spectrum antibiotics are all reasons for rapid response. case and control numbers do not have to be the same; the calculations compare ratios so equal numbers in each group are not needed. generally a 1:1 to 1:2 ratio of cases to controls is perfectly adequate. as interview data from cases and controls are accumulated, leading to formation of hypotheses about the source of the illness, human resources should be deployed in two additional essential tasks: (1) tracking down and confirming the hypothesized source of the illness, and (2) promptly issuing warnings to all affected groups about the possible risks from any source that is still accessible, with assurances that further bulletins will be issued as soon as confirmation is received. this precautionary principle is a vital component of risk management in modern public health. waiting for absolute confirmation before releasing warnings and advisories should not be an option in the twentyfirst century. the principle holds that while false alarms can be quickly forgiven, further illness should be avoided at the highest priority. failure to heed this step has contributed to needless suffering and severe damage to reputation, trust and credibility. 1 a case-control approach is necessary because unlike the data in table 5b , we rarely have full information about all the attendees, and therefore the true incidence/attack rate is not available. very rarely, when all cases and controls are available for interview, we would have the true incidence rates for ill and for not-ill persons and this would allow a "retrospective cohort study" to be carried out. under such circumstances, and using table 6 as an illustration, we could state that of 33 persons exposed to item x, 25 persons (75.8%) had become ill compared to 12 ill of 34 not exposed (35.3%). where incubation times are longer than a day, there is increasing likelihood that only a small proportion of the non-ill people will be available for interview, and on many occasions, not even all the ill persons can be contacted. the point here is that the investigator is usually working with sub-sets of the true cases and controls. the 30 controls in table 6 and possibly even the 37 cases may have been drawn from larger groups, and therefore we cannot state the incidence rate, for example, as: "…25 of 33 exposed were ill," because the "33" had been artificially assembled, and may not resemble the true incidence at all. we can, however, use exposure rates, for example: "…of 37 ill persons, 25 (67.6%) had been exposed to x," and, "…of 30 who were not-ill, only 8 (26.7%) had been exposed to x." the overwhelming majority of waterborne or foodborne illness investigations are run as "case-control" studies (or to be more accurate, "case-comparison" studies, as very little true "controlling" is accomplished during the selection of the comparison group). a broadcasted invitation to all who might have been exposed to come forward, typically results in few non-ill persons volunteering information, because nonaffected individuals believe they have little if anything to contribute. this reduces validity even further, and more active recruitment is often necessary to convince them that their information is just as essential for the investigation as are the contributions from the less-fortunate attendees. let us examine a waterborne illness suspected as being due to the consumption of water bottled from a certain spring. you have found 60 people who meet the case definition of illness, and another 29 non-ill people in same neighborhood who report no symptoms at all, and who will be your controls. in table 7 we display the data and ask the question: "is drinking this water related to the risk of illness?" whenever a 2 × 2 table appears, the first step is to calculate the odds ratio (or). an odds ratio tells us if there is a relationship (where or ≠ 1), and the strength of the relationship (the or value itself). it also clearly indicates the direction of the relationship: was drinking or not-drinking the dangerous activity? this is easily determined by finding the dominant pair from (a × d) or (b × c). in the example above, (a × d) is greater, so cell "a" links the row "drank" with the column "ill," while cell "d" links "not drink" with "not-ill." this assumption is not as obvious as it may seem; the cause of the illness may have been whatever "other" thing was drunk by those who avoided spring water! it is important to clarify that the odds ratio yields the strength of the association, not the statistical significance. most or values (where many exposures are being assessed) will be close to 1.0 (= "no association"), while an or clearly exceeding 1.0 signifies a positive association between this exposure and illness, such that this exposure increased the risk of illness. an or < 1.0 is protective, meaning that exposure to this factor reduced the risk of illness compared to the other group. for example, an or of 0.25 means that the exposed group had only one-quarter the risk of illness compared to the non-exposed group. the non-exposed group therefore has a greater risk (by a factor of 4). while this protective effect can be due to true therapeutic protection (e.g., exposure to antibiotics when you have an infection), it is frequently explained as "statistical" protection. as an example, consider an outbreak where everyone consumed only one of two possible types of bottled water. one source, a, contains a pathogen, and b does not. if the ill people were found to be five times more likely to have consumed type a (odds ratio = 5.0) then the not-ill would have five times the rate of consuming water b, and only one-fifth of the rate of choosing water a (or = 0.20 or 20%). this can also be read as the risk of illness for the non-exposed group, or as the risk of staying well by the exposed group. an easier way to interpret an or less than 1 is to place 1 over the or to reveal a value greater than 1, but clearly labeled "protective." the or is a ratio between numbers, and therefore not sensitive to the actual numbers of people in individual cells, an important consideration when the numbers of subjects are relatively small. this is illustrated by the common question: "how large does an odds ratio have to be before it is considered evidence of an association?" a popular response is "at least 2.0," but this must be considered with extreme caution. for instance, with very large studies, an or of 1.12 (barely more than 1.0) can be shown to be very highly significant statistically (p = 0.001), whereas in a small-n study, an or of even 5.0 may not achieve statistical significance. the odds ratio is certainly a useful measurement, and should always be used when a 2 × 2 table is encountered. it will quickly advise you (1) that there is an association, (2) the strength of that association, and (3) the direction of the association, none of which are specifically measured by a test of statistical significance. unfortunately, it is not reliable with small cell sizes, and is unable to answer the question: "how likely is it that these numbers could happen just due to chance?" for this, we need to test the statistical significance. the best advice is to use the or (or relative risk where appropriate) together with a test of statistical significance. most online statistic calculators or laptop versions of sas, spss, epiinfo, etc. will give a selection of useful statistics (odds ratio, relative risk, several versions of chi-square, and fisher's exact test, both onetailed and two-tailed.) in keeping with all scientific enquiry, we begin by advancing the notion (the "null hypothesis") that there is no association between the exposure and the illness, and attempt to support that notion. if insufficient evidence is found to support the null hypothesis, we reject it and cautiously consider that an association may exist between the two variables. this can be described as a statistically significant association. two methods of testing are presented: the chi-square test (written χ 2 and pronounced "ky"-square) for most 2 × 2 (or larger) tables, and the fisher's exact test (only for 2 × 2 tables) when chi-square is not valid due to the numbers in the cells being too small (the following sections give advice about this decision). the original data value in each cell we call the observed, or "o" value, and these are compared with the numbers that you would expect ("e" values) if there were no relationship at all; that is, if the variables were not related, and the data were arranged purely by chance (as stated by the null hypothesis). the chi-square test measures the difference between the o and e values. if they are close, we have to accept that there may be no real relationship; if far apart, we can reject the null hypothesis and cautiously declare that exposure and illness were probably related. numerous online statistical calculators can be used to yield ors, rrs, and chisquare values. 2 if you prefer to do the calculation by hand, construct a 2 × 2 table as shown, with "observed" data, marginal totals, and the grand total. the expected "e" values are found from: to make sure the chi-square analysis is appropriate for your table, you must be sure that all "e" numbers 3 are more than 5. the quickest way is to first calculate for the cell with the smallest e value; (this will be the cell with the smaller column total and the smaller row total.) in table 8 , the smallest e value will be cell "d," and this is calculated as (19 × 29)/89 = 6.2. as this is >5, all other e values will be greater than this, so chi-sq. is valid. (note that the smallest e value did not coincide with the smallest o value). . for all four cells the sum (χ 2 ) is: 1.64 + 3.40 + 6.05 + 12.49 = 23.58 an online statistical calculator will give you this same chi-square (χ 2 ) value. to verify by hand whether the o vs. e difference is statistically significant, compare your chi-sq. value (for a 2 × 2 table only) with 3.841. if your calculated value exceeds 3.841, then this is unlikely to be due to chance, and thus you can begin to believe that this exposure did influence the risk of illness, and you can reject the null hypothesis. statistical results usually include a probability (p) statement. this is the probability that the null hypothesis ("no association") is correct. the 3.841 value is the minimum needed for statistical significance, where the p is less than 5% (p < 0.05). recall that the p is the probability that no real association exists between exposure and illness. by convention, if p > 0.05 (more than 5%) then the relationship is declared not statistically significant. where p = 0.05 or <0.05, then the relationship is statistically significant. the smaller the p value, (p < 0.01, p < 0.001, etc.) the more confidence you have that a relationship really exists. other critical values exist for assessing calculated chi-sq. values, from larger tables than 2 × 2, and at more extreme levels of significance. a further chi-square calculation is shown as an appendix. where a table greater than 2 × 2 is found to have more than 20% of the cells with an e value less than 5, chi-square is not valid. the solution is to collapse either columns or rows to allow the e values to increase. for example in table 9a , two cells out of six (33%) have e values less than 5, but if "high dose" is merged with "medium dose" the resulting increase in observed (o) cell sizes is also reflected in greater e values, while the table becomes 2 × 2 (table 9b) . some outcome information has been lost, but the chi-square analysis can proceed. if, after trying to collapse cells and/or rows, a 2 × 2 table is reached still with an e value <5, the fisher's test is indicated. this procedure is reserved only for 2 × 2 tables where one or more expected (e) values is less than 5, making the chi-square test not valid. our example is taken from an investigation into an outbreak of shigellosis presumed to be due to water from a well ( table 10 ). the odds ratio has been calculated as (8 × 6)/(4 × 2) = 6.0, meaning ill persons were six times as likely to have drunk well water compared to non-ill the "!" denotes a factorial, meaning that number multiplied by the next smallest number, and so on down to 1. (e.g.: 6! = 720) p 1 10 10 12 8 8 4 2 6 20 0 075018 persons. an attempt to use chi-square is prevented by at least one e value less than of 5. (cells c and d both show e values as (8 × 10)/20 = 4.0). the starting null hypothesis is "that no relationship exists." this is not quite the end of the calculation however. the goal is to calculate the probability of the original data occurring plus all more extreme probabilities. the original data have to be adjusted by increasing the "dominant" pair of cells by +1 and the others by −1, while leaving the margin totals the same (table 11) . because no zero has yet appeared in the matrix of cells, we continue to increase the "dominant" pair by +1 and obtain a zero. the next calculation is the last. (by convention, 1! and 0! = 1) (table 12) . no reference table is required. the total calculated probability (0.085) is exactly the probability that the null hypothesis ("that there was no relationship"), is correct: 8.5%. 4 by convention, for a result to be significant statistically, that probability (p) must be less than 5% (<0.05), so in this instance we are not able to reject the null hypothesis and must conclude that the relationship could have occurred by chance alone more than 5% of the time. the odds ratio of 6.0 is explained as the number of times more likely it was for a shigellosis victim to have drunk well water than for a non-ill person. this increased risk would normally be impressive, but because of the small number of persons in the study, it has been found not to pass the test of statistical significance. a basic write up of the results might read: "a relationship exists between drinking well water and developing shigellosis. a shigellosis patient is six times more likely to have drunk water from the well compared to a non-ill person. this relationship is not statistically significant, however, and could have occurred by chance alone more than 5% of the time. the null hypothesis of no-relationship cannot be rejected." [1 df, p > 0.05, or: 6.0, not statistically significant.] with the odds ratio (or) calculated for all the suspected exposures, and the chisquare test or fisher's exact test calculated for the strongest of these, all the results can be displayed in a composite table. earlier protocols for the investigation of waterborne and foodborne diseases encouraged the use of the "factor-specific attack rate table" (for example the "foodspecific attack rate table"), but where only a "convenience sample" of controls and cases are available, we are unable to derive valid incidence/attack rates. investigators are discouraged from using it as it may produce misleading results. the exposurerate table for cases and controls is preferred in all case-control studies, and compares the rates of exposure to each factor between both the ill and non-ill people. table 13 displays six exposure factors from a hypothetical outbreak involving water contamination. exposure rates are calculated from both cases and controls. the "spring-water" data that we used for the odds ratio calculation example in table 8 appears as the first exposure in table 11 . the column headed "differences in exposure rates" subtracts the exposure rate among the non-ill from the exposure rate among the ill. [use: exp. rate (cases) minus exp. rate (controls), keeping the signs correct]. you are looking for a large positive difference to indicate the most likely culprit. the spring water shows the largest positive difference at +45%. the odds ratio of 15.0 supports this, again the largest value, indicating that ill persons were 15 times more likely to have drunk the spring water compared to non-ill persons in this group. hence both the large positive difference in exposure rates and the large odds ratio point to the spring water being the likely source of the illness, and it is certainly the strongest association between illness and any of the exposures shown. the chi-square value has also been added (23.6), as well as the associated p value. taken together, the evidence clearly points to this factor as the culprit. in those less-common circumstances in which all the ill and non-ill persons can be contacted for interview, the table can be rearranged to show attack rates (incidence rates) for each of the suspect factors (table 14) . here, the column of "differences" shows the attack rate (exposed) minus the attack rate (non-exposed), (i e − i n ), and again a large positive difference will point to the culprit. this measure is called the attributable risk and for the spring water example we obtain +59%, the largest value of all the risk factors. also, because of the availability of valid attack rates (incidence rates), the true relative risk (rr) is available, and can be substituted the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant the ("true") relative risk (also called the risk ratio) is only used when we have the true incidence data. it is calculated as the attack rate (exposed) over the attack rate (non-exposed), or i the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant for the odds ratio. the data in table 14 shows the same data as table 13 rearranged for easy comparison. values in the column of "differences" are not the same as for table 13 and of course the relative risks are not the same as the odds ratios in table 13 , but both of these results still point clearly to the suspect exposure. in both analyses, spring water is clearly the factor most strongly associated with illness. it is important to note that in both tables a high rate (exposure-or attack-) on the left side taken by itself is meaningless until it is compared with the rate from the right side of the table. this again underscores the importance of gathering complete data from the non-ill as well as the ill. an interesting phenomenon is visible in the second factor listed (soft drink). the or is listed as 0.18, which is "protective," meaning that this factor is strongly associated with not being ill. it is the equivalent of or equal to 5.55 (1/0.18), and the chi-square is seen as quite large, although not enough for statistical significance. this is sometimes seen where two factors are "in competition" with each other; if everyone had drunk one item, and the spring water was the contaminated source, then those drinking the other item would be strongly "protected" because they did not drink the spring water, and this shows clearly. all other factors have or values very close to 1.0. most attack rate tables record some persons who did not ingest the suspect vehicle but who nevertheless became ill. plausible explanations are that (a) some people forget which beverages or foods they ingested; (b) some might have become ill from other causes; or (c) some may have exhibited symptoms with a psychosomatic rather than a physiological origin. it is also not unusual for the table to include some persons who ingested contaminated water or food but did not become ill. plausible explanations are that (a) organisms or toxins are not always evenly distributed in water or food and consequently some persons ingest small doses or perhaps none at all; (b) some persons eat or drink larger quantities than others; (c) some are more resistant to illness than others, and (d) some will not admit that they became ill, or fail to report it. whichever table is used, the combined totals for cases (ill) and controls (well) are fixed and should not change for each exposure unless there are "missing" responses from interviewees. while some procedure manuals include confidence limits around both rr and or, this may be omitted here as the use of the chi-square test or fishers exact test yield the statistical significance for both tables. illness caused by ingestion of waterborne toxicants and some pathogenic organisms can be dose-related in that the risk of developing symptoms, and their severity varies with the quantity ingested. where the suspect water is no longer available (for example, the well may have been quickly super-chlorinated to break the chain of infection before samples were taken), attack rates can be based on the amount of water usually drunk per day by each person. this is easily extended to other non-treated sources of water such as ice cubes, water-reconstituted fruit juices, and flavored crystals. a comparison of attack rates at various water intake levels may provide valuable evidence that water is, or is not, the vehicle responsible for the outbreak. for an example, see table 15 . here, the entire group was 210 people and we have interviewed them all, so we are justified in calculating the attack/incidence rates: in this example, the attack rate increased as the consumption of water increased, which suggests that the illness was directly related to water and the agent it contained. this is a trend established from the group as a whole, and an individual's experience may vary with factors such as (a) preferences of water ingestion, (b) intermittent contamination, (c) unequal distribution of the contaminant, or (d) varying susceptibility of individuals. these data can be compared with rates from persons who ingested no water, but only hot tea, hot coffee, soups, and/or other safe sources of liquids. if unheated water was indeed the vehicle, and the agent was a living biological agent, these persons should have attack rates showing no increase in risk of illness. (outbreaks from a toxic agent may be unaffected by chlorination, boiling, and some types of filtering.) the data can be displayed in a contingency table as follows for analysis using chi-square procedure (table 16) . for this example, chi-square equals 8.90 and if calculated by computer or online, p will be shown as p = 0.031. reference to form j1 confirms that for a 2 × 4 table (3 df), the calculated chi-square (8.90) exceeds the critical value for statistical significance at the 0.05 level (7.82), allowing us to claim statistical significance at p < 0.05. odds ratios are normally associated only with 2 × 2 tables, but here, the or can usefully be calculated on selective cells or groups of cells as long as you clearly explain the selection process. for instance, persons who were ill were 2.8 times more likely to have drunk three or more glasses of water per day compared to those who were well. for this calculation we collapse cells into a 2 × 2 table and crossmultiply: (a + c) × (f + h)/(b + d) × (e + g) = (38) × (72)/(89) × (11) = 2736/979 = 2.79. alternatively, because we have all people involved, we can compare the attack rates (ar) for each intake level, and observe the increasing attack rate as the intake increases: for five or more glasses/day, ar: 33%, for 3-4/day, ar: 28%, for 1-2/ day, ar: 16%, and for <1/day, ar: 8%. we might summarize as follows: "there was a relationship observed between the quantity of water consumed each day and the risk of illness. the incidence rate increased with the quantity consumed from 8% for <1 glasses/day to 33% for five or more glasses/day. this relationship is statistically significant. the null hypothesis of no association can be rejected." , 3 df, p < 0.05] water as a vehicle can deliver pathogenic organisms in many ways beyond simply drinking a glass of water, or using a drinking fountain. investigators should be sure to ask about the preparation of ice-cubes, the mixing of fruit flavored crystal drinks, reconstituting concentrated orange juice, brushing and rinsing teeth, and washing hands, utensils, or containers. swimming or playing in muddy pools or even swimming pools have caused waterborne poliomyelitis, and naegleriasis, while swimming in saltwater inlets have allowed inadvertent infections from vibrio parahaemolyticus and v. vulnificus. unwashed plastic jugs containing poster paint residue have caused rapid illness when drink crystals are reconstituted in them, while refillable plastic containers and bottles have a long history of contamination from biological and chemical agents. in the late 1970s, an increase of viral ear, nose, and throat infections among people who were using parkland next to a river was hypothesized to have been due to people waterskiing on the river and creating an aerosol. the river was the receiving body for effluent from a water treatment plant upstream. record all laboratory results on form i, laboratory results summary. compare epidemiological and statistical results with on-site observations, laboratory results and the information summarized on form i. the agent responsible for the outbreak can be determined by (a) isolating and identifying pathogenic microorganisms from patients, (b) identifying the same strain and/or pfge pattern or genetic sequence of pathogen in specimens from several patients, (c) finding toxic substances or substances indicative of pathological responses in specimens, or (d) demonstrating increased antibody titer in sera from patients whose clinical features are consistent with those known to be produced by the agent. when implicating the water as a likely (or presumptive) vehicle of transmission, ideally identification of a pathogen in samples of suspect water will correspond to the one found in clinical specimens from ill persons or that produces an illness that is compatible with the incubation period and clinical features of the ill who were exposed to the water. for organisms that are common in the gastrointestinal tract or that have multiple strains, compare strains isolated from ill persons with strains isolated from the suspected water. additionally, specific microbial markers (e.g., serotype, phage type, immunoblotting, plasmid analysis, antibiotic resistance patterns, restriction endonuclease analysis, nucleotide sequence analysis) or chemical markers identified by chromatography or spectrophotometry can be used for this purpose. for confirmation of water-related transmission, the same pathogen strains should be found in both the ill persons and the epidemiologically implicated water. however, due to the period of time that may have passed after the outbreak was actually reported, and to methodological issues, such as the need for concentrating pathogens in water samples, it is often unlikely that the outbreak-associated pathogen will be found in the water samples. laboratories frequently test water samples for indicator organisms, such as fecal coliforms, escherichia coli, or enterococci, rather than pathogens. the finding of these bacteria in high densities in the water may indicate contamination (from a fecal source) and implicate the water was a possible vehicle. however, the finding of increased indicators in water samples alone is insufficient evidence to confirm the water as the source of an outbreak. the probable source of contamination or the situation that allowed contamination to reach and survive in a water supply (e.g., water supply not disinfected or inadequately disinfected, inadequately filtered, or upstream to sewage or agricultural discharges; cross connection between sewerage and drinking water pipes; well improperly constructed; nearby septic tank system; or livestock in water supply) can often be identified, but the etiologic agent in the water may never be found. success in finding the etiologic agent is most likely where (a) the incubation period of the illness is short, (b) the agent is stable in water and the system is static, or (c) large amounts of the agent are being continually added to the water supply. try to recover and identify the specific agent whenever a water supply is suspected to be the vehicle of transmission, even if finding the etiologic agent is likely to be difficult and not considered practical for routine monitoring of water supplies. if water samples do not reveal a likely causal agent, clinical data as well as time, place, and person associations can cast strong suspicion on a water supply, particularly if indicator organisms are found in the water. tests other than those for pathogens, however, are frequently used to evaluate water supplies on a routine basis. organoleptic tests attempt to evaluate the total effect of all compounds present in water that can be measured by the senses of taste, smell, or sight. results cannot be expressed in terms of specific compounds present, and the measured qualities are usually a result of a mixture of compounds. these tests are often empirical and arbitrary, but changes in the physical qualities of water (such as ph, turbidity, color, odor, or taste) can indicate abnormalities of the water. outbreaks have occurred, however, when turbidity readings have met present standards and when water appeared and tasted good. chemical examination of water is useful for (a) detecting pollution (especially from industrial wastes and pesticides), (b) determining effectiveness of treatment processes, (c) evaluating the previous history of the water, (d) determining hardness, and (e) detecting the presence of specific toxins. results are usually expressed in milligrams per liter (mg/l = ppm, parts per million), or micrograms per liter (μg/l = ppb, parts per billion). historically, acute water-related outbreaks seldom involve chemical substances, so chemical tests are not requested routinely unless either (a) circumstances indicate possible chemical contamination or (b) clinical symptoms suggest chemical poisoning. flowing water in a distribution system can be monitored to determine chlorine residual. free available residual chlorine refers to that portion of the total residual chlorine remaining in chlorinated water at the end of a specific contact period that will react chemically and biologically as hypochlorous acid or hypochlorite ion. the reaction is influenced by ph and temperature. total or combined residual chlorine refers to chlorine that has reacted with ammonia or other substances and is not available for further reactions, as well as the free available chlorine. a chlorine demand exists in a chlorinated water until a free available residual is produced. a free available chlorine residual, e.g., 1 mg/l (1 ppm) or higher, maintained throughout the distribution system of a community supply is an indicator of safety from enteric bacteria but not necessarily from pseudomonads, viruses or parasites. outbreaks have occurred when chlorine residue levels have met present standards. analyses for microbial indicator organisms provide information on the microbiological quality of water and guidance as to its safety for consumption or contact. indicator organisms are easier to test for than pathogenic organisms, and some serve as a surrogate measure of fecal contamination in water. the absence of indicator organisms in the water, however, does not guarantee water safety; numerous outbreaks of water-related disease have occurred from water in which no indicator organisms were detected. evaluation of the safety of water should be based upon a combination of results of (a) an on-site study to identify sources and modes of contamination and means by which contaminants survived treatment and (b) appropriate laboratory analyses. microbiological results should be compatible with observed sources of contamination and/or treatment failures found during the investigation. although all natural waters contain bacteria, the number and kind vary greatly in different places and under different climatic and environmental conditions. the number of bacteria isolated and reported, however, often represents only a fraction of the total number present, for several reasons. colonies seen on agar plates develop from either single organisms or clusters or chains of organisms. heterotrophic bacteria represent only those that can use organic matter and grow at the selected temperature (30-35°c) within 48-72 hours under aerobic/microaerophilic conditions in/on a defined medium when the standard test (spread plate, membrane filter or pour plate) is used. the hpc may also be done using different media under different incubation times/conditions. (higher counts are usually found when the longer incubation periods are used.) also, certain microorganisms are unable to grow aerobically either in or on the medium used. because of these variables, the terms total plate count (tpc), standard plate count (spc) and aerobic plate counts (apc) should not be used. hpcs serve as an index of changing sanitary conditions. in general, counts of good-quality well water are fewer than 200-500 colonies per ml. densities in surface water are higher, but quite variable, depending on water temperature, sources of pollution, amount of organic matter present, and soil that washes into the water. the sources of pathogens, toxic substances, or fecal contamination may not increase the hpc of a surface water sample as much as washings from soil. nevertheless, marked changes in the number or kind of microorganisms should be viewed with concern, at least until the reason for the change is discovered. heterotrophic plate counts greater than 1000/ml and some specific antagonistic species may interfere with the growth or recovery of pathogenic or indicator organisms. some heterotrophic species are opportunistic pathogens that may pose a health threat to immunocompromised persons. the coliform group of bacteria comprises those from non-fecal environmental sources, and those from animal and human intestines, including escherichia coli. the environmental species of non-fecal bacteria are found in soil, on fruits, leaves, and grains, and in run-off water, especially after heavy rains. some of these species are capable of surviving in water longer than e. coli. furthermore, some coliform strains and can multiply on decaying vegetation in water, in biofilms in pipelines, or on pump packings, washers, and similar materials. therefore, finding coliforms may not be indicative of fecal contamination, although most water utilities have standards for coliforms in water. fecal coliforms are present in large densities in all human and animal feces, normally much higher than pathogens which are typically only present in infected persons and normally at lower levels. as such, high populations of fecal coliforms can indicate recent sewage pollution of water, but are not always indicative of pathogens present, particularly viruses and parasites. none of the coliform group, however persists as long as most viral or protozoan pathogens in water, and indicator bacteria described below (fecal streptococci and clostridium perfringens). typical chlorination or ozonation of water inactivates coliform bacteria. presence of the coliform group or even a high population of coliform bacteria is not proof that a treated water supply contains pathogens. however, coliforms can provide a warning that either the water treatment was inadequate or contamination occurred after treatment, and that some pathogens may be present. as mentioned above, under some conditions, pathogens may be present where there are few or no coliforms. furthermore, unlike coliforms, many parasites and viruses are resistant to normal levels of disinfectants. coliforms have little or no correlation with the presence of parasitic protozoa or pathogenic viruses. the standard test for the coliform group may be carried out by a membrane filtration technique, a multiple-tube fermentation technique (presumptive test, confirmed test, or completed test), or a presence-absence test. results of the membrane filtration technique are reported as colony forming units (cfu) per 100 ml of water. results of the multiple-tube fermentation technique are reported as the most probable number (mpn) per 100 ml of water. this is a statistical estimation of the total number present, but the actual number can fall within a considerable range. counts derived from these two methods are not necessarily the same, but they have the same sanitary significance. false-negative or false-positive results can also occur with the membrane filtration technique because of interfering background growth of nonfecal microorganisms. results of the presumptive test of the multiple-tube fermentation technique can be misleading, because other microorganisms frequently found in water also produce gas in laboratory media, and may thereby give false-positive results. also, especially in waters containing a large number of microorganisms, some coliforms present may produce gas slowly, leading to false-negative results. the presence of coliform bacteria is corroborated by means of the second phase of the multiple-tube fermentation technique, known as the confirmed test. positive results are usually considered confirmation of the presence of coliforms. a third phase of this test, known as a completed test, further ensures the correct identification of coliform bacteria. a simple modification of the coliform test is to analyze for the presence or absence of coliforms in a 100-ml drinking water sample. the "presence-absence (p/a) coliform test" allows for simple examination of a larger number of samples. when a positive sample is detected, it is advisable to measure coliform densities in repeat samples by one of the other methods to determine the magnitude of the contamination. thermotolerant coliform (fecal coliform). coliform bacteria will frequently grow at a relatively high temperature, 44.5°c, unlike species or strains normally encountered in the environment, which usually have an optimal temperature near 30°c. this thermotolerant characteristic has been used in an attempt to separate coliform bacteria into those of so-called fecal and non-fecal origin. this test may provide better indication of fecal contamination than the coliform test, but it is however, unreliable. positive results are not proof that either organisms of fecal origin or pathogens are present. the number of thermotolerant coliforms is considerably lower than the number of total coliforms in contaminated water; therefore, the test is less sensitive for testing treated drinking water. furthermore, escherichia coli o157:h7, which has been implicated as causing water-related illness, does not grow well at 44.5°c. escherichia coli. e. coli is common in feces of human beings, other mammals, and birds. it can also be found to grow naturally in the environment, specifically in tropical waters. comprised of the larger coliform group, its detection in water is a more definitive indicator of fecal contamination, compared to total or fecal coliforms. however, a positive test result does not identify if the fecal source is human or nonhuman. rather, the finding of e. coli in water serves as an indicator that fecal matter reached the water and provides a warning, but not proof, that pathogenic organisms may also be present. it should be noted that some strains of e. coli are pathogenic (see table b ). simple commercial p/a and quantitative tests have been developed to detect the presence of total coliforms and e. coli in 24 hours by observing color changes and fluorescence of the media under daylight and uv light. such tests may be useful for field evaluation of microbiological water quality. another group of organisms, collectively known as fecal streptococci, is also used as an indicator of fecal contamination. enterococci (enterococcus faecalis, enterococcus faecium) are particularly used for testing recreational waters. like coliforms, enterococci are normal inhabitants of the intestinal tract of human beings and other animals. in human feces, they occur in considerably lower numbers than e. coli. some members of the group, such as e. faecalis, subsp. liquefaciens, however, have been associated with vegetation, insects, and certain types of soils. enterococci generally survive longer than coliforms in fresh water, and therefore the source of contamination may be distant in either time or place from the site where samples were obtained. their resistance is, however, less than that of clostridium perfringens, enteric viruses, and parasites. like e. coli, simple commercial p/a and quantitative tests have been developed to detect the presence of enterococci in 24 hours by observing color changes under uv light, which may be useful for field evaluation of microbiological water quality. clostridium perfringens (sulfite reducing clostridia). c. perfringens is also of fecal origin, but it occurs in feces in much lower densities than e. coli and can also be found in soils. being a spore-former, it can survive for long durations in soil and water, and persist when all other bacteria of fecal origin have disappeared. therefore, it is a useful indicator of remote or intermittent contamination in wells that are not frequently examined by the coliform test; but, it is not, by itself, evidence of recent contamination. chlorine, in the concentration typically used in water treatment, does not inactivate all spores; and thus c. perfringens is not valuable in assessing the efficiency of chlorination for bacterial vegetative cells. its long persistence and its resistance to chlorine make this organism a potential indicator for viral and parasitic organisms that have similar resistance and disinfectant susceptibility. coliphage. coliphages, which are viruses that infect e. coli, are simpler to detect and enumerate, compared to other viruses, and are generally associated with fecal contamination. they have been considered as possible indicators of treatment effectiveness for human enteric viruses. coliphages are categorized into two groups: the somatic phages, which enter e. coli via the cell wall and the male-specific phages, which enter e. coli through the sex pili. the somatic and male-specific phages are common in sewage and the feces of human beings and other animals, but in lower densities than the common fecal indicator bacteria, fecal coliforms, e. coli, and enterococci. some strains appear to be more resistant to chemical disinfection than water-related pathogens or indicator bacteria. be aware of local standards for water distribution systems, private water systems, and recreational water. although drinking water standards, such as the total number of coliforms allowed in a water sample, vary from jurisdiction-to-jurisdiction, it is generally agreed that any fecal contamination (e.g. fecal coliforms, escherichia coli) render the water unacceptable for human consumption and may close down recreational bathing waters. there are numerous pathogens that can be transmitted by water, many of which are also able to cause respiratory symptoms, in addition to the classical gastrointestinal symptoms. for a comprehensive summary of waterborne pathogens see "american waterworks association manual of water supply practices, m48 waterborne pathogens, 2nd edition (2006) ." for several reasons, analyses for pathogens are not usually conducted during routine water testing, or are only conducted by specialized laboratories. first, tests for pathogens are pathogen-specific, expensive, and often difficult to perform because they may require specialized trained personnel. secondly, the etiologic agent of the outbreak is often unknown at the time of analysis; hence, many analyses would have to be done blindly. thirdly, pathogens are not always recovered because they are heterogeneously dispersed and diluted in the environment, and their numbers decline in water over time. as a result, they may be absent or present in low densities by the time samples are collected following an outbreak. fourthly, recovery efficiencies are often poor because microorganisms are stressed by disinfectants or the method is sensitive to interferences from the source waters environment, hence not easily recovered by routine methods. additionally, recovery efficiencies for viruses and protozoa may be poor because of the interferences of substances within the sample matrix with method reagents (concentrating 1800 l of water down to 200 μl will also concentrate inhibitory chemicals and substances). finally, the time required for isolation and identification is often long, and the number of samples is usually too small to allow the investigator to have much statistical confidence in the results when pathogens are not found. negative results should be reported as "not detected" because they do not ensure that the water sampled was not the source of the pathogen. procedures used for many bacterial pathogens are qualitative because enrichment procedures are used. quantitative procedures (e.g., mpn) require considerable work and are less reliable than those used for coliforms because small populations may be present, and these may be unevenly distributed. despite these difficulties, pathogens that cause a syndrome similar to the one being investigated should be sought. see tables b, c, and d, for descriptions of the disease syndrome associated with the pathogens described in the following material. finding the same pathogen in specimens from patients and in water samples confirms water as a vehicle. summarize investigative data in a narrative report. describe in this report situations that led to contamination of the water and survival of etiologic agents up to the time of consumption. include all events that contributed to the outbreak to guide control and preventive measures. compare your data with the listings in table g (guidelines for confirmation of waterborne outbreaks) and table h (guidelines for confirmation of water responsible for illness), and criteria for confirmation of vehicle responsible for waterborne illness before assigning the etiologic agent and the vehicle. outbreak confirmation is based on (a) time, place, person associations, (b) recovery of etiologic agents from clinical specimens from cases and samples of water, and (c) identification of sources and modes of contamination and means by which pathogens or toxic substances survived treatment. all three of these, however, might not be found in any one investigation. complete form k (waterborne illness summary report). attach the narrative and the epidemic curve. also attach form d2 (case history summaries: water/laboratory data), all applicable parts of forms g, forms h, form i, and other data that will provide supplemental information to reviewers. send this report through administrative channels to the appropriate agency responsible for waterborne disease surveillance. make the final report as complete as possible, so that the agency can accurately interpret the results and develop a meaningful waterborne disease data bank. in the interest of continuing cooperation, give all participants in the investigation due credit and send each a copy of the report. also, send copies of the report through administrative channels to agencies (a) that have jurisdiction over the implicated water, (b) that initiated the alert, and (c) that participated in the investigation. those concerned with water sources, treatment and recreation, as well as with public health, should make every effort to ensure the complete investigation and reporting of waterborne diseases. without reliable, complete information, trends in waterborne disease incidence and causal factors of the disease are difficult to determine. good surveillance is essential for detecting and evaluating new waterborne disease hazards. the primary purposes of a waterborne disease investigation are to identify the cause, establish control measures, and take actions to prevent future illness. prudence may require some action before all the hypotheses regarding the water supply involved and the source of contamination are confirmed. frequently the local health authority will issue a boil water advisory if a microorganism is suspected to have contaminated the water. refer to "possible precautionary control actions" section for a discussion of these precautionary control measures. if these measures have not already been considered, consider them now. once control measures have been implemented, continue to monitor for disease to evaluate whether the measures were effective. in a waterborne event in sydney, australia (see box 1) sydney water severely overestimated levels of cryptosporidium and giardia present in the water raising public alarm. boil water advisories were announced and rescinded several times. however, it is better to announce boil-water advisories than to have thousands ill, as has happened in the past, such as the cryptosporidium outbreak in milwaukee in 1993. deficiencies in treatment must be corrected and defective parts of distribution systems must be repaired, beginning with those that either contributed to or had a high potential for contributing to the outbreak. the effectiveness of these efforts will be directly related to the thoroughness of the investigation. document the source and the manner of contamination and survival of the etiologic agent through the water treatment process. provide clear documentation of contributory factors, so that preventive measures taken will be specific to the problem. if previous sanitary surveys have revealed, or if subsequent ones reveal, that conditions which contributed to the outbreak are widespread, initiate a training and education program. these programs can be developed for water treatment plant or recreational water operators and employees, engineers, homeowners, or other appropriate groups. impress upon them the importance of proper construction and operation of facilities and proper protection, treatment, storage, and distribution of water. follow up with periodic inspections and surveys and verify by sampling, as appropriate, to determine whether faulty conditions have been corrected or allowed to be reintroduced. legal action may be necessary to ensure compliance with official standards and accepted sanitary practices. formulate solutions to problems found during outbreak investigations, and incorporate these into regulations for drinking, agricultural, industrial, domestic, and recreational waters. inform the public, through mass media and other means available to your agency, of hazardous conditions that can affect their water supply, but do so only after hypotheses are confirmed. the public must be told of any potential or actual harm that may result from ingesting or contacting contaminated water and must also be informed of measures that they can take and that official agencies are taking to correct these conditions. the water supply and recreational water facilities must be verified periodically to determine whether critical processes are being monitored and operated within limits of appropriate public health standards (see box 2, the walkerton outbreak). most waterborne illnesses are preventable, but prevention requires that those in the water treatment industry and in health and water-protection regulatory agencies be constantly vigilant to ensure that the hazards are understood and that questionable water treatment or delivery system construction or practices are avoided. examples investigation guidelines and investigative forms iafp manual, "procedures to investigate waterborne illness, 3rd ed"; 50 copies of form c; one dozen copies each of forms e and f; two copies of form d and all parts of form g, epi-info software (cdc, atlanta). water sample bottles (bottles for chlorinated water should contain enough sodium thiosulfate to provide a concentration of 100 mg of this compound per l of sample), plastic bags (whirl-pak ® type), 250 ml, 1-l and 1-gal sized jars and jugs. sterile and wrapped sampling implements moore swabs (compact pads of gauze made from strips 120 cm [4 ft] by 15 cm [6 in.] tied in the center with a long, stout twine or wire-for sewer drain, stream or pipeline samples), fiberglass-epoxy bacterial filter cartridge, 0.3 μm; tongs, scoop or similar utensils for collecting ice. specimencollecting equipment (for human specimens from cases and controls) sterile containers (with lids) for stool specimens, bottles containing a bacterial preservative and transport medium, mailer tubes or styrofoam box, sterile swabs, rectal swab units, tubes of bacterial transport medium, stool preservative medium for parasites, phlebotomy supplies for blood specimens. kits for testing chemical disinfectants and ph dpd (n,n-diethyl-p-phenylenediamine) chlorine comparator with color disc for chlorine (0.1 ppm) and chlorine test papers; field-type ph meter or ph comparator with color disc or ph test papers; applicable ph indicator solutions and dpd reagent solution; dissolved oxygen testing unit. dye tracing study equipment fluorescein (yellow-green fluorescent) dye in powder form (10 packages containing 300 g each), in tablet form (100 tablets), or in liquid form (prepared by mixing 300 g in 1 l of water); fluorometer; filters (primary and secondary) for use with fluorometer; sample holder for continuous sampling or individual sampling; fluorometer recorder. disinfectant and neutralizer 0.5% w/v solution of calcium hypochlorite or 5.25% household liquid bleach; 50% w/v sodium thiosulfate. virus filtration equipment for viruses and parasites b large plastic container for storing water sample prior to concentration; portable electric or gasoline powered water pump with quick disconnect brass or stainless steel plumbing adapters or hose couplings; two filter holders for 10-in. water filter cartridges fitted for adapters or couplings; portable water meter fitted for adapters or couplings; four lengths of fiber-reinforced garden hose fitted with adapters or couplings; one length of a strong-walled supply hose fitted with adapters or couplings; 10-in. prefilter (3 μm nominal porosity wound polypropylene yarn filter with hollow perforated stainless steel core) cartridge filter; 10-in. virus absorbing filter pleated 0.2 μm porosity nylon membrane type (positively charged) for waters of ph values up to 8.5, or pleated 0.45 μm porosity glass fiber membrane type (positively charged) for waters of ph value of 7.5 or lower (e.g., virosorb, 1-mds, amf/cuno meriden, pleated, 0.45 μm, glass filter); 1600 ml sterile, ph 7, 3% beef extract solution in 1 gal wide-mouth screw capped autoclavable polypropylene container for each sample to be collected; stands to support filter holders during filtration; for parasites 10-in. polypropylene yarn-wound cartridge filter, 1.0 μm porosity (e.g., micro wynd ii™, amf/cuno; meriden, ct. 1.0 μm normal porosity). (continued) laptop or tablet, with software; thermocouples of varying lengths with either recording potentiometer, data logger, or digital indicator; devices to take samples below surface and sediment samples; chemical smoke kit and/ or micromanometer; occupational safety and health administration (osha) or equivalent approved respirator; sterile plastic gloves; plastic container liners for ice; waterproof marking pens; waterproof test tube rack; pencils, note pad; roll of adhesive or masking tape; labels; waterproof cardboard tags with eyelets and wire ties; flashlight; matches; test tube rack to fit tubes used; insulated chest or styrofoam container; packing material; camera with flash; spare batteries for all equipment; 95% ethyl alcohol; propane torch; refrigerant in plastic bags, liquid in cans, rubber or heavy plastic bags that can be filled with water and frozen; heavy-duty bags for ice, "canned ice," or cold-packs (blue ice). a assemble a kit to be kept in the agency responsible for investigating waterborne illness. it should include at least ten water sample bottles; ten 1-l, or gal jars or jugs; ten specimen collection containers or devices; and one each of the following supporting equipment and sterilizing equipment. date of sterilization should be marked. periodic resterilization or replacement of sterile supplies, media, or transport media is required to maintain the kit in a ready-to-use condition b similar equipment for sampling for either viruses or parasites may be available from national water, environmental, or health agencies how much to collect two rectal swabs or swabs of fresh stool from ten ill persons; samples from ten controls also can be submitted. whole stool is preferred if nonbacterial stool testing considered a fresh stool sample from ten ill persons; samples from ten controls can also be submitted. to enhanced detection, three stool specimens per patient can be collected >48 h apart as much stool sample as possible from ten ill persons (a minimum of 10 ml of stool sample from each); samples also can be obtained from ten controls. a fresh urine sample (50 ml) from ten ill persons; samples from ten controls also can be submitted. label each specimen in a waterproof manner and put the samples in sealed, waterproof containers (i.e., plastic bags). batch the collection and send in overnight mail to arrive at the testing laboratory on a weekday during business hours unless other arrangements have been made in advance with the testing laboratory. contact the testing laboratory before shipping, and give the testing laboratory as much advance notice as possible so that testing can begin as soon as samples arrive. when etiology is unclear and syndrome is nonspecific, all four types of specimens may be appropriate to collect c for more detailed instructions on how to collect specimens for specific parasites, please go to http://www.cdc.gov and search the website of key words d for more detailed instructions on how to collect specimens for viral testing, please go to http://www.cdc.gov and search the website of key words e the containers have been tested for the presence of the chemical of interest prior to use f unused specimen collection containers that have been brought in to the field and subjected to the same field conditions as the used containers. these containers are then tested for trace amounts of the chemical of interest table e (continued) isolation of agent from ill persons and from water and laboratory criteria for confirming etiologic agent as stated in table g . combination of on-site investigation, statistical evidence and laboratory analysis. (see entries below) presumptive vehicle on-site investigation demonstrating source and mode of contamination of water and survival of etiologic agent in water. also, desirable to have laboratory isolations from water of etiologic agent that causes syndrome similar to that observed during the investigation and other supportive epidemiologic data. if so, this might provide sufficient evidence for confirmation. or p-value for water <0.05 when other epidemiologic data supports water hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. or odds ratio or relative risk for water greater than 2 and the lower limit of the 95% confidence level greater than 1 when other epidemiologic data supports the water vehicle hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. for calculation of data when all persons who may have been exposed to the suspect vehicle (but not all persons will have ingested every beverage/food) have been identified and interviewed (retrospective cohort) form j1: chi-sq. analysis can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps: ill well totals step 1: create a 2x2 table as shown with observed data (o values) and marginal totals step 2: calculate odds ratio: (axd)/(bxc) = (18x16)/(12x3)= 8.0 step 4 . . chi-sq. (all four cells) = 2.057 + 1.543 + 3.248 + 2.436 = 9.284 step 5: compare your calculated chi-sq. value with the critical value to determine significance (table 17) : begin at column p < 0.05 … your calculated chi-square value must meet or exceed the critical value to be considered statistically significant at that level. if you fail to meet or exceed the minimal value for p < 0.05, the result is p > 0.05, and the relationship is declared "not significant". step 6: summarize: exposure was related to illness. ill persons were eight times more likely to have been exposed to this factor than non-ill persons. this relationship is statistically significant. the probability of these data occurring by chance alone is less than 0.5%. reject the null hypothesis of "no association." [odds ratio: 8.0, 95% cl: 1.64 < or < 44.23, chi-sq.: 9.28, 1 df, p < 0.005] form j2: fisher's exact test can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps. ill well totals step 1 step 2: calculate the probability directly... here, cell 'a' has smallest e value at 4.2 p 1 = 10! x 21! x 13! x 18! = 0.0044 8! x 5! x 2! x 16! x 31! step 5: the final probability (p) is the sum of all probabilities (in this case p 1 + p 2 + p 3 ) or approximately 0.0047. step 6: summarize: exposure was related to illness. ill people were almost 13 times more likely to have been exposed to this factor compared to non-ill people. the relationship is statistically significant. the probability of these data occurring by chance alone is less than 0.5% (<0.005). reject null hypothesis of "no association". [odds ratio: 12.8, 95% cl: 1.6 < or < 131.1, p = 0.0047]. 1. when deciding which cells to increase by +1, always multiply (a) × (d) and compare with (b) × (c). increase each cell of the pair with the higher product and decrease each cell of the pair with the smaller product, while keeping all marginal totals unchanged. 2. the final p is an "exact" p (probability) and may be reported as such (p = 0.0047). in this example, it is also <0.005 of course, and can be reported in this way if preferred. 3. the fisher's test is used when the chi-square test is invalid due to any "e" values <5 in a 2 × 2 table. in all other circumstances, chi-sq. is an excellent approximation for the fe test. 4. if original data include a zero in one of the cells, you will calculate only one p value. (the o.r. will be reported as "undefinable" but the direction of the effect will be very clear). 5. this p is calculated for a one-tailed fe test. it is adequate for this application. two-tailed fe test will require further calculation. 6. should a relationship not meet the critical value for significance (that is, p > 0.05), it is described as "not statistically significant". note that a relationship may be observed, but this result is telling you that it could have occurred by chance alone more than 5% of time if you were to repeat the analysis. that may still require further investigation, but from a statistical standpoint, it cannot be claimed as a statistically significant relationship. water treatment plant design, fifth edition american waterworks association. manual of water supply practices, m48 waterborne pathogens, second edition the flint water crisis confirms that u.s. drinking water needs improved risk management produce safety-what's going on here? national environmental health association neha-cert ep0704 global health -division of parasitic diseases and malaria page last reviewed surveillance reports for recreational water-associated disease & outbreaks. page last reviewed on internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce environmental protection agency technical guidance manual lt1eswtr disinfection profiling and benchmarking environmental protection agency guidance manual for the compliance with filtration and disinfection requirements public water systems using surface water sources environmental protection agency. manual of individual and non-public water supply systems. epa no. 570991004 epa no. 570991003 water treatment manual: disinfection. environmental protection agency health canada risk analysis of the walkerton drinking water crisis algal bloom-associated disease outbreaks among users of freshwater lakes-us microbial resistance to disinfectants: mechanisms and significance. environ health perspect safe drinking water: lessons from recent outbreaks in affluent nations risk management for assuring safe drinking water ensuring safe drinking water: learning from frontline experience with contamination. american water works association cyanotoxin management and human health risk mitigation in recreational waters a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply private water supplies: technical manual good practice guide to the operation of drinking water supply systems for the management of microbial risk: research project 1074 two cases of keratosis follicularis squamosa (dohi) caused by swimsuit friction part two report of the walkerton inquiry: a strategy for safe drinking water public health inspector's guide to the principles and practices of environmental microbiology acute otitis externa: an update collect representative distribution system samples guidance on water supply and sanitation in extreme weather events. who regional office for europe effect of irrigation method on transmission to and persistence of escherichia coli o157:h7 on lettuce transmission of escherichia coli o157:h7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization acknowledgments the committee and association thank and cite the following persons for chloramines refer to all forms of chloramine. the ct values may be assumed to achieve greater than 99.99% inactivation of viruses only if chlorine is added and mixed in the water before addition of ammonia. if this condition is not met, the system must demonstrate by on-site studies or other information that it is achieving at least this much inactivation of viruses key: cord-282530-55lhjfm8 authors: carsana, luca; sonzogni, aurelio; nasr, ahmed; rossi, roberta simona; pellegrinelli, alessandro; zerbi, pietro; rech, roberto; colombo, riccardo; antinori, spinello; corbellino, mario; galli, massimo; catena, emanuele; tosoni, antonella; gianatti, andrea; nebuloni, manuela title: pulmonary post-mortem findings in a series of covid-19 cases from northern italy: a two-centre descriptive study date: 2020-06-08 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30434-5 sha: doc_id: 282530 cord_uid: 55lhjfm8 background: covid-19 is characterised by respiratory symptoms, which deteriorate into respiratory failure in a substantial proportion of cases, requiring intensive care in up to a third of patients admitted to hospital. analysis of the pathological features in the lung tissues of patients who have died with covid-19 could help us to understand the disease pathogenesis and clinical outcomes. methods: we systematically analysed lung tissue samples from 38 patients who died from covid-19 in two hospitals in northern italy between feb 29 and march 24, 2020. the most representative areas identified at macroscopic examination were selected, and tissue blocks (median seven, range five to nine) were taken from each lung and fixed in 10% buffered formalin for at least 48 h. tissues were assessed with use of haematoxylin and eosin staining, immunohistochemical staining for inflammatory infiltrate and cellular components (including staining with antibodies against cd68, cd3, cd45, cd61, ttf1, p40, and ki-67), and electron microscopy to identify virion localisation. findings: all cases showed features of the exudative and proliferative phases of diffuse alveolar damage, which included capillary congestion (in all cases), necrosis of pneumocytes (in all cases), hyaline membranes (in 33 cases), interstitial and intra-alveolar oedema (in 37 cases), type 2 pneumocyte hyperplasia (in all cases), squamous metaplasia with atypia (in 21 cases), and platelet–fibrin thrombi (in 33 cases). the inflammatory infiltrate, observed in all cases, was largely composed of macrophages in the alveolar lumina (in 24 cases) and lymphocytes in the interstitium (in 31 cases). electron microscopy revealed that viral particles were predominantly located in the pneumocytes. interpretation: the predominant pattern of lung lesions in patients with covid-19 patients is diffuse alveolar damage, as described in patients infected with severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses. hyaline membrane formation and pneumocyte atypical hyperplasia are frequent. importantly, the presence of platelet–fibrin thrombi in small arterial vessels is consistent with coagulopathy, which appears to be common in patients with covid-19 and should be one of the main targets of therapy. funding: none. since the earliest reports of cases in china in december, 2019, countries around the world have faced outbreaks of covid-19, the disease caused by the novel coronavirus severe acute respiratory syndrome corona virus 2 (sars-cov-2). italy was the first country in europe to document a large number of cases of covid-19, and lombardy in particular was severely affected, with a total of 17 713 people testing positive for sars-cov-2 and 1593 admitted to intensive care units between feb 20 and march 18, 2020. 1 luigi sacco hospital in milan and papa giovanni xxiii hospital in bergamo were the first hospitals in lombardy faced with managing the epidemic crisis. sars-cov-2 is the seventh member of the coronavirus family identified to cause disease in humans. coronaviruses are enveloped, positive-sense, single-stranded rna viruses. 2 two other members of this family, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), cause acute diffuse alveolar damage, pneumocyte hyperplasia, and interstitial pneumonia. [2] [3] [4] at the mild end of the clinical spectrum, covid-19 can be asymptomatic or manifest as mild upper respiratory disease with fever and cough, while severe cases can result in pneumonia, leading to acute respiratory distress syndrome (ards) in around 15% of hospitalised patients. 5 to the date of our last literature review (may 3, 2020), the only published reports of the pathological features of covid-19 lung involvement were from small case series or isolated cases, both from china and other countries. in those reports, the main histological fea tures comprised exudative diffuse alveolar damage with massive capillary congestion, often accompanied by microthrombi. [6] [7] [8] [9] we describe the lung histopathological findings from a large series of patients who died from covid-19 in northern italy, with the aim of reporting the main micro scopic pulmonary lesions associated with sars-cov-2 infection and severe respiratory failure. we histologically analysed lung tissue samples from 38 consecutive patients who died from covid-19 between feb 29 and march 24, 2020, in two referral centres for the management of the covid-19 outbreak in northern italy: luigi sacco hospital in milan (20 autopsies) and papa giovanni xxiii hospital in bergamo (18 autopsies). all patients had sars-cov-2 infection confirmed by real-time pcr analysis of throat swab samples taken at the time of hospital admission, and all had undergone molecular tests for common respiratory viruses and bacteria by the microbiology laboratories at the respective hospitals, with negative results. the ethics committees of luigi sacco hospital and papa giovanni xxiii hospital approved the use of personal and sensitive patient data for scientific research related to the disease in this study. the study followed the italian general rules used for scientific research purposes (regulation n.72-26/03/2012). autopsies were done in airborne infection isolation autopsy rooms, with personnel using personal protective equipment in accordance with the italian recommendations. 10 a team of pathologists (lc, as, an, rsr, ap, pz, ag, and mn) with extensive experience in the field of infectious diseases was involved in the autopsy procedures in both hospitals. a median of seven tissue blocks (range five to nine) were taken from each lung, selecting the most repre sentative areas identified at macroscopic examin ation. tissues were fixed in 10% buffered formalin for at least 48 h. paraffin-embedded sections of 3-µm thickness were stained with haematoxylin and eosin. to better characterise the inflammatory infiltrate, immunohistochem ical staining was done on the most representative areas of randomly selected cases, and included staining with antibodies against cd45 (clones 2b11 and pd7/26) for the identification of leucocytes, cd3 (clone 2gv6) for t lymphocytes, cd68 (clone kp-1) for mono cytes, and cd61 (clone 212) for megakaryocytes. tissues were also stained with antibodies against ttf1 (clone 8g7g2/1) and p40 (clone bc28) to identify squamous metaplasia of pneu mocytes, and ki-67 (mib-1) to determine the proliferative index of epithelial cells. all antibodies were ready-to-use monoclonal antibodies (ventana, roche diagnostics, basel, switzerland) and staining was done with the benchmark ultra ihc/ish system (roche, basel, switzerland) in accordance with the standard protocols supplied by the manufacturer. masson trichome staining was applied to char acterise collagen and fibrin deposition, epithelial cells, and fibrosis. additionally, in cases with histological suspicion for bacterial or fungal infections, periodic acid-schiff staining and grocott methenamine silver staining were done to confirm morphological findings. additional samples from ten consecutive cases were assessed for the presence of viral particles with use of electron microscopy: two samples from each lobe were selected, fixed in glutaraldehyde, and examined with use of a zeiss em-109 transmission electron microscope (zeiss, cologne, germany) and an olympus megaview g2 transmission electron microscopy camera with an integrated imaging platform (item; olympus, munich, germany) evidence before this study we searched pubmed up to may 3, 2020, using the search terms "autopsy" and "covid-19", with no language restrictions. the search revealed ten reported cases. lung morphological features of covid-19 in deceased patients were described in a few isolated case reports and in small case series, including some reports in chinese. histological descriptions of lung pathology were extremely concise, without any analytical report of morphological details, and lacking any highlighting of distinctive lesions compared with other forms of interstitial pneumonia. furthermore, as only quantitative and aggregate data were available, the possible clinical implications of the findings could not be determined. we histologically examined the lung tissues of 38 patients who died from covid-19-to our knowledge the largest post-mortem series so far reported-in two main hospitals providing care to patients with progressive breathing failure in a peak epidemic area in italy. we focused on the detailed analysis of histological features in these patients to elucidate any distinctive lesions associated with covid-19. to our knowledge, these data represent the first relevant provisional information regarding tissue damage specifically induced by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), besides the previously described diffuse alveolar damage, a feature that characterises interstitial pneumonia regardless of infectious agent. although our observations are provisional, they were obtained in a large cohort of patients and revealed that histopathological lung damage was characterised by expected features of diffuse alveolar damage, as well as diffuse thrombotic vascular involvement. this latter finding could be relevant in the management and targeted treatment of patients infected with sars-cov-2, with the potential to modify outcomes. histological evaluation was done independently by two pathologists from each hospital, from the same team involved with the autopsy, who were masked to patient characteristics, symptoms, and diagnoses. each pathologist analysed all the slides from both hospitals, and any discrepant results were jointly reviewed. the histological features of cellular and interstitial damage were described and graded on a semiquantitative scale on the basis of the percentage of tissue involved: absent (0%), rare (<5%), focal (5-25%), multifocal (26-50%), plurifocal (51-75%), or diffuse (>75%). 11 to quantify pulmonary intracapillary megakaryocytes, each tissue sample was scanned at low magnification to identify the hotspot area in which megakaryocytes were most easily recognisable, and cd61-positive cells in these areas were counted. a high value was defined as the presence of more than four cd61-positive cells per 25 high-power fields, which is considered to be the average number of intracapillary megakaryocytes in the lungs of people without diffuse alveolar damage. 12 this study had no funding source. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. patients were 33 men and five women, with a mean age of 69 years (sd 12; range 32-86). time spent in the intensive care unit or intermediate medical ward (subintensive care) ranged from 1 day to 23 days (mean 7 days [sd 6]). regarding past comorbidities, data were available for 31 patients: nine (29%) had diabetes, 18 (58%) had hypertension, four (13%) had past malignancies, 11 (35%) had cardiovascular disorders, and three (10%) had mild chronic obstructive pulmonary disorders. at the time of hospitalisation, all patients had clinical and radiological features of interstitial pneumonia. of the 26 patients with available d-dimer results, all had high values (>10 × the upper reference limit). mean time from symptom onset to death was 16 days (sd 6; range 5-31). upon macroscopic examination, the lungs of all patients were heavy, congested, and oedematous, with patchy involvement. in all cases, histological examination revealed features corresponding to the exudative and early or intermediate proliferative phases of diffuse alveolar damage (figure 1). these features were also focally associated with patterns of interstitial pneumonia (presence of inflammatory lymphomonocytic infiltrate along the slightly thickened interalveolar septa), organising pneumonia (alveolar loose plugs of fibroblastic tissue), and acute fibrinous organising pneumonia (some alveolar spaces containing granulocytes and fibrin, with the formation of balloon structures). features indicative of the fibrotic phase of diffuse alveolar damage, such as mural fibrosis and microcystic honeycombing, were observed to be focal, suggesting that none of the patients had progressed to the fibrotic phase, possibly because of the short duration of the disease. histological examination of the main bronchi and bronchiolar branches revealed mild, non-specific alterations: focal squa mous metaplasia and mild transmural lymphocytic and monocytic infiltrates. lumina often contained residual dense mucoid material, and granulo cytes were present in ten cases. four (11%) patients also had bacterial abscesses (one or two per lung, <5 mm in diameter), and one (3%) had a single fungal abscess (<7 mm in diameter). the abscesses were presumed to have formed after hospital admission. morphological findings and their corresponding semiquantitative grades are reported in the table. the predominant histological pattern of the exudative phase of diffuse alveolar damage, which was observed in all cases, included capillary congestion, interstitial and intraalveolar oedema, dilated alveolar ducts and collapsed platelet-fibrin thrombi in small arterial vessels (<1 mm diameter) were found in 33 (87%) cases. moreover, type 2 pneumocyte hyperplasia, showing various aspects of cellular atypia, was present to some extent in all patients; interstitial myofibroblastic reaction was observed in 25 (66%) cases and alveolar granulation tissue in 22 cases (58%), whereas septal collagen deposition was found in 15 (39%) cases and alveolar loose plugs of fibroblastic tissue in 11 (29%). mural fibrosis was sometimes observed (24 [63%] cases), as was microcystic honeycomb ing (15 [39%] cases, most often with a focal pattern of distribution). although no clinical history of pre-existing fibrosing interstitial lung diseases was found in patients with mural fibrosis and microcystic honeycombing, the presence of mild fibrotic alterations not in continuum with other pre-fibrotic fea tures (myofibroblastic proliferation or organising pneu monia), as expected in a context of disease progression, might suggest that fibrosing interstitial lung diseases were pre-existing in some cases. the inflammatory component was represented by cd45-positive and cd3-positive lymphocytes infiltrating the interstitial space; a large number of cd68-positive macro phages were also found, mainly localised in the alveolar lumina. immunohistochemistry with anti-cd61 antibodies identi fied an increased number of megakaryocytes in the lung capillaries in 33 (87%) cases. ultrastructural examination (figure 2) revealed particles suggestive of viral infection in nine (90%) of the ten cases analysed. the particles had a mean diameter of about 82 nm and projection of about 13 nm in length. the particles, assumed to be virions, were mainly localised along plasmalemmal membranes and within cytoplasmic vacuoles, as described for other corona viruses. 13 infected cells were type 1 and type 2 pneumocytes; however, in two cases, particles were observed in alveolar macrophages, albeit scarcely. no particles resembling viruses were observed in multinucleated cells. ultra structural analyses of alveolar capillaries frequently showed platelet and fibrin plugs within the lumina, but no particles resembling virions were detected in endothelial cells. to our knowledge, we report the largest series of covid-19 autopsies focusing on pulmonary lesions. in all samples, a diffuse pattern of exudative and early proliferative phases of diffuse alveolar damage was alveolar multinucleated giant cells 19 (50%) 6 (16%) 9 (24%) 1 (3%) 1 (3%) 2 (5%) tissues were categorised on the basis of the percentage of tissue involved, as follows: absent (0%), rare (<5%), focal (5-25%), multifocal (26-50%), plurifocal (51-75%), or diffuse (>75%). *absent was defined as fewer than four cells per 25 high-power fields. found, while the fibrotic phase was rarely observed. the distinctive histopathological findings were atypical pneumocytes (reactive atypia) and diffuse thrombosis of the peripheral small vessels. sars-cov, mers-cov, and sars-cov-2 infections show many similarities in clinical presentation. 2 sars-cov and mers-cov particles have been observed and described in pneumocytes, macrophages, and lung interstitial cells by electron microscopy, immunohistochemistry, and in situ hybridisation. 3, 4, 11, 14 in two autopsy studies of patients who died from sars (eight cases from singapore 11 and 20 cases from toronto), 3 the predominant pattern of lung injury was diffuse alveolar damage, including the exudative and proliferative phases. inflammatory infiltrate, oedema, pneumocyte hyperplasia, fibrinous exudate, and organisation were found. the toronto case series 3 included a comparison group of matched control patients who presented with respiratory symptoms and signs, died in the same period as those with sars, and were negative for sars-cov. com pared with non-sars lung lesions, sars lesions were distinguishable by a prominence of vascular endothelial injury and extensive acute lung injury in varying stages of exudation and organisation. 3 autopsy studies of patients who died from mers are limited. in the only complete report available, 4 the authors described lung injury characterised by exu dative diffuse alveolar damage, pneumocyte hyperplasia, and septal inflammatory infiltrate. despite the relevance of lung involvement in patients with covid-19, few data regarding lung pathology are available. in a case report of a patient who died from covid-19 in china, the histological findings in the lungs included desquamation of pneumocytes, diffuse alveolar damage, and oedema. 7 in addition, tian and colleagues 9 described the pulmonary pathology of early-phase covid-19 in two patients with lung carcinoma; both patients showed signs of the exudative phase of diffuse alveolar damage. in our study, fibrin thrombi in the small arterial vessels (<1 mm diameter) were observed in 87% of cases, around half of which had involvement of more than 25% of the lung tissue as well as high levels of d-dimers in the blood. these findings might explain the severe hypoxaemia that characterises ards in patients with covid-19. 1 vascular micro thrombi are often identified in areas of diffuse alveolar damage, and are associated with diffuse endothelial damage. these features, although not pathognomonic, were frequent in our series, widespread in the lung samples of the patients examined, and the predominant distinctive vascular component. our data support the hypothesis proposed in clinical studies 15 that covid-19 is complicated by coagulopathy and thrombosis. furthermore, d-dimer values of greater than 1 µg/ml have been associated with fatal outcomes in patients with covid-19. 16 for these reasons, the use of anticoagulants has been suggested to be potentially beneficial in patients with severe covid-19, owing also to their anti-inflammatory properties, although their efficacy and safety are being closely monitored. [17] [18] [19] in this study, we looked for virions in a subset of patients, and found particles resembling virions to be present, albeit rarely, in the cytoplasm of pneumocytes and macrophages. the morphology of the observed particles (about 80 nm in diameter, enveloped, with spike-like projections, and an electron-lucent core with peripheral electron-dense granules of the sectioned nucleocapsid) and their intravacuolar cytoplasmic location are consistent with the reported ultrastructural features of coronaviruses, including sars-cov-2. 20 despite the low number of cases assessed, these findings might suggest that the virus remains in the lung tissue for many days, even if in small quantities, and might trigger the mechanism that leads to lung damage and causes it to progress. further histological and molecular analyses and extension of the case series are ongoing to better define the cellular and tissue distribution of the virus as well as inflammatory responses in different organs. although this report represents the largest european study of lung autopsy findings from cases of covid-19 to date and is based on the analysis of a large number of lung samples, it is limited by the absence of controls. future pathological studies should include an extensive analysis of cases of ards associated with other viral pneumonias. baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov pulmonary pathology of severe acute respiratory syndrome in toronto histopathology of middle east respiratory syndrome coronavirus (mers-cov) infection-clinicopathological and ultrastructural study clinical features of patients infected with 2019 novel coronavirus in wuhan, china covid-19 autopsies pathological findings of covid-19 associated with acute respiratory distress syndrome histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer management of the corpse with suspect, probable or confirmed covid-19 respiratory infection-italian interim recommendations for personnel potentially exposed to material from corpses, including body fluids, in morgue structures and during autopsy practice lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore megakaryocytes and platelet homeostasis in diffuse alveolar damage the intracellular sites of early replication and budding of sars-coronavirus immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan incidence of thrombotic complications in critically ill icu patients with covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy anticoagulant therapy in acute respiratory distress syndrome thromboembolic risk and anticoagulant therapy in covid-19 patients: emerging evidence and call for action sarscoronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology we declare no competing interests. key: cord-282561-t1edr9gp authors: gershengorn, hayley b.; warde, prem r.; nguyen, dao m.; suarez, maritza m.; merchant, nipun b.; ferreira, tanira; shukla, bhavarth title: pre-procedural screening for covid-19 with nasopharyngeal polymerase chain reaction testing date: 2020-08-15 journal: br j anaesth doi: 10.1016/j.bja.2020.08.011 sha: doc_id: 282561 cord_uid: t1edr9gp nan 1 example, symptoms concerning for . the low test positivity rate precluded multivariable modeling. among cases with >1 test, we evaluated the predictive accuracy of the first test for the second test. irb approval was obtained from the university of miami (#20200739). our cohort consisted of 4,176 cases (3,804 patients in sum, we found that very few (1 in 220) cases had a positive covid-19 pcr within 7 days preprocedure; this rate was substantially lower (1 in 442) if testing was specifically for "pre-procedural" indications. pre-procedural testing is intended to protect patients from morbidity and clinicians and staff from exposure. however, when prevalence is low, testing costs (i.e., financial, resources) coupled with procedural delays from false positive results cannot be ignored. the exact point prevalence of covid-19 active infection in miami during the study period is not known; yet, antibody testing of residual sera collected by a commercial laboratory from april 6-10, 2020 estimated covid-19 prevalence of past j o u r n a l p r e -p r o o f infection in south florida to be 1.9% (95% confidence interval: 1.0%-3.2%). 5 with 5% community prevalence, a positive result from a test with 70% sensitivity and 99% specificity will be found in likely uninfected people ~1 in every 5 times (supplemental figure 2 ). our data are limited by their single-centre nature. we were unable to reliably assess the indication for covid-19 testing in all cases; however, ineffectively excluding symptomatic patients likely biases us towards overestimating screening positivity rates. our analysis shows that there is little role for obtaining more than 1 test pre-procedurally. and, as we learn more about the incubation period, risk of asymptomatic transmission, and exposure potential of covid-19, it will be important to reconsider policies advocating for testing every patient pre-procedurally, even once. all authors were responsible for the conception and design of the study, revising the article critically for important intellectual content, and final approval of the submitted manuscript. hbg, prw, and mms were responsible for data acquisition. hbg was responsible for data analysis and primary manuscript drafting. funding to support this project was provided by the university of miami through support for the uhealth-dart research group. no conflicts of interest exist for any author. j o u r n a l p r e -p r o o f vascular surgery 79 (1.9%) 2 (10.5%) 54 (1.5%) 0 (0.0%) iqr, interquartile range; pcr, polymerase chain reaction a p<0.05 for comparison between "no positive test" and "≥1 positive test" in full cohort; none of the comparisons in the subcohort of cases including only tests marked to be "pre-procedural" had p<0.05 b comorbidities were determined from international classification of diseases, 10 th revision codes from all patient encounters within our health system since 2012 based on the strategy outlined by elixhauser and colleagues. 6, 7 the median number of patient encounters across cases for the full cohort was 7 (iqr 2-22) with 191 cases (4.6% of cohort) with no prior patient encounters; for the subcohort of cases including only tests marked to be "pre-procedural", the median number of patient encounters across cases was 7 (iqr 3-21) with 110 cases (3.1% of subcohort) with no prior patient encounters c includes diabetes mellitus with and without chronic complications d includes chronic pulmonary disease and pulmonary circulation disorders e cases on patients without prior encounters within our health system (n=191, 4.6% of full cohort; n=110, 3.1% of subcohort of cases including only tests marked to be "pre-procedural") were assumed to have 0 comorbidities f "ambulatory" describes outpatients who come in for their procedure and are discharged following it (never admitted as inpatients); "direct-admit for case" describes outpatients who come in for their procedure and are admitted as inpatients post-procedurally g all other service lines: anaesthesiology (full cohort n/subcohort n) n=4/1; cardiology, n=195/94; cardiothoracic, n=78/65; colorectal, n=174/143; dermatology, n=1/1; endocrine, n=13/12; gynaecology, n=19/19; gynaecology oncology, n=54/42; gynaecology urology, n=11/11; head and neck, n=1/1; hepatology, n=13/12; obstetrics, n=1/0; oculoplastic, n=49/43; ophthalmology, n=906/881; oral surgery, n=6/5; pain, n=11/11; plastics, n=32/29; podiatry, n=16/11; pulmonary, n=2/2; radiation oncology, n=16/11; sports medicine, n=58/58; thoracic, n=49/32; and urology, n=305/278 had no cases with ≥1 positive tests; these service lines were included in chi-square testing clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection surgical outcomes after systematic preoperative sars-cov-2 screening the impact of preoperative screening system on head and neck cancer surgery during the covid-19 pandemic: recommendations from the nationwide survey in japan early institutional head and neck oncologic and microvascular surgery practice patterns across the united states during the sars-cov-2 (covid19) pandemic seroprevalence of antibodies to sars-cov-2 in 10 sites in the united states comorbidity measures for use with administrative data healthcare cost and utilization project (hcup) key: cord-282338-u01qv3uc authors: cherry, james. d. title: the chronology of the 2002–2003 sars mini pandemic date: 2004-11-05 journal: paediatr respir rev doi: 10.1016/j.prrv.2004.07.009 sha: doc_id: 282338 cord_uid: u01qv3uc severe acute respiratory syndrome (sars) was a new human disease in the autumn of 2002. it first occurred in southern china in november 2002 and was transported to hong kong on february 21, 2003 by an infected and ill patient. ten secondary cases spread the infection to two hospitals in hong kong and to singapore, toronto and hanoi. in march 2003 a novel coronavirus (sars-cov) was found to be the causative agent. within 11 weeks from the first sars case in hong kong it had spread to an additional 27 countries or special administrative regions. the mini pandemic peaked during the last week of may 2003 and the last new probable case was on july 13, 2003. there were a total of 8096 probable cases and 774 deaths. sixty-six per cent of the cases occurred in china, 22% in hong kong, 4% in taiwan and 3% in both singapore and canada. twenty-one per cent of all cases occurred in healthcare workers. it is an honour to have been asked to contribute the lead paper in this symposium on paediatric sars. of all the participants in this symposium i have the distinction of having had no first-hand experience with sars. nevertheless, i do have some credentials. in march of 2003 i travelled to and through hong kong, shanghai, taipei, seoul and narita and realised on my trip back to los angeles on march 17 th that i needed a chapter on sars for the 5 th edition of our book 'textbook of pediatric infectious diseases' which was soon to be published. so, with the help of four hong kong colleagues this chapter was written in under 2 weeks and is the most complete reference to date relating to paediatrics. 1 sars was apparently a new human disease which first occurred in southern china in the autumn of 2002 and spread to 29 countries or regions with a total of 8096 cases and 774 deaths. [1] [2] [3] the aetiologic agent of sars (a previously unrecognised coronavirus; sars-cov) was isolated and its genome sequenced in record time. [3] [4] [5] [6] [7] [8] following an incredible global public health effort, the mini pandemic was brought under control within 7 months of its initial occurrence. 9 the overall effort demonstrated unprecedented international cooperation and this was facilitated by electronic transmission of information. in general, worldwide media coverage was unusually accurate and provided pictures to augment scientific data. as of june 1 st , 2004 there were approximately 2000 citations related to sars in the medical literature. however, of these publications only about 0.1% are related to children. during the third week of march 2003 both the who and the cdc published preliminary case definitions for severe acute respiratory syndrome (sars). table 1 contains the who initial definitions of suspect and probably cases and table 2 the cdc original definition of a suspected case. 10, 11 following the rapidly expanding knowledge about sars paediatric respiratory reviews (2004) (table 3) . 12 the cdc case definition was updated on four occasions throughout the epidemic as understanding of the clinical, laboratory, and transmission characteristics of sars associated coronavirus increased. 13 the most recent version from the cdc, which is exceedingly complex, is presented in table 4 . transmission of sars apparently first occurred in fosham city, guangdong province, china. 1, 9 the primary case may have been a 46-year-old man who had a fever for 9 days and respiratory distress. he was hospitalised and treated in an intensive care unit. he recovered but his wife, aunt, and aunt's daughter all became ill. 14 [15] [16] [17] [18] viral particles were seen on electron microscopic examination of swabs and sputum specimens from sars patients. the sars virus grew in vero cell and fetal rhesus kidney cell tissue cultures. cytopathic effect was noted in the vero cell cultures in 5 to 6 days and in the fetal rhesus kidney cell cultures in 2 to 4 days. 16, 17 this virus, which was a novel coronavirus (sars-cov), was rapidly identified using both conventional virologic methods and cutting edge molecular biology. the genome was completely and rapidly sequenced in several laboratories. [4] [5] [6] [7] [8] this was a significant accomplishment when one realises that coronaviruses have the largest genomes (30 000 bases of positivestrand rna) found in any rna virus. sequence data allowed the rapid development of highly specific diagnostic tests and also helped in the epidemiologic study of the mini pandemic. the genome sequence data from human isolates assisted in the search for the origin of the disease when similar viruses were isolated from himalayan palm civets and raccoon dogs in a live animal market in shenzhen, china. 19 the viral genomes recovered in nasal swabs from the palm civets were 99.8% homologous to the human sars-cov isolates. early in the epidemic orf8 reading frame sequences from human isolates were identical to those from the palm civets, which suggested animal to human transmission. as noted above, sars originated in southern china in november 2002. presumably the first case or cases were the result of transmission from palm civets to humans. subsequently, human to human transmission occurred. apparently, human to human transmission did not occur outside of china until february 21, 2003 when the adult physician from guangdong province, whose illness commenced on february 15, stayed at the metropole hotel in hong kong. 15 the chronology of events related to the metropole hotel is presented in fig. 1 . case a (the primary case) stayed on the ninth floor of the hotel on february 21, 2003 and was then admitted to hospital #2 the following day and died on february 23, 2003. secondary cases related to case a, in addition to cases b to k, were four healthcare workers at hospital #2 and two of the patient's family members. and -cough or breathing difficulty and one or more of the following exposures during the 10 days prior to onset of symptoms: -close contact b with a person who is a suspect or probable case of sars -history of travel to an area with recent local transmission of sars -residing in an area with recent local transmission of sars 2. a person with an unexplained acute respiratory illness resulting in death after 1 november, 2002, a but on whom no autopsy has been performed and one or more of the following exposures during to 10 days prior to onset of symptoms: -close contact, b with a person who is a suspect or probable case of sars -history of travel to an area with recent local transmission of sars -residing in an area with recent local transmission of sars a case should be excluded if an alternative diagnosis can fully explain their illness. as sars is currently a diagnosis of exclusion, the status of a reported case may change over time. a patient should always be managed as clinically appropriate, regardless of their case status -a case initially classified as suspect or probable, for whom an alternative diagnosis can fully explain the illness, should be discarded after carefully considering the possibility of co-infection -a suspect case who, after investigation, fulfils the probable case definition should be reclassified as 'probable' -a suspect case with a normal cxr should be treated as deemed appropriate and monitored for 7 days. those cases in which recovery is inadequate should be re-evaluated by cxr -those suspect cases in whom recovery is adequate but whose illness cannot be fully explained by an alternative diagnosis should remain as 'suspect' -a suspect case who dies, on whom no autopsy is conducted, should remain classified as 'suspect'. however, if this case is identified as being part of a chain transmission of sars, the case should be reclassified as 'probable' -if an autopsy is conducted and no pathological evidence of rds is found, the case should be 'discarded' a the surveillance period begins on 1 november 2002 to capture cases of atypical pneumonia in china now recognized as sars. presence of two or more of the following features: fever (might be subjective), chills, rigors, myalgia, headache, diarrhea, sore throat, or rhinorrhea mild-to-moderate respiratory illness temperature of >100.4 8f (> 38 8c) * and one or more clinical findings of lower respiratory illness (e.g. cough, shortness of breath or difficulty breathing) severe respiratory illness meets clinical criteria of mild-to-moderate respiratory illness and one or more of the following findings: --radiographic evidence of pneumonia or --acute respiratory distress syndrome or --autopsy findings consistent with pneumonia or acute respiratory distress syndrome without an identifiable cause possible exposure to sars-associated coronavirus (sars-cov) one or more of the following exposures in the 10 days before onset of symptoms: travel to a foreign or domestic location with documented or suspected recent transmission of sars-cov a or close contact b with a person with mild-to-moderate or severe respiratory illness and history of travel in the 10 days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov a likely exposure to sars-cov one or more of the following exposures in the 10 days before onset of symptoms: close contact b with a person with confirmed sars-cov disease or close contact b with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the 10 days before onset of symptoms tests to detect sars-cov are being refined and their performance characteristics assessed c ; therefore, criteria for laboratory diagnosis of sars-cov are changing. the following are general criteria for laboratory confirmation of sars-cov: detection of serum antibody to sars-cov by a test validated by cdc (e.g. enzyme immunoassay) or isolation in cell culture of sars-cov from a clinical specimen or detection of sars-cov rna by a reverse transcription polymerase chain reaction test validated by cdc and with subsequent confirmation in a reference laboratory (e.g. cdc) information about the current criteria for laboratory diagnosis of sars-cov is available at http://www.cdc.gov/ncidod/sars/labdiagnosis.htm a case may be excluded as a sars report under investigation (sars rui), including as a cdc-defined probable sars-cov case, if any of the following apply: an alternative diagnosis can explain the illness fully d or antibody to sars-cov is undetectable in a serum specimen obtained > 28 days after onset of illness e or the case was reported on the basis of contact with a person who was subsequently excluded as a case of sars-cov disease; then the reported case is also excluded, provided other epidemiologic or laboratory criteria are not present case classification sars rui reports in persons from areas where sars is not known to be active sars rui-1: cases compatible with sars in groups likely to be first affected by sars-cov f if sars-cov is introduced from a person without clear epidemiologic links to known cases of sars-cov disease or places with known ongoing transmission of sars-cov 8c) is expected. however, clinical judgment may allow a small proportion of patients without a documented fever to meet this criterion. factors that might be considered include: patients' self-report of fever; use of antipyretics; presence of immunocompromising conditions or therapies; lack of access to healthcare; or inability to obtain a measured temperature. initial case classification based on reported information might change, and reclassification might be required. a types of locations specified will vary (e.g. country, airport, city, building, or floor of building). the last date a location may be a criterion for exposure is 10 days (one incubation period) after removal of that location from cdc travel alert status. the patient's travel should have occurred on or before the last date the travel alert was in place. transit through a foreign airport meets the epidemiologic criteria for possible exposure in a location for which a cdc travel advisory is in effect. information about cdc travel alerts and advisories and assistance in determining appropriate dates are available at http://www.cdc.gov/ncidod/sars/travel.htm. b close contact is defined as having cared for or lived with a person with sars or having a high likelihood of direct contact with respiratory secretions and/or body fluids of a person with sars (during encounters with the patient or through contact with materials contaminated by the patient) either during the period the person was clinically ill or within 10 days of resolution of symptoms. examples of close contact include kissing or embracing, sharing eating or drinking utensils, close (i.e. <3 feet) conversation, physical examination, and any other direct physical contact between persons. close contact does not include activities such as walking by a person or sitting across a waiting room or office for a brief time. c the identification of the aetiologic agent of sars (i.e. sars-cov) led to the rapid development of enzyme immunoassays and immunofluorescence assays for serologic diagnosis and reverse transcription polymerase chain reaction assays for detection of sars-cov rna in clinical samples. these assays can be very sensitive and specific for detecting antibody and rna, respectively, in the later stages of sars-cov disease. however, both are less sensitive for detecting infection early in illness. the majority of patients in the early stages of sars-cov disease have a low titre of virus in respiratory and other secretions and require time to mount an antibody response. sars-cov antibody tests might be positive as early as 8-10 days after onset of illness and often by 14 days after onset of illness, but sometimes not until 28 days after onset of illness. information about the current criteria for laboratory diagnosis of sars-cov is available at http://www.cdc.gov/ncidod/sars/labdiagnosis.htm. d factors that may be considered in assigning alternate diagnoses include the strength of the epidemiologic exposure criteria for sars-cov disease, the specificity of the alternate diagnostic test, and the compatibility of the clinical presentation and course of illness with the alternative diagnosis. e current data indicate that > 95% of patients with sars-cov disease mount an antibody response to sars-cov. however, health officials may choose not to exclude a case on the basis of lack of a serologic response if reasonable concern exists that an antibody response could not be mounted. f consensus guidance is in development between cdc and cste on which groups are most likely to be affected first by sars-cov if it re-emerges. sars-cov disease should be considered at a minimum in the differential diagnoses for persons requiring hospitalisation for pneumonia confirmed radiographically or acute respiratory distress syndrome without identifiable aetiology and who have one of the following risk factors in the 10 days before the onset of illness: (1) travel to mainland china, hong kong, or taiwan, or close contact with an ill person with a history of recent travel to one of these areas, or; (2) employment in an occupation associated with a risk for sars-cov exposure (e.g. healthcare worker with direct patient contact or worker in a laboratory that contains live sars-cov) or; (3) part of a cluster of cases of atypical pneumonia without an alternative diagnosis. guidelines for the identification, evaluation, and management of these patients are available at http://www.cdc.gov/ncidod/sars/absenceofsars.htm. g during the 2003 sars epidemic, cdc case definitions were the following: suspect case; (1) meets the clinical criteria for mild-tomoderate respiratory illness and the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria or; (2) unexplained acute respiratory illness that results in death of a person on whom an autopsy was not performed and that meets the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria; probable case; (3) meets the clinical criteria for severe respiratory illness and the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria. was investigating the outbreak in hanoi became ill and returned to bangkok. patients c, d, and e were the primary cases in singapore and their illnesses led to 70 cases in singapore and three cases in germany. patients f and g were the initial cases in canada and case f was subsequently linked to a cluster of 16 cases in toronto. patients h and j were linked to outbreaks among healthcare workers in hong kong hospitals #1 and #3. patients i, l, and m were suspect cases in the united states. case k was a potential primary case in ireland. on february 28, 3003 the hanoi office of the world health organization was contacted by officials at the vietnam french hospital. 20 they were concerned about the unusual influenza-like illness that patient b had which they suspected could have been due to an avian influenza virus. dr. carlo urbani from the who office answered the call and rapidly determined that the illness which had spread throughout the hospital was unusual. he carried out an extensive on-site investigation and worked with the hospital staff to set up quarantine procedures. his efforts led to the containment of sars in hanoi. unfortunately, he became ill with sars on march 11, 2003 and died in an isolation room in a bangkok hospital on march 29, 2003. he did not live to see the worldwide successes of sars control which resulted to a great extent from his early detection of sars. following fig. 2 were india, japan and mongolia. of these countries, no secondary transmission occurred in india, japan was later removed from the list and in mongolia there were nine cases with eight of these being importations. as of may 12, 2003 new countries or administration regions included bulgaria, china, macao sar, columbia, finland, poland and the republic of korea. of these countries or regions only macao sar and the republic of korea had accepted cases and in both regions the cases were imported. in the june 2nd report one case in the russian federation was noted but this was subsequently removed from the data set. eighty-seven per cent of all probably sars cases occurred in hong kong or china and information relating to these cases in presented elsewhere in this symposium. only five other countries (canada, taiwan, singapore, the united states and vietnam) had more than 20 probable cases. in canada there were 257 cases with the first case occurring on february 23, 2003 and the last case on june 12, 2003. the vast majority of cases in canada (98%) were secondary to five primary importations and 43% of the cases occurred in healthcare workers. in taiwan 6% of the cases were importations and 20% occurred in healthcare workers. in singapore 3% of the cases were importations and 41% of the locally acquired cases were in healthcare workers. in singapore the epidemic was controlled within 13 weeks of the initial importation. all 27 probable cases in the united states were importations. however, only eight of these were laboratory confirmed. in vietnam, 62 of the 63 cases were the result of local transmission. the outbreak was controlled within 7 weeks of the first and only imported case. in summary, the sars mini pandemic of 2002-2003 probably does not deserve to be called a pandemic. however, the death rate in adults was sobering. in reality, significant epidemic disease occurred in only five countries and the special administrative region of hong kong. nevertheless, sars was an eye opener in regard to the number of healthcare workers who became infected. hopefully the clinical lessons relating to isolation practices, personnel protection and the effective use of quarantine will be retained. personally i will be less cavalier in my approach to patients with fever and respiratory illness. severe acute respiratory syndrome (sars) world health organization. summary of probable sars cases with onset of illness from 1 world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) the genome sequence of the sars-associated coronavirus identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the sars coronavirus: a postgenomic era world health organization. severe acute respiratory syndrome (sars) outbreak news: severe acute respiratory syndrome (sars) trends in tuberculosis morbidity -united states world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) tracing the path of sars: a tale of deadly infection update outbreak of severe acute respiratory syndrome -worldwide coronavirus as a possible cause of severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada world health organization. summary on major findings in relation to coronavirus by members of the who multi-centre collaborative network on sars aetiology and diagnosis isolation and characterization of viruses related to the sars coronavirus from animals in southern china key: cord-273913-xem3alih authors: marraha, farah; al faker, ibtissam; gallouj, salim title: a review of the dermatological manifestations of coronavirus disease 2019 (covid-19) date: 2020-08-11 journal: dermatol res pract doi: 10.1155/2020/9360476 sha: doc_id: 273913 cord_uid: xem3alih coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has affected 210 countries and territories around the world. the virus has spread rapidly, and the disease is still extending up to now. the pathophysiology for sars-cov-2 has not been well elucidated, and diverse hypotheses to date have been proposed. initially, no skin manifestations were observed among patients with covid-19, but recently a few cases have been described. in this review, we discuss these various cutaneous manifestations and skin problems related to personal protective equipment, as well as different cutaneous anti-covid-19 drug-associated reactions. we also focus on the currently proposed managements of these rare manifestations. e first case infected by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) was reported in wuhan, china, in late november 2019. in december 2019, several patients have been presented to the hospital for acute pneumonia of unknown origin [1] . later, sars-cov-2 has been found in the lower respiratory tract of hospitalized patients, and the world health organization (who) recognized the disease as covid-19 (coronavirus disease 2019) on january 12, 2020. e virus has spread rapidly, and the condition is still extending up to now. on march 11, the who declares the covid-19 outbreak as pandemic [2] . we are still counting to this day 11,577,979 confirmed cases with 537,103 deaths worldwide. initially, no skin manifestations were observed among patients with covid-19 [2, 3] . e first case was described in ailand with petechiae rash mimicking a dengue fever [4] , and then a few other cases followed [2] [3] [4] [5] . ese skin lesions can guide clinicians for diagnosis if the patients present other covid-19 symptoms; however, viral infection cannot be the only cause; mediated inflammatory responses and drug reactions can also be suspected. e aim of our literature review is to report the various cutaneous manifestations described to date associated with covid-19, the skin problems related to personal protective equipment, and the different cutaneous anti-covid-19 drug reactions [6, 7] . we also discuss the different interventions proposed to manage these patients [8, 9] and solutions to protect skin and mucous membrane barriers for healthcare workers [10] [11] [12] . since the epidemic began in europe, some countries have presented virus-spreading indicators higher than others. in italy, the first cases were detected in the last ten days of february. its case fatality rate at present is 14.4%. in france, the mortality rate has continued to increase, reaching 17.9% (july 6, 2020). in the united states, the first cases appeared around march 10. e country represents more than 25% of the cumulative cases worldwide. as for morocco, the first case emerged on march 2; official indicators seem to display volatile trends. it is tough to demonstrate a correlation between them. e growth factor can vary from a value of 2 to a value of 0.5 within 24 hours. e contamination rate displayed every day (new cases in 24 hours compared to the number of tests carried out in 24 hours) is also very volatile ranging from 9% on march 28 to 30% on april 8 and 2.2% on july 5. as for the mortality rate, it decreased from 6.2% to 1.6% lately (july 6, 2020) [13] . e pathophysiology of covid-19 has not been well elucidated, but it seems similar to that of sars-cov [14] . sars-cov has the appearance of a crown under electron microscope. it is composed of genomic rna and four structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n). it can attach to the host cell by binding its s protein to the receptor protein membrane [15] [16] [17] [18] . a number of studies showed that angiotensinconverting enzyme 2 (ace2) is a receptor on the host cell membrane, with a high affinity to sars-cov-2 protein (s) [19, 20] . yet, the significance of this binding affinity is still under intensive research study [17] . ace2 is shown to be expressed by epithelial cells of the intestine, kidney, blood vessels, and most abundantly in type ii alveolar cells of the lungs [19, 20] . importantly, hamming et al. [20] demonstrated that ace2 is also present in the basal cell layer of the epidermis extending to the basal cell layer of hair follicles, which might explain the cutaneous manifestation of covid-19. decreased ace2 function can cause dysfunction of the renin-angiotensin system (ras) and intensify inflammation, vascular permeability, and neutrophil accumulation [14, 21] . rapid viral replication can also cause cellular apoptosis and triggers a cascade of inflammatory reaction, as well as an increase in cytokine and chemokine blood levels [21] . erefore, the inflammatory response of the body also plays a crucial role in sars-cov-2-induced lung injury cases, since the high production of these cytokines is responsible for the accumulation of cells and fluids (cytokine storm) [18] . ese cytokines arriving at the skin and reaching the various cells of the cutaneous immune system can cause the dermatological lesions described during covid-19 infection (urticarial lesion, erythema, and vesicles) [22] . wenzhong et al.'s latest hypothesis suggests that lung damage and inflammation are secondary to extreme lung cell poisoning [23] . in fact, orf8, a sars-cov-transcribed nonstructural protein, could bind to porphyrin as well as other proteins and can attack the heme on the 1-beta chain of hemoglobin. consequently, this will decrease hemoglobin and gas exchanges, which eventually results in ground-glasslike lung images. several cases of patients with covid-19 showed leucopenia, lymphocytopenia, increased d-dimer level, and prolonged prothrombin [1, 17] . besides, abnormal findings of different organs function assessment may indicate multiorgan failure [17] . it has been suggested that covid-19 is a zoonotic infection as the first infected people were exposed to the wet animal market in wuhan city [1, 17] . however, the intermediate host is still unknown. e virus is transmitted by inhalation of aerosol droplets from the infected patients [1, 17] . e fecal-oral transmission was also hypothesized [21] . erefore, it is essential to strictly adhere to environmental and hand hygiene to control the infection [24] . so far, the golden clinical diagnosis methods to confirm cases of covid-19 include the detection of nucleic acids in the nasal and throat swab sampling by real-time polymerase chain reaction (pcr) [25] . e identification of sars-cov-2specific igm and igg antibodies can also be useful for diagnosis [17] . 19 patients e most common symptoms in covid-19 are fever, fatigue, and dry cough, succeeded by other symptoms, such as headache, nasal congestion, sore throat, myalgia, and arthralgia [1, 26] . initially, no skin involvement during covid infection was observed, but more recently, some cases (table 1 ) have been reported [2, 4, 5, 11] , as well as the skin problems related to personal protective equipment. secondary skin reactions to the different treatments suggested are also possible. e frequency of the skin lesions associated with covid-19 infection varies according to the series; in a chinese study of 1099 positive cases, the incidence was only 0.2%, while in an italian series of 88 patients it was 20.4% [42] . joob and wiwanitkit described in ailand, where the first case of covid-19 outside of china was reported, a skin rash with petechiae in a case of covid-19 [4] . because of the frequent dengue cases in this country, it was the first diagnosis to be mentioned. after the appearance of other respiratory problems, covid-19 has been diagnosed by rt-pcr. in italy, in a series of 148 cases, recalcati reported, after the exclusion of 60 patients who have started new drugs in 15 previous days, that 20.5% of the 88 patients developed skin manifestations [2] . eight of the 18 (44%) had skin eruptions with symptoms in the beginning and the rest after hospitalization. erythematous rashes were the most common sign (78%) and then urticarial (3 cases) and chickenpox-like vesicles (1 case). however, no documentation has been assembled in these cases (photos or biopsy) to prevent the spread of infection. at the end of march, the french society of dermatology launched a call for a case, after discovering erythematous maculopapular lesions on the faces of three patients with a very probable or confirmed covid-19 infection [5] . finally, 113 cases have been reported, of which 74% of them had frostbite-like lesions. an american team with a series of 505 patients with dermatological lesions tried to study the characteristics of pernio lesion or chilblain lesions in acral skin. despite the absence of a confirmation test of covid-19 infection in all these patients, the results showed that 318 of them had pernio-like lesions and most of them were young. e localization of these lesions was in the feet in 84% of the cases, in the hands in 5.1% of the cases, and in both in 10% of the cases [27] . in another spanish study [30] , including 20 children and adolescents with acral lesions, four clinical patterns were described: acral erythema (30%) (figure 1 ), dactylitis (20%), purpuric maculopapules (35%), and mixed pattern (15%). rt-pcr could not confirm the presence of the virus. e authors believe that it is secondary to a realization of a diagnostic test at an early stage of the disease. coagulopathy and thrombocytopenia are also common complications for covid-19 infection [17, 26, 43] . in wuhan [26] , patients with covid-19 might present acroischemic lesions; there were 7 patients, in a critical situation, hospitalized in the intensive care unit with different clinical presentations, including finger/toe cyanosis, skin bulla, and dry gangrene. four patients among them were diagnosed with definite disseminated intravascular coagulation (dic). a team from italy [28] tried to collect cases from a website, and 63 cases were collected, but only 54 pictures were analyzed: 31 of 54 patients had erythematous-edematous lesions and 23 of 54 had blistering lesions. a report by fernandez-nieto et al. [36] describing the characterization of acute acro-ischemic lesions in 132 patients showed two different clinical presentations: chilblainfernandez-nieto et al. [31] described a patient in madrid with an urticarial rash that appeared six days after the first symptoms. ey also mentioned the presence of other cases with different skin manifestations, and for that, a prospective study is in progress. in france, henry et al. [32] reported a similar case; however, the urticarial rash in this patient was inaugural. e only associated symptoms were odynophagia and arthralgia. is eruption was in the form of disseminated erythematous plaques with facial and acral localization. estébanez et al. [33] published another type of cutaneous manifestation: lesions in the form of yellowish erythematous papules located in the heels. a very recent publication in italy [34] suggested that papulovesicular eruption (varicella-like) was rare but specific to covid-19. twenty-two patients have been reported with the same clinical presentation; itching was present in 40.9% of patients. and the eruption was generally localized at the trunk. another article by the same authors [35] detailed the case of a little girl who has papulovesicular skin eruption on the trunk, face, and genital tract ( figure 2 ); after 6 days, the patient had a moderate fever and was tested positive for sars-cov-2. sanchez et al. [37] described a new clinical presentation of skin lesions associated with covid-19. ey reported a skin eruption similar to that of pityriasis rosea: erythematous and squamous plaque, in the trunk, upper arms, and periumbilical. a spanish publication has classified the skin lesions observed during covid-19 into 5 types: maculopapular eruptions in 47% of cases, urticarial lesions in 19% of cases, acral areas of erythema with vesicles or pustules in 19%, other vesicular eruptions in 9%, and livedo or necrosis in 6% [29] . in the pediatric population, the frequency of coronavirus infection is estimated to be less frequent than adults (<1%) [22, 44] . a particular presentation has been reported in children in europe and north africa, with a lesion similar to kawasaki syndrome [38, 44, 45] . hence, the who established a preliminary definition and case for multisystem inflammatory disorder in children and adolescents [44] to help define and recognize these cases for better management and possible surveillance. clinical presentation includes fever, rash, mucosal inflammation associated with gastrointestinal problems, myocardial dysfunction, and sometimes even hypotension or shock [44] . according to a french series, concerning 16 children with this syndrome, the skin rash was present in 81% of patients, edema with plantar and palmar redness was present in 67% of patients, and 87% of them had dry lips [38] . as part of this syndrome, the skin eruption can be very varied; for example, an erythema multiforme rash has been described in a 13-year-old patient [39] . other lesions have also been reported, such as androgenic alopecia (aag) [40] , sebopsoriasis, herpes, and exanthem [41] . also, nonspecific erythematous lesions in the trunk and face have been described [2] . to control this infection, all personnel must wear personal protective equipment (ppe) for an extended period in addition to other safety and health measures. eczema is the most common problem among healthcare workers [10] , often secondary to frequent hand hygiene and long-time gloves wearing [46] . besides, humidity, prolonged contact with masks, and goggles may cause a variety of cutaneous diseases such as contact and pressure urticaria or contact dermatitis and pigmentation of the nasal bridge [3, 11] . in a survey study [12] , lan et al. found that 526 participants among healthcare professionals mentioned that they had damaged skin. e nasal bridge was the most affected area and then hands, cheek, and forehead. dryness and desquamation were the most common symptoms, and these damages depended on the hours of work, especially for gloves wearing. another questionnaire distributed among 66 patients objected that wearing of masks has caused in 95.1% of cases side effects including first, nasal bridge scar. wearing of gloves has caused in 88.5% of cases skin reactions [47] . we are currently carrying out a study to clarify the consequences of wearing personal protective equipment on the skin. to date, 268 health personnel have completed the questionnaire. because of frequent handwashing to prevent the disease, dry skin was present in 68.3%, with erythema in 31.3% of cases (figure 3) . exacerbations of preexisting dermatoses such as acne, rosacea, atopic dermatitis, and neurodermatitis have been observed [3, 7] . is can be explained by the prolonged wearing of masks during the epidemic. e occlusion generated by protective hats may provoke pruritus and folliculitis or exacerbate seborrheic dermatitis [11] , and the frequent use of disinfectant and soap can impair the hydrolipid mantle of the skin and increase the risk of contact dermatitis [7] . e current stress generated by the global situation and confinement can also exacerbate dermatoses [12] . treatments. preventive measures are currently the best strategy to fight covid-19. while vaccines and monoclonal antibodies against sars-cov-2 are under development, several other therapies are proposed [48, 49] , hence the interest to review the skin side effects of these molecules. chloroquine/hydroxychloroquine (hcq), a widely used antimalarial and autoimmune disease drug, has been demonstrated to inhibit sars-cov-2 and blocks viral activity by increasing endosomal ph [48] [49] [50] . however, the use of this drug remains controversial; some studies have tried to prove the effectiveness of this treatment [48, [50] [51] [52] . nevertheless, each had methodological deficiencies [53] . other studies are underway with a large sample. chloroquine is widely used in dermatology disorders and autoimmune disease; its toxicity is infrequent [54] , and reactions can be mainly gastrointestinal and cutaneous (pruritus or urticaria), usually mild [54, 55] . few cases of hcq toxidermia have been reported. acute generalized exanthematous pustulosis (agep) was the most common presentation [56, 57] ; it appears like a sterile nonfollicular pustule with an erythematous base [58] . also, drug reactions with eosinophilia and systemic symptoms (dress) have been described in four reports [58] . besides, rare cases of hcq and sweet syndrome have also been reported [6, 59] . e moroccan health ministry and national technical committee for prevention and control of covid-19 decided on march 23, 2020, to adopt the therapeutic protocol based on using hydroxychloroquine under medical supervision, and no problem has been noticed yet. other prescription drugs such as otc, antibiotics, healthcare products, and a variety of plants can also cause a skin reaction. zheng and lai [7] reported cases of urticaria, urticarial vasculitis, and other pruritus lesions in coronavirus pneumonia patients after accepting anti-covid-19 medicines. healthcare institutions and policymakers developed various plans to control the pandemic, including preparing hospitals and clinics, developing a strategy for the identification of suspected covid-19 cases, and implementing a strategy to reduce the spread [24] . prevention of nosocomial infection should be another priority; this will require specialized protocols and training of the medical staff. in most countries, several dermatological services have been transformed into a structure for the treatment and isolation of patients with covid-19 infection, and many private dermatology practices have closed doors [3] . different solutions have been proposed. [64] revealed that over 75% of the patients were interested in using telehealthcare (medical consultations and electronically transmitted prescriptions). during a patient visit, the dermatologist and the patient should both wear a surgical mask with hand hygiene. e dermoscopy should be performed with caution and only if necessary to prevent dermatoscope from becoming a possible source of nosocomial spread [65] . in addition, this exam should be avoided for specific sites such as hands, nails, face, eyes, and mucosa, and for all patients with signs and symptoms suggestive of covid-19 [65] . several skin diseases, such as psoriasis, as well as depression and anxiety, can increase the risk of 2019-ncov infection [61, 66] . phototherapy and the other hospital treatments are needed to be adjusted to avoid displacement [7] . up until today, there is not enough evidence or guidelines for managing the patients with severe inflammatory skin disorders treated with immunomodulators during this pandemic, and there is no proof that these persons may have a more increased risk of becoming infected with covid-19 [8, 9, 61] . e first treatment option in this particular period must be the one who has lower effects on personal immune functions such as il-17 inhibitor [7] . other treatment options include decreasing the dosage of traditional immunosuppressants (methotrexate and cyclosporine) [8] . on the other hand, there has not been any recommendation or indication justified to stop or reduce the systemic corticosteroid dose [8] . in the case of skin cancer, surgical treatment, if indicated, should not be delayed, especially in critical localization [67] . e wearing of the ppe for an extended period and the cutaneous complications that generate with stress and anxiety can reduce the efficiency of the health workforce [10] . as such, it has been recommended to use latex-free gloves or use of cotton gloves inside it to avoid cutaneous hand problems. moreover, the use of cleansing products containing moisturizing ingredients and the application of hand cream frequently are highly recommended [3, 11, 68] . e alcohol hand sanitizers may be used more frequently as they have been shown to be less irritating than washing hands with soap [69] . to avoid friction and pressure from wearing the mask, it has been suggested to wear a properly fitted mask, in different ways, and apply moisturizers or gel just before [10, 11] , or combine a paper towel with facial mask [67] . e medical training of future doctors and the scientific communication between dermatologists have also been affected. in morocco, the ministry of national education has deployed an online platform for continued remote instruction for students. e department of dermatology and allergology of the ludwig maximilian university hospital [70] has presented numerous online case studies and offered opportunities for continuous education. other similar projects with different features and focus already exist on the internet [71] . to date, several skin manifestations related to covid-19 have been reported, but additional efforts are needed to collect further data. it is essential not to forget the patients who suffer from other skin diseases and to insist on preventive measures for healthcare professionals. no data were used to support this study. e authors declare no conflicts of interest. e epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak cutaneous manifestations in covid-19: a first perspective covid-19 pandemic and the skin: what should dermatologists know? covid-19 can present with a rash and be mistaken for dengue covid-19 et lésions cutanées-point d'étape covidskin de la sfd-15 avril 2020 syndrome de sweet médicamenteuxà l'hydroxychloroquine:à propos de 2 cas dermatology staff participate in fight against covid-19 in china advice regarding covid-19 and use of immunomodulators, in patients with severe dermatological diseases european task force on atopic 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online with interactive technology (doit) key: cord-261256-iwdusvrw authors: gandolfi, alberto title: planning of school teaching during covid-19 date: 2020-10-01 journal: physica d doi: 10.1016/j.physd.2020.132753 sha: doc_id: 261256 cord_uid: iwdusvrw learning and education are two of the biggest world issues of the current pandemic. unfortunately, it is seen in this work that, due to the length of the incubation period of covid-19, full opening of schools in the fall of 2020 seems to be impractical unless the spread of the virus is completely under control in the surrounding region (e.g. with fewer than 5 active cases every million people). in order to support the possibility of some in-person learning, we model the diffusion of the epidemic within each single school by a seair model with an external source of infection and a suitable loss function, and then evaluate sustainable opening plans. it turns out that blended models, with almost periodic alternations of in-class and remote teaching days or weeks, are generally (close to) optimal. in a prototypical example, the optimal strategy prescribes a school opening of 90 days out of 200 with the number of covid-19 cases among the individuals related to the school increasing by about 71%, instead of the about 390% increase that would have been a consequence of full opening. as clinical fraction is low in children, these solutions could lead to very few or no symptomatic cases within the school during the whole school year. using the density of active cases as a proxy for the number of preand asymptomatic, we get an indication for each country of whether either full opening, or blended opening with frequent testing, or no school opening at all, are advisable. more than one billion students are out of school because of covid-19 [1] ; most of them are now using remote learning practices that present several drawbacks [2] and, in most cases, have been hurriedly arranged. in addition, in most schools, school districts, and countries there is uncertainty on how to plan school activities for the 2020-2021 school year [3] . as a result, learning and education are two of the biggest world issues of the current pandemic [4, 5] . as we see below, the characteristics of covid-19, and in particular the length of its incubation period of about 5 days [6, 7] , are such that cases within each single school tend to undergo a multifold increase should the school be completely open in the upcoming school year. this makes full opening of schools impractical unless the epidemic in completely under control in a region, or till a vaccine is available: difficulties have been already experienced by the first reopening attempts [8, 9] . on the other hand, remote teaching has been put together in an emergency situation, and it appears to have badly affected most students [10] [11] [12] , especially those from less advantaged contexts [13] . at the same time, it has imposed, and it likely to impose again, an additional burden on many families. therefore, the hypothesis of a fully remote 2020-2021 school year is also impractical for different reasons. to help reconcile such conflicting circumstances, there is thus a need for studies and methods that suggest sustainable opening strategies; they should be aimed at supporting the possibility of some in-person learning while simultaneously containing extra infection transmissions, both at the level of each single school, and in a broader context. in this first work, we focus on a single school. what each school needs is a flexible instrument that incorporates some of the specific details of the school, the students, the personnel, and the surrounding area, and provides indications of the outcome of in-person school activities in terms of the potential spread of covid-19 within the school. some of the difficulties in this process are that it is hard to estimate the transmission rate within schools; that the individuals involved in the school are subject to potential external infectious transmissions; that asymptomatics are hard to detect; and that policy makers have to establish a relative importance of the two targets (in-person teaching and covid-19 containment). in this paper, we provide a model whose set-up and parameters incorporate several features of covid-19 dynamics related to a single school, and then outline the optimization process. we then study optimal solutions for a wide variety of plausible values of the parameters of the model, obtaining both an adaptable procedure that can be conformed to specific situations, and general policy indications. blended solutions appear to be extremely efficient in reducing the potential spread, and together with some detection of asymptomatic cases could offer the possibility of a sustainable planning for the whole year. besides containing the covid-19 diffusion, these solutions could be pedagogically acceptable, and could also become a driving model for the society at large. in this work we focus on a single school. to model the diffusion of covid-19 among the personnel and the students of the school, we set-up a suitable system of ordinary differential equations. in particular, we consider seair, a version of the sir model [14] with an external source of infection [15, 16] , and a control. the population is divided into: susceptible (s), pre-symptomatic or, equivalently, exposed (e), asymptomatic (a), infected (i) and recovered (r). variables are normalized so that s + e + a + i + r = 1. we assume that susceptible individuals might become pre-symptomatic or exposed (e) at transmission, entering a latency period in which they have contracted the virus and are contagious, without showing symptoms. the contagion can be caused by contact with other individuals with viral load, at a rate βc(t) (c(t) is the control, as described below); or, alternatively, it is caused by an infectious contact outside the school taking place at rate α. the asymptomatic members of the school community present a challenge, as they cannot be easily detected while still being infectious for a relatively long time: we assume accessibility to some type of fast screening with sufficient sensitivity [17] : the contacts with asymptomatics within the school are then reduced by a parameter η, the false negative rate of the screening procedure. exposed individuals either develop symptoms at a constant rate δ, becoming infected, or progress into being asymptomatic with rate γ. the viral load is carried by exposed (e), asymptomatic (a) or infectious (i) individuals; however, infectious individuals are assumed to be isolated, while a large fraction of asymptomatic (a) is supposed to be detected by the screening procedure. both asymptomatic and infected individuals recover at rate ρ. as covid-19 mortality is particularly low in a mostly young population [18] , we disregard mortality. the key differences with the usual seir model [14] are: an external source of infection [15] , [19] , the possibility of transmission limited to 7 hours per working day; a control indicating from the start whether, for each day, schools are open or closed; and the presence of asymptomatic individuals. with these assumptions, the system of ode modeling infectious transmissions of concern for the school is: exposed: infected: recovered: 3 j o u r n a l p r e -p r o o f for a certain time interval [0, t ]. we consider 40 weeks, and count time in hours, so that t = 6720. the initial population at the beginning of the school year might be partly immune due to previous infections, but to avoid issues about efficacy and duration of the immunity, we assume that the initial population consists primarily of susceptible, s(0) ≈ 1, and a small fraction of exposed, so that s(0) + e(0) = 1. the function c(t) describes the control variable, and c(t) = 0 for all times when the school is closed; these include all hours except from 8 am to 3 pm of those working days in which it has been decided that the classes are in person. for each of the 200 working days, c(t) can be 0 again if remote activities have been decided for that day, or 1 if teaching is in person. formally, with t measured in hours, let m indicate the class of controls c(t) such that for some c i,j ∈ {0, 1}, i = 1, . . . , 40, j = 1, . . . , 7, and 0 ≤ t ≤ 6720. lemma 2.1. for each solution of (1)-(5), the total population s +e +a+i +r is preserved. in addition, 0 ≤ s, e, a, i, r ≤ 1 proof. let φ(t) = s(t) + e(t) + a(t) + i(t) + r(t); we have that φ(0) = 1 and dφ dt = 0 for all t from (1)-(5); it follows that φ ≡ 1 by uniqueness of solutions of linear differential equations. in addition, r(0) ≥ 0 and r ≥ 0, so that r(t) ≥ 0 for all t. the other functions have to take the value 0 before becoming negative by continuity. if i(t) = 0 for some t, then i (t) > 0 unless e(t) = 0, and the same applies to a(t), therefore let's consider the case that e(t) = 0. but then e (t) > 0 unless s(t) = 0. in this last case, (1) implies that all derivatives d n s(t) dt n = 0, in which case s(t) ≡ 0; on the other hand, the system (2)-(5) with s = 0 and nonnegative initial condition has an explicit, unique solution, which is seen to be non negative. proof. let x(t) = (s(t), e(t), a(t), i(t), r(t)) and express (1) where 168 is the number of hours in a week. take k = 0. extending c(t) = c 0 for t ≤ 0, the conditions of peano's theorem are satisfied ( [20] , ch. 6, th. 9), so that there is a solution to the initial boundary value problem (1)-(5) for some positive t's. by considering r 0 as above, one can see that the solution can be continued to the boundary of r 0 ( [20] , ch. 6, th. 11), which is t = 168. in addition, as each component of x(t) satisfies x i ≤ 1 by lemma 2.1, f (c, x) is seen to be lipschitz in [0, 1] × { x : x i ≤ 1 for i = 1, . . . , 5}. hence, the solution is unique (see [20] , ch. 6, th. 1). the same reasoning can now be repeated from t = 168 and with initial conditions given by the limit t → 168 of the unique solution determined in the previous time interval. as c(t) has only jump discontinuities, the same procedure can be repeated in each interval, (see [21] ), to show that there always exists a unique solution for all times t of the system (1)-(5). we are, however, interested in a finite time interval t , and hence in the transient solution up to time t . in the first part of the analysis, we will explore the effect of the opening policies on the number of remote teaching days and infection spreading. for this, we need an appropriate parameter selection. we make a first choice based on current observations, and present results in section 3, and then evaluate the modifications for a wide range of possible values in the sensitivity analysis section. as time is counted in hours, we need to scale all available parameter estimates, usually expressed for time counted in days, by a factor of 24. estimates of the infection rate β are typically around 0.1-0.4 [22] ; however, as the school is likely to elicit more frequent contacts, we adopt the somewhat higher value of β = 0.9/24 ≈ 3.75 × 10 −2 . the value of β can also be computed from contact matrices and susceptibility. [23] has contact matrices in school for most countries: averaging the contacts for class ages 0 − 19 with a factor 16 for the number of classes, one gets an average number of daily contacts varying between 6 (germany) and 20 (italy); the contacts need to be revised by the recent containment measures, and related awareness of the population; a reasonable figure would be a factor of 1/2. covid susceptibility (probability of infection upon contact) in school is estimated around 0.01-0.02, see [18] figure 1 (b) as our equations are written for the school population only, which is about 1/6 of the total population, one needs to use a scale factor 6; in addition, one needs to divide by 7 to have the hourly contact number during school hours. in the end, estimates of hourly β range from β m ≈ 0.01×1/2×6×6/7 ≈ 2.5×10 −2 to β m ≈ 0.02 × 1/2 × 20 × 6/7 ≈ 1.7 × 10 −1 (this is a range of 0.175-1.2 for the daily β), in line with our choice above. in the stability analysis we consider the other values in this range. the duration of the latency period after infection and before symptoms are developed has been estimated at about 5 days (see for example [6] and [7] ), so that γ + δ ≈ 1/5 × 1/24 = 0.2/24; the clinical fraction, which is the fraction of symptomatic cases, depends on many factors [24] , and it is substantially lower for younger individuals [18] . we take a ratio of about 100, which is γ = 0.198/24 ≈ 8.25 × 10 −3 , and δ = 0.002/24 ≈ 8.33 × 10 −5 similarly, the average recovery period is about 7 days, for mild cases [25] , suggesting ρ = 0.14/24 ≈ 5.8 × 10 −3 ; more severe cases (i) are excluded from contacts, so their recovery rate is irrelevant: we use the same value of ρ. the parameter η describes how asymptomatic individuals are excluded from contacts with the other individuals; such a separation depends on the availability of detecting tests: we assume that a sufficiently reliable test is available, with a 90% test sensitivity, so that η = 0.1. this assumption is subject to parameter sensitivity analysis in section 4 below, where we see that a test sensitivity of at least 45% is needed for our calculations to make sense. we finally consider the external rate of infection. this is due to encounters of infectious individuals from the schools with others outside of the school; it is primarily based on the number of presymptomatic and asymptomatic individuals in the region around the school. this number is clearly unavailable, but an approximation can be obtained from the number of active cases, which is a well established figure [26] : since about half of the infected shows symptoms, and the symptomatic period lasts longer than (perhaps 3 times as long as) the asymptomatic, which itself lasts as the presymptomatic period of everyone infected, the fraction of presymptomatic and asymptomatic together is about the same as the fraction of active cases; the asymptomatics are not assumed to be detected outside of the school, so we take the density of active cases as a proxy for the density of infectious individuals that can be met outside of the school. a survey of the current situation in about 200 countries shows that the fraction of active cases to the population ranges between 1.7 × 10 −7 and 1.2 × 10 −2 (see [26] and supplementary material). this number must then be multiplied by the average number of contacts, similar to what done above to estimate β. as external contacts are, for the most part, between students and other individuals outside schools, we use the corresponding matrices in [23] , with entries averaged over the various age classes: the averages vary between 6.02 for germany and 10.5 for italy; we then add a factor 1/2 for reduced mobility and awareness. susceptibility is taken again at 0.01-0.02, and the scale factor is 6. plausible values of α are then in the range from 0.01 × 6.02 we take an intermediate external infection rate α ≈ 1.8 × 10 −5 . with this value, in the observation period of 40 weeks there is a moderate, but non negligible number of cases even if the school is totally closed; with our selection of parameters the individuals infected outside of the school will be about 12.2% of the total. the parameter α will be carefully monitored in the sensitivity analysis, where it is seen that our optimality results are insensitive to the specific 6 j o u r n a l p r e -p r o o f value of α provided that this lies in a specific range. to aid readability, one can consider an approximate conversion factor of 0.18 (to be adapted to local characteristics) between α and the density of active cases: α ≈ 1.8×10 −5 corresponds to an overall density of active cases of approximately 1.8 × 10 −5 /0.18 ≈ 1 × 10 −4 active cases, or 1 active case in 10000 inhabitants. this conversion factor and the above range of α will also be used to give a tentative indication of which countries can reopen schools and which cannot. table 1 : recap of the model parameters and their estimated values. as initial condition, we consider two possibilities. in the first one, we assume that there are a few (about 1%) exposed individuals already present at the start of the school year: we start from e(0) = 0.01. all others are susceptible, so s(0) = 0.99. in the second, we assume that a careful screening is carried out at the beginning of the school, so we take s(0) = 1. in the second part of the analysis, we introduce a loss function which incorporates the aim of the planner to maximize the number of days with in-class vs. remote teaching, and simultaneously minimize the number of extra infectious transmissions due the opening of the school. for this, it is essential that the planner determines the relative weight τ of these two objectives. once a value of τ is fixed, the aim of the planner becomes that of minimizing a functional that combines these two objectives by the relative weight. to achieve her/his targets, the planner can fix a school calendar, deciding ahead of time which days are in-class and which are restricted to remote teaching; for simplicity, we consider here a plan for the whole year, but monitoring and en route adjustments can also be incorporated. we remain agnostic as to the value of the relative weight τ , and develop tools to analyze all possible scenarios. let s 0 (t) be the fraction of susceptibles at time t in the hypothesis that teaching is completely remote, that is with c ≡ 0; s 0 (0)−s 0 (t ) is then the fraction of individuals who receive an infectious transmission even if the school never opens; then, let s(t) = s c (t) be the fraction of susceptibles with opening plan c(t); we is the fraction of extra infectious transmissions due to the opening of the school 7 j o u r n a l p r e -p r o o f journal pre-proof according to plan c(t). in addition, let n (c) = 400 − t 0 c(t)dt/7 be the number of remote teaching days (the factor 1/7 is due to the number of hours that the school is open in a regular day). the loss function combining the two effects is then where τ is the relative weight of a day of in-class teaching to infectious transmissions: τ /100 can be interpreted as the number of days of in-class teaching that the planner considers equivalent to a 1% increase in the infectious transmissions in the school. the planner's objective becomes then with m as in (6). it is easy to see that using the system (1)-(5) the optimization problem can be cast in a more standard form [27, 28] , in which where e 0 (t) is the exposed component of the solution of the system (1)(5) for the case c ≡ 0; however, as we optimize over the very restricted class of functions (6) , which are piecewise constant and depend on the finite number of parameters c i,j 's, the general theory of control optimization is not needed here: (8) becomes a discrete optimization problem. once the parameters have been selected, for instance with the values given in table 1 , we have optimized for each fixed value of τ ∈ [0.5, 2000] by simulated annealing [29] . for simplicity, we assume here that the decision about remote or in-class teaching is taken for each week, so the control c i = c i,j depends only on i = 1, . . . , 40. this still includes 2 40 possible policies. for a fixed value of τ , we take vanishing "temperatures" k = 0 e −k with 0 ∈ (0, 1], and a random initial configuration c (0) (τ ) = {c 1 , . . . , c 40 } ∈ {0, 1} 40 . at iteration k = 1, . . . , 50, a random position m between 1 and 40 is selected and c m is changed intoc m = 1 − c m , whilec m = c m for all other m 's. the system is solved using the opening rulec, and the loss function is computed in the solution. if the loss decreases, then c (k+1) (τ ) =c; else, we set c (k+1) (τ ) =c with probability k and c (k+1) (τ ) = c (k) (τ ) with probability 1 − k . we then repeat by drawing a new random integer m. this is repeated 50 times for each initial configuration. the sequencec (k) (τ ) tends to a (local) minimum of the loss functional; the outputc(τ ) =c (50) (τ ) of 50 iterations is retained as a candidate (close to) optimal strategies for the given value of τ . in addition to the above, all the configurations c (k) (τ ), k = 1, . . . , 50, not only the (close to) optimal ones, are recorded for several values of τ , to get an 8 j o u r n a l p r e -p r o o f idea of the possible scenarios. figure 1 reports remote teaching days vs. fraction of infected in school at the end of the school year for each c (k) (τ ), k = 1, . . . , 50 and for various values of τ . as benchmark cases we consider the full closure and the complete opening of the school, which is to say, the two most extreme choices of a fully remote teaching or regular 7 hours a day in-class activities for the whole year. as we have assumed that there is a constant source of external infection in the region surrounding the school, even if the school is completely closed, which is c ≡ 0, the simulations based on (1)-(5) determine a number of cases. we have simulated the fraction of cases at school year end, and the number of cases in a school of 1000 individuals in the first two months. the parameters are those selected in the section 2.3, except that we consider a variety of possible rates for the external source of infection. for example, for α = 2×10 −5 , we have that with the school completely closed there would be about 12.45% cases (a+i). in a school with 1000 individuals this means that about 125 individuals of the school would be infected (with or without symptoms) in the course of the year from contacts outside of the school. at day 60 the number of infected individuals showing symptoms is about 28, and we assume that this is in line with the rate of infection in the overall community. if the school is completely open, on the other hand, simulations with c ≡ 1 show a fraction of infected of about 38.7% at the end of the school year, with already 17% of new cases, infected or asymptomatic, in the first 60 days. in a school of 1000, one would observe about 173 cases, with perhaps a several symptomatic, in the first 60 days, vs. the expected 28 and very likely no symptomatic; a deviation which would not pass unnoticed. table 2 compares the percentage of cases at the end of the school year, and the number of likely cases in the first two months, in the two cases of completely remote and fully in-class teaching. the values of α can be either determined from open cases in the area, contact matrix and susceptibility, as done in section 2.3, or from the fraction of infected (a+i) that it causes at the end of the observation period. this last fraction is called seropositivity, and it is discussed in [30] and [31] ). seropositivity at the end of the school year, assuming the school is closed. is in table 2 , column 2. the range of α covers all cases of interest. in table 2 we assume that there are no active cases in the school at the start of the school year due to a sufficiently accurate initial testing. there is not much difference if we assume a 1% active cases for the largest values of α, but this last assumption would not be appropriate for small values of α and hide the possibility of opening the schools with almost no restrictions should one can see that complete opening of the school determines about a four fold increase in the number of cases, except for the extreme cases. if the virus is completely under control, which is less than 5 active cases per million, or α < 0.18 × 5 × 10 −6 ≈ 10 −6 , then the number of cases is so limited that schools can be safely reopen completely (with an initial screening and maintaining the physical distancing measures that are controlling the spread of the virus). if, on the other hand, there are more than about 550 active cases per million in the surrounding region, or α > 10 −4 then opening the school would not particularly aggravate the situation; but the number of cases is already so high that drastic measures need to be taken, such as keeping all schools closed. the conclusion is that, except in the extreme situations of almost complete elimination of covid-19, or uncontrollable outbreak, the number of cases registered in a fully open school is likely to raise concerns from authorities before the end of the second month of school opening in any reasonable scenario; this is very likely to lead to the application of containment measures to the school, such as a forced, unplanned remote teaching. for these reasons, since this situation is likely to occur in most schools and in almost every external evolution of the epidemic, simulations indicate that it is advisable to attempt a sustainable, partial opening. this is the question faced here below. journal pre-proof with the reference parameters identified in section 2.3, we have explored various opening strategies; for each policy c we have recorded the total fraction of infected, measured by the marginal decrease in susceptible s c (0) − s c (t ), and the total number of remote teaching days n (c). recall that we consider policies with opening or closure applied for entire weeks. one can notice that, due to the form of the loss functional, some policies c are uniformly better than others regardless of the value of τ : define c 1 as strictly preferable to c 2 if s c1 (0) − s c1 (t ) ≤ s c2 (0) − s c2 (t ) and n (c 1 ) ≤ n (c 2 ) with at least one inequality strict; if a policy admits another one which is strictly preferable, the first policy can clearly be disregarded. we say that a policy is perfect with respect to the loss functional if there does not exist a strictly preferable one. on the other hand, once a value of τ is fixed, an optimal policy for τ is a policy minimizing the functional in (8) . clearly, an optimal policy for some τ is perfect with respect to the loss functional, and, vice versa, a perfect policy with respect to the loss functional can be optimal for an interval of values of τ . in figure 1 we plot remote teaching days vs. fraction of infected in school at the end of the school year for each c (k) (τ ), k = 1, . . . , 50 and for various values of τ ∈ [0. 5, 2000 ]. such range corresponds to between 0.5 and 20 days of in-class teaching rated equivalent to a 1% increase in the number of infected. notice, though, that figure 1 does not allow one to retrieve the value of τ . one of the (likely) perfect opening strategies is represented by the red dot: it is very likely optimal for τ around 800. notice, finally, that the figure reports some, but very few of the strategies that are far from perfect or optimal. there are many others that our runs of the simulated annealing algorithm never visited. figure 1 : fraction of extra infected, next to the 12.2%, vs days of remote teaching, for several opening strategies. the potentially perfect, and hence optimal ones for some values of τ , are close to the continuous curve. one special possibly perfect and hence optimal solution corresponds to the red dot before turning to the exploration of specific optimal strategies, let us discuss 11 j o u r n a l p r e -p r o o f the solid line in figure 1 . experimentally, we have noticed that the outcomes of the perfect policies seem to lie close to a specific curve c, which corresponds approximately to where x is the percentage of covid-19 cases in the school population, and y is the number of remote teaching days. the curve c can be thought of as parametrized by τ , the relative importance of in-class teaching and covid-19 cases; in an abstract sense, each point on the curve c corresponds to one value of τ , although, as mentioned above, by the discrete nature of the opening strategies, more values of τ give the same perfect strategy. as indicated before, the red dot corresponds to a perfect strategy which is optimal for τ ≈ 800 (see below). this curve c is of both theoretical and practical importance. in fact, 0.76 turns out to be a characteristic exponent of the comparison of opening days vs. extra infected; theoretically, it is quite interesting to explore the analytical reasons behind the appearance of such a curve; its role in the analysis of the solutions of the differential system (1)-(5); and the functional dependence of the value of the exponent on the values of the parameters in the system. in practice, if one was able to have some information about the dependency of the exponents from the parameters of the model different from τ , this would give an explicit benchmark for perfect strategies: for instance, if one fixes the number of opening days, then the curve indicates the best result that can be ideally achieved in the containment of the infection, or vice versa, without the need of an explicit numerical minimization. in this section we show one example of (nearly) optimal policy. as mentioned, a choice has been required by the policy maker: such a choice could have consisted of the minimal number of opening weeks, or of the maximal fraction of cases which is allowed by the circumstances. however, we adopted the less intuitive, but more intelligible possibility of using the relative weight of remote teaching vs. percentage of cases, as incorporated by the parameter τ (see section 2.4). we consider the parameters identified in section 2.3 and τ = 800, which means that 8 days of remote teaching are considered equivalent to a 1% increase in the number of cases. the aim of the planner becomes then to solve the optimization problem (8) , with the functional l defined in (7). this can be achieved by a discrete optimization in {0, 1} 40 . we use here simulated annealing with 50 iterations from a random seed, repeated for 100 random initial conditions. the (possibly) perfect opening strategy indicated by the algorithm is (0, 0, 1, 0, 1, 0, 1, 0, 1, 0, 0, 0, 1, 0, 1, 0, 1, 0, 1, 0, 1, 0, 0, 1, 0, 1, 0, 1, (10) 0, 1, 0, 1, 0, 1, 0, 1, 0, 1, 0, 1) where 0 indicates a week of remote, and 1 a week of in-class activities; notice that there is an initial period of closure, before progressing to a regular alternation j o u r n a l p r e -p r o o f journal pre-proof of openings and closures. this is due to the assumed presence of 10% of cases at the start of the school year, and could be avoided with a very efficient screening before the start of the school year. the solution in (10) has 90 days of in-class teaching, with the additional infectious transmissions contained to 7.9%, which is an increase of 64.75% of the cases with respect to remote only activity. in a school of 1000 individuals this corresponds to creating an extra 79 cases of covid-19 in the school year, in addition to the 122 who would have been infected in any case. the administrators can decide to realize a stricter or more relaxed containment of the extra cases by assigning more or less weight to infectious transmissions. notice that the solution in (10) is quite periodic, with alternated weeks of in-class and remote activities; figure 2 shows the evolution of the epidemic functions under the policy in (10) . table 3 summarizes the change in infectious table 3 : performance of optimal and random solutions with 90 days of in-class teaching. first 4 columns: total number of infectious transmissions; last 3 columns: percentage increase with respect to remote only solution. "optimal" refers to the policy in (10) identified by simulated annealing. we have also explored the possibility of selecting at random which weeks to open or close, with the constraint of having 90 days of in-class activities, which is to say 18 open out of the 40 weeks. a simulation of the distribution gives that the increment in the number of cases is 84% on average, with a standard deviation of 10%, hence the solution in (10) is more than 2 sd's below what the average random selection would provide; in addition, it is also not subject to possible adverse fluctuations, so it is definitely preferable to a random, unjustified selection. it is, however, the case, that adopting a blended model with alternated open and closed weeks does provide in any case, even without optimization, a sizeable containment of the infection within the school. in section 4 we carry out a sensitivity analysis, which shows that the optimal planning is pretty stable in case of some extra opening or remote days, and is only moderately affected by errors in parameter selection, within certain ranges. the optimal solutions turn out to be blended models, with approximately one week of in person teaching, and one week of remote learning. the pedagogical aspects of such models have been examined in various researches, for instance [32, 33] it turns out that there are several advantages in blended learning: • in the current context, it seems to allow a sustainable programming of school operations; • it permits to take advantage of both in person and remote teaching, limiting drawbacks of both; one can, for instance, concentrate in person tests and labs for the in-class weeks, and less participative moments for remote teaching; • in general, it allows the use of a variety of learning modes and methods; • it can provide a pedagogical experience that can be replicated and expanded in the future in order to exploit new technologies while keeping regular personal contacts. of course, there are challenges, in particular concerning the ability of teachers and students to master the needed technologies and the alternating formats [32, 33] . in addition, as schools could attempt planning in advance, especially if this is coordinated for most schools in some location, the regular economic and social activities could be somewhat adapted to the school calendar; for instance, more working hours could be planned during the weeks in which schools are open. clearly, there are several difficulties in implementing blended strategies, but the alternative, which is already unfolding in many situations, seems to be chaotic schedules, with schools attempting full openings, and then suddenly going back to closure as the first covid cases surface. we provide a sensitivity analysis in four parts. the first part concerns errors in the determination of the optimal solution. if one of the weeks for which remote teaching is planned by the optimal solution is instead changed to an in-class week, then the increment in the number of cases due to school opening ranges from an extra 72% to an extra 78%, compared to the extra 68% of the optimal solution. if one week is moved remote from inclass, then the increment in the number of cases is reduced to about 64%. this suggests that altering the number of weeks of opening is not optimal, but does not substantially change the final outcome: therefore, once an optimal policy has been determined, extra openings or closures can be adopted if needed without major changes in the final outcome. in addition, once the flexible blended model has been adopted, one can quickly and easily adapt to major changes in the external conditions, such as a sudden outbreak or a vaccine. the second part of the sensitivity analysis concerns each of the parameters of the model used to determine the optimal solution in (10). we assume that the optimal policy has been determined, and observe deviations from the expected performance due to possible errors in the parameter determination. for each possible value of the parameters, we compute the fraction of cases in the cases of complete closure f cc , full opening f f o , and optimal solution f opt . then compare the percentage increase of optimal solution with respect to complete closure (f opt − f cc )/f cc with the analogous increment for the full opening (f f o − f cc )/f cc . such comparison is reported in table 4 for the largest and the smallest values of (f opt − f cc )/f cc in the given range of the parameter. in addition, we indicate of each parameter the range of values for which the optimal solution achieves a substantial reduction with respect to complete opening, but limited to determining at most an 100% increase in the number of cases with respect to school closure. wee see that the most critical parameters are α, β and η. the range of α in which the optimization process has a change of being effective is 10 −6 -10 −4 , which corresponds to about 5 to 555 active cases per million in the area around the school. for the infection rate β, the range in which optimization is effective is 1.25-5 × 10 −2 ; this is a very critical parameter, as observations in different countries lead to a range of 2.5 × 10 −2 -17 × 10 −2 . so, each region or country should monitor this rate and introduce strict measures to keep β within the above limits. finally, the fraction of undetected asymptomatic η must not exceed 45%: we assume availability of an easy, rapid and cheap test with a rate of false negative not exceeding 55%, such as crispr [34] . the third part is about the selection of τ . in fact, the sensitivity to τ has already been discussed in section 3.2: figure 2 shows that the optimal strategies lie approximately on the curve c with equation (9); even if the equation is not exact and the dependency on τ is not known analitycally, it nonetheless gives an idea of the variability of the optimal solutions as function of τ . the data plotted in figure 2 has been obtained with τ ranging from 200 to 1400, which corresponds to a range between 2 and 14 days of in-class teaching equivalent to a 1% of extra cases. there is at most doubling of the cases for τ ≥ 700, and, correspondingly, for at least 85 of remote teaching. this seems to indicate that next school year will allow no more than 115 days of in-class teaching without pharmaceutical progress; recall that possibly one of two extra weeks can be gained with an efficient initial screening. the last part of the sensitivity analysis is about the weekly schedule. a possible alternative is to schedule opening or closure of the schools for each single day (still with a calendar fixed in advance). the optimization is now over 2 200 possible policies. optimization by simulated annealing in this case determines a plot similar to that of figure 1 . for instance, the optimal policy for τ = 800 gives an increase of 63.24% in the number of cases, vs. the 64.75% increase of the weekly schedule; the daily schedule consists of 89 of in-class teaching, instead of the 90 which are considered by the weekly schedule. in summary, the daily schedule turns out to offer little advantage with respect to j o u r n a l p r e -p r o o f journal pre-proof the weekly option, but a more complicated implementation process. we have considered the issue of planning in-class activities for the school year '20-'21. education is, in fact, one of the areas in which the measures to contain the current covid-19 pandemic have had a negative impact, with many countries and local authorities struggling to find acceptable plans for the next academic year. in this work we have focused on a single school. to aid the planning of teaching for the school, we have modeled the evolution of the covid-19 epidemic in the school by an extension of the sir model, with asymptomatic; an external, constant, source of infection; interactions within the school happening only during the 7 hours of opening during week days; a reduced rate of encounter with asymptomatic due to a somewhat efficient screening system applied weekly. we have then determined realistic values for the remaining parameters of the model. next, we have set up an optimization problem, based on a preliminary assessment by the planner of the relative importance of ensuring in-class teaching vs. containing the epidemic spread. for each value of such relative importance and of the other parameters, we have carried out a discrete optimization by simulated annealing to obtain a (close to) optimal strategy within the space of all possible weekly opening policies. modeling of epidemic outbreaks by differential equations dates back to the original paper by kermack and mckendrick [35] , with an enormous number of recent works on the current covid-19 outbreak. among these, [36] discusses the impact of reopening various combinations of schools in england vs. keeping schools closed; results in [36] are expressed in terms of the reproduction number r 0 , and show that, in many cases, opening of all schools would bring the reproduction number above the critical value of 1. our model has many fewer categories and variables, and it is focused on one single school. in any case, our results also confirm that opening all schools is not advisable in uk, even with extra testing and blended models (see supplementary material). as far as optimization is concerned, methods similar to those used in this paper have appeared in many other contexts. in particular, several recent studies have considered optimal planning strategies in response to general epidemics [14, 37] or, more specifically, to the covid-19 pandemic [27, 38] ; to our knowledge, however, none has considered the current optimization problem for school planning. limitations. there are several limitations to our study. we use a variation of the sir model, in which we have averaged encounter rates and susceptibilities over several age classes and different countries to obtain reasonable parameters. it could be that local deviations from the values we have used might lead to sensibly different outcomes. the model includes no randomness, which, however, is an essential feature of both human interactions and virus spreading; randomness would also allow us to assess the variability of the predictions. as a result, there are no error bars in our predictions, except for the case of a random selection of the weeks of in-person teaching. the external infection rate is taken to be constant, as focusing on a single school does not allow us to model the evolution of the epidemic in the overall area. this rate, however, is not likely to be constant over a long period of time, but is subject to many variations due to expansion or containment of the outbreak. in addition, our results are only a first indication of a modeling methodology for the search of an optimal trade off between in-class teaching and containment of infectious transmissions. even if parameters are carefully and realistically selected, the values are based on information known at the time of this study; when a parameter has a wide range of variability we settle for one representative value; in addition, the worked out examples refer to an ideal school: for each particular concrete situation, one needs to adapt the model to the specific case. we also did not consider other alternatives, such as reducing the number of in-class students for each lesson. many possible alternatives could be conveniently incorporated in a more elaborate model. conclusions. first, we have seen in section 3.1 that the regular opening of a typical school would cause a multifold increase in the number of cases among the individuals involved with the school itself, both at 60 days and at school year end. the only exception would be if the virus is under tight control in the region around the school, which is to say with less than 5 active cases per million, in which case one can safely plan a complete reopening. if there are more than about 550 active cases per million in the surrounding region, then the opening of the school would not particularly aggravate the situation; in that case it would, however, not be advisable to carry out any in person activity as the number of cases would be out of control (at least 35% of the school population in the first 60 days of opening). the second conclusion, discussed in section 3.2, is that careful planning of the opening weeks is capable of making a drastic reduction on the number of extra cases induced by opening the school. optimal or close to optimal solutions consist of blended models, with an alternation of weeks of remote and in-class 18 j o u r n a l p r e -p r o o f activities; with about half of the weeks in class, for instance, one can reduce the number of cases by a factor of 4; we have also seen that schedules with a choice between in-class and remote on a daily basis would not improve the reduction much. the likely cause of the need for the weekly alternation is the duration of covid-19 incubation period. the third result, in section 3.3, is that to identify an optimal opening strategy suitable for a specific situation, planners are first required to explicitly incorporate into their objective the relative value of in-person teaching vs. reduction of covid-19 spread; once this is done, optimal or close to optimal solutions can be numerically estimated to produce one of the optimal or close to optimal strategies mentioned above. a random choice of the weeks of in-class activities would not reduce the number of new cases as effectively as an optimal choice, but would nonetheless achieve a substantial reduction with respect to full opening. in section 4 we have also seen that the achieved reduction is preserved by small changes in the weeks of in-person activities. as such, one school could identify the number of in-class days or weeks, and slightly adjust the calendar to other needs. finally, in section 4, we have carried out a sensitivity analysis of the results. these have turned out to be rather stable for small errors in the selection of the model parameters. the more critical parameters are • the contact rate during school opening, a parameter which should be very carefully monitored and controlled via physical distancing and other measures; • the fraction of detected asymptomatic, for which schools should adopt some frequent and possibly not very expensive testing procedure; there is no need to have an exceptionally high reliability of the test itself, and we have shown viable opening policies based on reliability above 55%; • the external risk rate: as mentioned, one can achieve some control on the spread of the virus in the school only if there are no more than about 550 active cases per million in surrounding area. based on the above broad indications, and pending adaptation to specific situations, we have determined for each country whether full opening is feasible, or whether a blended opening of schools for 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covid-19 lockdown key: cord-024564-ff5ex004 authors: ricoca peixoto, vasco; nunes, carla; abrantes, alexandre title: epidemic surveillance of covid-19: considering uncertainty and under-ascertainment date: 2020-04-09 journal: nan doi: 10.1159/000507587 sha: doc_id: 24564 cord_uid: ff5ex004 epidemic surveillance is a fundamental part of public health practice. addressing under-ascertainment of cases is relevant in most surveillance systems, especially in pandemics of new diseases with a large spectrum of clinical presentations as it may influence timings of policy implementation and public risk perception. from this perspective, this article presents and discusses early evidence on under-ascertainment of covid-19 and its motifs, options for surveillance, and reflections around their importance to tailor public health measures. in the case of covid-19, systematically addressing and estimating under-ascertainment of cases is essential to tailor timely public health measures, and communicating these findings is of the utmost importance for policy making and public perception. vigilância epidemiológica covid-19 · subindetificação · subdeteção · sensibilidade sistemas de vigilância epidemiológica a vigilância epidemiológica é uma parte fundamental da prática de saúde pública. considerar e avaliar a sub-deteção de casos é relevante na maioria dos sistemas de vigilância, especialmente em pandemias de novas doenças com um amplo espectro de apresentações clínicas, porque pode influenciar os momentos e de decisões em políticas de saúde e a perceção de risco da população. este artigo apresenta e discute evidência inicial sobre a sub-deteção do covid-19 e os seus motivos, opções de vigilância e reflexões sobre sua importância para informar medidas de saúde pública. no caso do covid-19, abordar e estimar sistematicamente a su-deteção de casos é essencial para adequar as medidas de saúde pública, e comunicar essas achados é de extrema importância para a formulação de políticas. epidemic surveillance is a critical component of public health practice. it gives us pictures of reality, informs policy and decision making, gauges health service demand, and feeds forecasts and models. however, surveillance is never perfect and diseases that present with a high proportion of mild, pauci-symptomatic, or subclinical cases can be hard to detect in most indicator-based surveillance systems and become harder to contain [1] . surveillance pyramids can take different formats and proportions of undetected cases (often mild or asymptomatic). the scientific community is making efforts to address uncertainty about where to draw the lines separating different clinical manifestations and between ascertained and unascertained cases. covid-19 surveillance is challenging because mild and subclinical cases may not seek health services, cases are advised to avoid health care unless necessary, and testing capacity may be limited (fig. 1 ). in the case of covid-19, addressing and estimating under-ascertainment of cases is essential to tailor public health measures. communicating these findings is of the utmost importance for evidence-based policy making and community engagement. in europe in late february there was a fast rise in the number of confirmed cases in italy, and the probability of importation of cases of covid-19 may have been high in europe until italy stopped movement from northern italy and later from the rest of the country. the rapid rise and characteristics of confirmed cases in northern italy indicated an earlier important undetected community transmission, especially among mild cases. as such, it is possible that in late february many cases from northern italy (and possibly from other european areas) were exported all over europe. these cases may have given rise to mostly undetected transmission chains that remained active in the next months, justifying early enhanced surveillance and control measures that were taken by different governments to buy time for preparedness. the generalized under-detection of imported cases has been confirmed in a study in singapore, which estimated that the overall ability to detect imported cases was 38% (95% hpdi: 22-64%) of singapore's capacity. in high-quality surveillance locations (based on the global health security index) it was 40% (95% hpdi: 22-67%) [2] . in line with these findings, a report by imperial college london estimated that 2/3 of covid-19 cases exported from mainland china remained undetected worldwide (until february 17), resulting in several undetected transmission chains. they found countries in europe had relatively low sensitivity for imported cases [3] . a relevant part of the fast growth in detected cases in italy and other countries could be related to undetected transmission chains from early introduced cases and accumulated a large pool of infected cases. it is likely that a high proportion of imported cases all around europe have not been found, and isolation, contact tracing, and quarantine could not be applied considering the who suspect case definition (as of february 27, 2020). although many countries advised travellers returning from certain areas to report (call a health line or medical doctor) if they had symptoms, it is possible that people with mild symptoms may not have followed this advice. consequently, many cases that might have followed from this would not be tested because they lacked a clear epidemiological link (fig. 2) . in fact, several articles suggest a major under-ascertainment/under-reporting of total cases in different countries. one study modelled the real dimension of the epidemic in italy from international case exportations and estimated a real size of 3,971 cases (95% ci: 2,907-5,297), compared to a case count of 1,128 on february 29, 2020, suggesting an under-ascertainment of 72% (61-79%) of cases. in sensitivity analyses, the range of variation was from 1,552 to 4,533 cases (implying the identification of 27-75% of cases) [4] . publicly available epidemiological data for hubei, from january 11 to february 10, 2020, revealed a significant decline in the fatality rate with time. as testing becomes widespread, the detection of milder cases that would not seek health care in the early stages will result in a lower case fatality rate [5] . another study used data from air travel and cases imported from iran in other middle eastern countries and estimated that there were 16,533 (95% ci: 5,925-35,538) covid-19 cases in iran as of february 25, which meant only 0.6% of all cases had been reported [6] . other models used reported infection within china, in conjunction with mobility data, a networked dynamic metapopulation model, and bayesian inference, and estimated that 86% of all infections were undocumented port j public health doi: 10.1159/000507587 (95% ci: 82-90%) prior to 23 january, 2020, travel restrictions and that undocumented infections were the infection source for 79% of documented cases [7] . adding to this we should consider specific surveillance system delays from infection and symptom onset to reporting. we should ask ourselves whether different choic-es at different timings would be made at an individual and policy level if we only looked to the confirmed cases. modelled estimations of total infected and counts of suspect/possible cases that were not laboratory confirmed should be considered and communicated together with uncertainty in model assumptions (fig. 3) . some countries initiated earlier and wider testing than others [8] . some started earlier to test admitted cases of bilateral pneumonia without other aetiology (allowing for earlier consideration of community transmission), and some tested contacts even if asymptomatic. criteria for testing in earlier stages was variable and resulted in very different testing rates [8] . the different testing strategies and consequent different levels of under-ascertainment may account for some of the differences in the fatality rate found in different countries in europe such as italy and germany (9 and 0.2%, respectively) [9] , though we should also consider the impact of overload of the health systems, characteristics of the specific surveillance system, and population demographics and behaviour. there are various options for surveillance to improve case detection or make estimations (table 1) . limited capacity for testing may play a role in testing strategies, forcing some countries to prioritize testing, but it makes discussion and research around under-ascertainment even more relevant in those contexts. underascertainment in surveillance should make us consider an under-ascertained need for specific early measures (for example of social distancing) but also, in later stages, that a much larger part of the population could be immune and social distancing strategies could change, which also raises the importance of serologic surveys in those circumstances to confirm these estimates. one document of the european centre of disease control (ecdc) [10] reports that "the detection of co-vid-19 cases and/or deaths outside of known chains of transmission is a strong signal that social distancing mea-sures should be considered." however, with restrictive testing strategies, these signals may be missed. the document states that "the early, decisive, rapid, coordinated and comprehensive implementation of closures and quarantines is likely to be more effective in slowing the spread of the virus than a delayed implementation" and that decisions "will very rarely be purely evidence-based" as social and political considerations will also need to be taken into account. evidence is always incomplete, especially in new pandemics. there is evidence of the effectiveness of public health measures such as travel bans, movement restrictions, and social distancing. a study used a seir (susceptible-exposed-infectious-removed) model to estimate epidemiological parameters before the implementation of measures in wuhan. if these measures had been initiated 1, 2, or 3 weeks earlier in china, cases could have been reduced by 66, 86, and 95%, respectively, together with a significant reduction in the number of affected areas [11] . another study reports that the effective reproduction number in wuhan fell from 2.35 (95% ci: 1.15-4.77) 1 week before the introduction of restrictions on january 23, 2020, to 1.55 (0.41-2.39) 1 week later and calculated that in sites with wuhan-like transmission potential in early january, when there were only 4 cases introduced, there is more than a 50% chance of the infection settling in this population [12] . other simulation [13] suggests that to control most outbreaks, for r0 (number of cases directly generated by one case in a population where all individuals are susceptible to infection) of 3-5, more than 70% of contacts had to be identified. in most scenarios, highly effective case identification and isolation of contacts could control a new outbreak of covid-19 within 3 months. however, importantly, the probability of control decreases with delays in the identification of cases and contacts, increased time from onset of symptoms to isolation, fewer cases identified, and increased transmission before symptoms. the oxford covid-19 government response tracker [14] describes variation in government responses and explores whether rising stringency of response affects the rate of infection. they calculate a stringency-risk ratio but warn that recorded cases partly depend on how much testing is done, which is likely to co-vary with the stringency of the government's response. regular updated results will be available for anyone. a recent study by the imperial college covid-19 response team shows that in the uk and us context, suppression requires a combination of social distancing of the entire population, home isolation of cases, and household quarantine of their family members, that may need school and university closures and reports other strategies that may involve social distancing of those > 70 years old. however, the study predicts that transmission will quickly rebound if interventions are relaxed and suggests that intermittent social distancing -triggered by trends in disease surveillance -may allow interventions to be relaxed temporarily [15] . this strategy needs robust surveillance systems that allow for acceptable sensitivity to timely decide on interventions. we believe these triggers must consider evidence and debate around surveillance system sensitivity and under-ascertainment. as suggested by another imperial college report [16] , this would be relevant to produce early evidence when exiting the co-vid-19 social distancing policy after achieving containment. due to different surveillance systems, different measures at different timings, different levels of stringency [14] , and different demographic and social dynamics, the outcomes within europe may be heterogeneous. undetected introduction of imported cases and many mild undetected cases making up those transmission chains may warrant early measures. by looking at the death epidemic curves (likely less sensitive to variations in surveillance system sensitivity) of different countries we see differences that can be related to earlier detection and implementation of measures (fig. 4) . notably, south korea has managed to keep new deaths by covid-19 low despite being in a relatively advanced stage of the epidemic and is one of the countries with a higher testing rate [8] . an early study [17] estimated the transmission potential of covid-19 in the country and suggested early sustained disease transmission in the region which contributed to the rapid early implementation of social distancing measures to contain the outbreak [14] . considering uncertainty and under-ascertainment is critical for decision when 500 cases could possibly mean 5,000. delay from exposure and infection to reporting must also be considered. there is at this stage high uncertainty about the behaviour of the disease in europe, the effectiveness of measures in different contexts, and the timing for their implementation. often, measures should be implemented early with imperfect scientific knowledge. on the other hand, estimated total infection numbers can be important to justify measures for public opinion, engage them in preventive action, and reinforce recommendations for those who are tested positive as well as to inform decisions on exiting social distancing policies. more robust models will be produced soon, but models make assumptions based on surveillance data and other sources with different levels of uncertainty. under-ascertainment should be considered, and the best available information and data should be included and pursued by improving surveillance strategies to reduce or better estimate under-ascertainment. with more epidemiological evidence, namely greater certainty in relation to the proportion of asymptomatic infections and their risk of transmission, different surveillance system sensitivity, effective r in different contexts and effectiveness of containment measures and with the results of the first few x cases ffx investigation proposed by who [18] for covid-19 models will better take into account underascertainment in different countries. understanding the extent of transmission in different regions can help policy makers tailor their response. acknowledging that "we can't fight a pandemic blindfolded," on march 16, 2020, the who urged countries to test. under-ascertainment may be of relevance for cov-id-19 policy. there is uncertainty on this subject and in the studies mentioned in this article and presented reflections. however, it is the role of the scientific community in public health and epidemiology not only to communicate what is certain and based in robust established science, but also of what is uncertain, when it may be of relevance for decision making and can be further researched, discussed, and shared. as in most infectious disease surveillance, under-ascertainment can be a relevant piece of the puzzle for covid-19 science, policy making, and public opinion and should be systematically addressed and communicated. a novel coronavirus emerging in china: key questions for impact assessment quantifying bias of covid-19 prevalence and severity estimates in wuhan, china that depend on reported cases in international travelers who collaborating centre for infectious disease modelling; mrc centre for global infectious disease analysis abdul latif jameel institute for disease and emergency analytics relative sensitivity of international surveillance estimation of covid-19 outbreak size in italy based on international case exportations siettos c. data-based analysis, modelling and forecasting of the novel coronavirus preliminary estimation of the novel coronavirus disease [covid-19] cases in iran: a modelling analysis based on overseas cases and air travel data. medrxiv. 2020. preprint substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2 our world in data. how many tests for co-vid-19 are being performed around the world? world health organization. coronavirus disease european centre for disease prevention and control. considerations relating to social distancing measures in response to the covid-19 epidemic effect of non-pharmaceutical interventions for containing the co-vid-19 outbreak: an observational and modelling study centre for mathematical modelling of infectious diseases co-vid-19 working group. early dynamics of transmission and control of covid-19: a mathematical modelling study feasibility of controlling covid-19 outbreaks by isolation of cases and contacts variation in government responses to cov-id-19: version 2.0. oxford: blavatnik school of government abdul latif jameel institute for disease and emergency analytics. imperial college london. impact of non-pharmaceutical interventions [npis] to reduce covid-19 mortality and healthcare demand report 11: evidence of initial success for china exiting covid-19 social distancing policy after achieving containment imperial college covid-19 response team transmission potential and severity of co-vid-19 in south korea the first few x [ffx] cases and contact investigation protocol for 2019-novel coronavirus [2019-ncov] infection the authors thank daniel thomas, sonia bounder, paula blomquist, and robert withaker for commenting on early drafts and aurora peixoto for artwork. the authors have no conflicts of interest to declare. the authors have no funding sources to declare. all authors contributed in a relevant way to the literature review and writing of the article. key: cord-263044-o8aosx2q authors: lipsitch, marc; donnelly, christl a.; fraser, christophe; blake, isobel m.; cori, anne; dorigatti, ilaria; ferguson, neil m.; garske, tini; mills, harriet l.; riley, steven; van kerkhove, maria d.; hernán, miguel a. title: potential biases in estimating absolute and relative case-fatality risks during outbreaks date: 2015-07-16 journal: plos negl trop dis doi: 10.1371/journal.pntd.0003846 sha: doc_id: 263044 cord_uid: o8aosx2q estimating the case-fatality risk (cfr)—the probability that a person dies from an infection given that they are a case—is a high priority in epidemiologic investigation of newly emerging infectious diseases and sometimes in new outbreaks of known infectious diseases. the data available to estimate the overall cfr are often gathered for other purposes (e.g., surveillance) in challenging circumstances. we describe two forms of bias that may affect the estimation of the overall cfr—preferential ascertainment of severe cases and bias from reporting delays—and review solutions that have been proposed and implemented in past epidemics. also of interest is the estimation of the causal impact of specific interventions (e.g., hospitalization, or hospitalization at a particular hospital) on survival, which can be estimated as a relative cfr for two or more groups. when observational data are used for this purpose, three more sources of bias may arise: confounding, survivorship bias, and selection due to preferential inclusion in surveillance datasets of those who are hospitalized and/or die. we illustrate these biases and caution against causal interpretation of differential cfr among those receiving different interventions in observational datasets. again, we discuss ways to reduce these biases, particularly by estimating outcomes in smaller but more systematically defined cohorts ascertained before the onset of symptoms, such as those identified by forward contact tracing. finally, we discuss the circumstances in which these biases may affect non-causal interpretation of risk factors for death among cases. the case-fatality risk (cfr) is a key quantity in characterizing new infectious agents and new outbreaks of known agents. the cfr can be defined as the probability that a case dies from the infection. several variations of the definition of "case" are used for different infections, as discussed in box 1. under all these definitions, the cfr characterizes the severity of an infection and is useful for planning and determining the intensity of a response to an outbreak [1, 2] . moreover, the cfr may be compared between cases who do and do not receive particular treatments as a way of trying to estimate the causal impact of these treatments on survival. such causal inference might ideally be done in a randomized trial in which individuals are randomly assigned to treatments, but this is often not possible during an outbreak for logistical, ethical, and other reasons [3] . therefore, observational estimates of cfr under different treatment conditions may be the only available means to assess the impact of various treatments. however, observational studies conducted in the early phases of an outbreak, when public health authorities are appropriately concentrating on crisis response and not on rigorous study design, are challenging. a common problem is that disease severity of the cases recorded in a surveillance database will differ, perhaps substantially, from that of all cases in the population. this issue has arisen in the present epidemic of ebola virus disease in west africa and in many previous outbreaks and epidemics [4] [5] [6] [7] [8] [9] and will continue to arise in future ones. here we outline two biases that may occur when estimating the cfr in a population from a surveillance database, and three more biases that may occur when comparing the cfr between subgroups to estimate the causal effect of medical interventions. we also briefly consider the applicability of these biases to a different application: comparing the cfr across different groups of people, for example, by geography, sex, age, comorbidities, and other "unchangeable" risk factors. such factors are "unchangeable" in the sense that they are not candidates for intervention in the setting of the outbreak, though some could, of course, change over longer timescales. the goal of estimating the cfr in groups defined by such unchangeable factors is not to understand the causal role of these factors in mortality, but to develop a predictive model for mortality that might be used to improve prognostic accuracy or identify disparities. such box 1. definition of the cfr. the cfr itself is an ambiguous term, as its definition and value depend on what qualifies an individual to be a "case." several different precise definitions of cfr have been used in practice, as have several imprecise ones. the infection-fatality risk (sometimes written ifr) defines a case as a person who has shown evidence of infection, either by clinical detection of the pathogen or by seroconversion or other immune response. such individuals may or may not be symptomatic, though asymptomatic ones may go undetected. the symptomatic case-fatality risk (scfr) defines a case as someone who is infected and shows certain symptoms. infection in many outbreaks is given several gradations, including confirmed (definitive laboratory confirmation), probable (high degree of suspicion, by various clinical and epidemiologic criteria, without laboratory confirmation), and possible or suspected (lower degree of suspicion). this paper describes issues in estimating any of these risks or comparing them across groups, but does not go into the details of each possible definition. furthermore, unlike risks commonly used in epidemiologic research (e.g., the 5-year mortality risk), the length of the period during which deaths are counted for the cfr is rarely explicit, probably because it is considered to be short enough to avoid ambiguity in the definition of cfr. however, a precise definition of the cfr would need to include the risk period, e.g., the 1-month cfr of ebola. clearly, the definition of cfr for a particular investigation should be specified as precisely as possible. predictions may be affected by survivorship bias and selection bias, but not by confounding, as we discuss. two biases that may affect the estimation of an overall cfr are presented in table 1 : for diseases that have a spectrum of clinical presentation, those cases that come to the attention of public health authorities and are entered into surveillance databases will typically be people with the most severe symptoms, who seek medical care, are admitted to hospital, or die. therefore, the cfr will typically be higher among detected cases than among the entire population of cases, given that the latter may include individuals with mild, subclinical, and (under some definitions of "case") asymptomatic presentations. laboratory confirmation as an inclusion criterion may reduce this bias if it is able to detect a wider spectrum of presentations, or may exacerbate it if the probability of receiving a laboratory test is higher for more severe cases and/or if test sensitivity is higher for more severe cases. the magnitude of this bias may be uncertain for a long period because the spectrum of clinical presentations is itself uncertain at the start of an outbreak of a new disease [12, 26] . all proposed approaches to estimate and correct for this bias (table 1 ) require auxiliary data sources to estimate how the reported subset of cases compares with the overall population of cases. the availability of such auxiliary data sources will depend on the context of the outbreak. during an ongoing epidemic, there is a delay between the time someone dies and the time their death is reported. therefore, at any moment in time, the list of cases includes people who will die and whose death has not yet occurred, or has occurred but not yet been reported. thus dividing the cumulative number of reported deaths by the cumulative number of reported cases at any moment will underestimate the true cfr. the key determinants of the magnitude of the bias are the epidemic growth rate and the distribution of delays from case-reporting to death-reporting; the longer the delays and the faster the growth rate, the greater the bias. heuristically, the underestimate will be proportionate to the expansion of the epidemic during the delay between the time a case enters the database to the time the death of that case enters the database (if it occurs). fig 1 illustrates an example where the delay is 3 weeks, the epidemic doubling time is 2 weeks, and the underestimate is by a factor of 2 3/2 2.8. this bias may be corrected for in various ways, and to varying degrees, using information on the growth rate of the epidemic, the distribution of times from case-report to death-report, and the distribution of times from case-report to recovery-report (i.e., report that the case is no longer at risk of dying of the infection). a simple approach is to limit analysis to those cases with sufficiently long follow-up for a death to have been recorded had a death occurred, but this approach may result in an exceedingly small sample size if applied early in the epidemic. several such strategies are described in table 1 . here, and in table 2 , we discuss the sources of three biases that threaten the validity of a causal interpretation of a difference in cfr between groups who have received different interventions. such a difference might be measured as a risk ratio (rr), the ratio of cfr in group a to that in group b, or as an odds ratio (or), the ratio of the odds of dying in group a and group b, or as table 1 . potential biases that can affect the estimation of cfr (and thereby also the comparison of cfr across groups). direction outbreaks in which analysts have noted this bias may be operating preferential ascertainment of severe cases: in an infection with a range of manifestations from relatively mild to highly severe, milder cases are less likely to appear in surveillance databases than more severe ones; therefore, the cfr among ascertained cases will be higher than that among all cases. spuriously increases estimate of cfr influenza h1n1pdm [10] [11] [12] , influenza h7n9 [6] , influenza h5n1 [7] (though this hypothesis has been refuted [8] ), middle east respiratory syndrome [4] , ebola (this article) [13] note: these solutions are listed in approximately the temporal order in which they may be practical, from early in the outbreak to later on; details will depend on the epidemiology of the outbreak. use sentinel surveillance sites to estimate multipliers between various levels of severity and extrapolate to a larger population [6] . survey-or health-facility-based surveillance for symptomatic infection [14] in a defined population, combined with enhanced surveillance for severe outcomes (particularly death) in the same population. use travelers from high-burden areas with low ascertainment to low-burden areas with higher ascertainment to estimate incidence of infection in source population [15, 16] , thereby providing a more accurate denominator for comparison to deaths in source population. surveillance pyramid approaches: reconstruct conditional probabilities of appearing at one severity level conditional on reaching a lower severity level; combine data sources that have relatively complete ascertainment of higher severity levels (e.g., hospitalization, icu, death) with those having relatively complete ascertainment of lower levels (e.g., seeking medical attention, hospitalization) [10, 11] . cfr can then be estimated as a product of conditional probabilities with associated uncertainties [17] . serologic ascertainment of infection [18, 19] to provide a population denominator for infections regardless of symptoms, combined with active surveillance for more severe outcomes. individuals ascertained by a different mechanism, e.g., named healthy contacts of cases who subsequently test positive, could be a more representative group in whom to assess severity [20] . bias due to delayed reporting. during an ongoing epidemic, at any week w the persons who have died up to time w will not be the only ones to die of the infection among those who became cases by w. the denominator of the cfr (cases) includes persons who have not yet died of the infection, but will do so in the future. thus the cfr by w will be less than the true cfr. this bias will be particularly severe for infections that are increasing rapidly in incidence and for which the infection-death time interval is long. spuriously decreases estimate of cfr sars [9] , influenza h1n1pdm [21] , ebola [22, 23] limit analysis to those cases with sufficiently long follow-up for a death to have been recorded had a death occurred. while this may lead to extremely small sample sizes near the beginning of an epidemic, this strategy is more feasible after a local epidemic wave, including reporting delays, has passed or nearly passed [10, 11] . limit analysis to those cases known either to have died or recovered, but exclude those with unknown outcome (biased if severity affects outcome ascertainment) [22] [23] [24] . apply a competing-risk kaplan-meier-like method or a parametric mixture model to the full dataset (biased if the times to death and time to recovery have different distributions) [24, 25] . fit the distribution of times to death and to recovery to estimate the true cfr [10, 11] , or inverse-probability weight deaths using the conditional probability of having survived by w, given that one dies [21] (biased if the probability distribution is incorrect). a risk difference (rd), the difference between the cfr in group a and group b. we use the term relative cfr to refer to any of these measures, and call a relative cfr non-null when it differs from 1 (ratio) or 0 (difference). when these biases are present, a relative cfr, different from the null value in group b compared with a does not imply a causal effect of group. for example, if group a is non-hospitalized patients and group b is hospitalized patients, an odds ratio of death less than 1 may not imply a beneficial effect of hospitalization on the odds of death. similarly, a relative cfr greater than 1 may not imply that hospitalization is harmful. we use the estimation of the causal impact of hospitalization on mortality as our example throughout this section. note that exactly the same reasoning applies to assessment of another intervention or to a comparison of two interventions, for example, a comparison of treatment at center a versus treatment at center b. the first bias arises in a naïve comparison of mortality between those who have and those who have not been hospitalized. if some individuals die before they can be admitted to a hospital, they will by definition not become hospitalized. therefore, even in the absence of any effect of hospitalization on the risk of death, there will be fewer deaths among those hospitalized than among those not hospitalized. we will refer to this bias as "survivorship bias." in an ongoing epidemic, there will typically be a delay between the reporting of a case and the reporting of the death of that case, if the infected person dies. thus, at any moment, there will be some cases reported who will die of the infection but who have not yet died, or whose deaths have not yet been reported. simple division of the number of deaths reported by week w (green), by the number of cases reported by week w (blue) will underestimate the cfr because the numerator does not include all those cases in the denominator who will eventually die. with a reporting delay of 3 weeks for deaths compared to cases, the reported deaths curve will be shifted 3 weeks to the right, relative to the curve of the total number of cases reported by week w who will die (red). if the epidemic doubling time is 2 weeks, as shown here, the underestimate of cfr will be by a factor of about 2 3/2 2.8, with the exponent being the number of epidemic doubling times that pass between case reporting and death reporting. in reality, there will be a distribution of reporting delays rather than a fixed delay, making this a heuristic rather than exact approach. the problem is ameliorated in an epidemic that grows more slowly or less than exponentially. for more details, see references in table 1 . this bias can be eliminated using data on the time d since the person became a case. the analysis would then compare the risk of death between those individuals who became table 2 . potential biases that can affect the comparison of cfr across groups (relative cfr), using the example of comparing the cfr among hospitalized and non-hospitalized persons to assess the relative cfr for hospitalization. direction outbreaks in which analysts have noted this bias may be operating possible solutions/means of detecting the bias survivorship bias: those who die before being hospitalized cannot, by definition, be hospitalized; a crude comparison of deaths among hospitalized and non-hospitalized cases will therefore reflect the "protective" effect of death against hospitalization. this is an example of reverse causality because for these individuals, death prevented hospitalization, rather than hospitalization preventing death. spurious protective effect of hospitalization on risk of death ebola (this article) conditioning analysis on survival up to day d of symptoms, and analyzing hospitalization on day d as the intervention, will avoid this bias, as individuals who die before hospitalization will not be included in the analysis. this analysis can be repeated for different values of d and potentially combined in a parametric model. individuals identified before becoming cases (e.g., as healthy contacts of infected persons) and actively followed regardless of clinical severity could be analyzed separately as a prospective cohort for whom the course of disease could be observed and this restriction readily made. confounding: if individuals are hospitalized in response to predictors of poor prognosis, hospitalization will be noncausally associated with poor outcome. this problem is common in the pharmacoepidemiology literature [27] . alternatively, in situations of triage, when beds or other resources are limited, individuals with better prognosis may receive hospitalization (or another intervention), creating a spurious beneficial effect of hospitalization. may be in either direction, depending on whether those receiving the intervention have better or worse prognosis. ebola (this article), h1n1pdm (effect of antiviral treatment on death) [28] , influenza h5n1 (effect of antiviral treatment on death) [29, 30, 31] in principle, analysis can adjust for prognostic factors that also predict hospitalization via matching, stratification, or multivariable analysis. in practice, such information may be unavailable [27] . such adjustments will be more readily made if data are obtained prospectively from a cohort of cases identified before becoming cases. selection bias occurring because mortality and hospitalization both affect the probability a case will appear in the database [32] : when inclusion in a database can occur as a result of either of two (or more) factors, the association between these two factors within the database will be biased relative to that in the source population. for example, if death and hospital admission are both means by which cases are ascertained and enter a database, as may be the case for ebola datasets, hospitalization will be spuriously associated with death in the dataset even if hospitalization has no causal effect in preventing death. direction of bias depends on the probabilities of inclusion in the dataset depending on exposure and outcome. ebola (this article) without knowledge of how cases came to enter a dataset, the magnitude of this bias cannot be evaluated. under assumptions about the proportion of cases entering the dataset for various reasons, a sensitivity analysis could be performed to assess the plausibility of assigning any observed protective effect to this bias [33] . this bias too may be avoided by prospectively following a cohort of individuals who are identified before becoming cases. the second source of bias is confounding. severity of disease will likely affect the probability of hospitalization and the probability of death. as a common cause of the exposure of interest (hospitalization) and the outcome (death), disease severity is a confounder of the causal effect of hospitalization on death. if hospitalization is offered to especially severe cases or-in the setting of extreme triage-to especially mild cases, then hospitalization would spuriously appear harmful (if hospitalization went to especially severe cases) or beneficial (if it went to especially mild cases). there may be other confounders of this effect besides disease severity. individuals living in rural areas may be at greater risk of mortality (e.g., due to malnutrition) and also less likely to be hospitalized (due to longer travel time to hospital). place of residence (or travel time to hospital) in this setting would be a confounder of the effect of hospitalization on death. the standard approach to reducing confounding is to stratify, restrict, or adjust for prognostic factors that affect the propensity to receive the treatment (in this case to be hospitalized) [27] . however, such information may frequently be limited or unavailable in databases compiled during outbreaks, especially in resource-limited settings. the third source of bias is selection occurring because mortality and hospitalization both affect the probability a case will appear in the database. during an outbreak, many cases may not appear in the database because they are not ascertained or because information about them is not obtainable. in particular, cases who are not hospitalized, and cases who do not die, may be less likely than other cases to appear in the database because they are less likely to come to medical or public health attention. if appearance in a database is the common effect of hospitalization and death, then the association between hospitalization and death among cases in the database may be non-null even if hospitalization and death were independent in the population of all cases. the direction and magnitude of the association between hospitalization and death among cases in the database will then be the result of combining the association due to this selection bias, the association due to a potential effect of hospitalization on mortality, and the association due to confounding. hypothetical examples are shown in tables 3-5 . in these tables, the association in the population between hospitalization on day 8 (an arbitrarily chosen day) and death is negative; individuals hospitalized on day 8 (an arbitrarily chosen day) of symptoms have a lower probability of death than those who are not hospitalized on day 8 of symptoms. if we assume that this analysis has avoided survivorship bias by limiting analysis to cases still alive on day 8, then the population-level association would reflect a combination of the causal effect of hospitalization on day 8 on risk of death, and confounding by severity or other factors. this population-level association is the same in tables 3, 4, and 5, but different probabilities are assumed for inclusion in the database, depending on whether an individual is hospitalized on day 8, dies, or both. relative cfrs on the rr, or, and rd scales for hospitalization on day 8 are calculated for each hypothetical example. the hypothetical data in these tables show that selection bias in such a circumstance may be either positive or negative on each of the three scales, depending on the specific probabilities of selection in each of the four states. table 3 shows an example of negative bias on the rr, or, and rd scales (overestimating the protective effect of hospitalization on day 8 expressed as a lower value of each relative risk measure). table 4 shows an example of a positive bias on the rr and rd scales and a negative bias on the or scale. table 5 shows an example of positive bias (underestimating the protective effect of hospitalization on day 8 expressed as a higher value of each measure) on all three scales. from experience, it seems that when databases are assembled in this way, it is rarely possible to tell why an individual case has come into the database. in the absence of such information, it is difficult to imagine how adjustments could be performed. however, sensitivity analyses could be performed to assess how strong such biases are likely to be [33] . we have stated already that survivorship bias can be avoided by limiting analyses of the intervention to those who remain alive on a certain day after becoming a case. one strategy that would help to resolve the other two sources of bias is to limit analysis to a cohort of cases who were identified before they became cases; for example those who were identified as healthy contacts of known cases, and were followed prospectively. confounding occurs because individual factors like severity of infection or place of residence (which could affect both the probability of exposure-receiving the intervention-and the probability of the outcome-mortality) are not accounted for in the analysis through stratification, restriction, or adjustment. selection bias in this setting occurs because the exposure and the outcome both affect the probability of inclusion in the database. follow-up of a cohort of contacts ascertained before becoming cases could eliminate hospitalization and mortality as predictors of inclusion in the database, thus eliminating the form of selection bias we have discussed. it would provide an opportunity for subscript p represents the population values, while subscript d represents the values measured for those cases included in the data base; selection bias produces the discrepancy. the extent of selection bias may be measured as or s ¼ s 00 s 11 s 01 s 10 , where s ij is the probability a case with exposure (hospitalization at day 8) i and outcome (mortality) j appears in the database. in this example, selection bias spuriously enhances the negative association between hospitalization on day 8 and death, on all scales: rr, or, and rd. gathering data on severity and other predictors of exposure and outcome, which would facilitate control of confounding, though not guarantee to eliminate it. such a cohort would also provide a natural setting for analyses that avoid survivorship bias. the cost of such improvements in inference would be the need to ascertain such contacts and maintain surveillance of those individuals, following them to obtain data on relevant covariates. such a strategy-which has been followed in cases of exposed health-care workers in settings with high resources and few cases-would likely have benefits for the individuals followed (e.g., increasing the probability they receive care if infected) and for reducing transmission (if such individuals were promptly isolated upon evidence of infection). however, it has not been possible so far in the large ebola outbreaks in west africa to do this routinely. it is often of interest to predict the probability of mortality for an individual case of an infectious disease based on that individual's demographic and clinical data, without placing any causal interpretation on the factors used to predict outcome. for example, in 2009, there was much interest in whether morbid obesity (or obesity in general) was predictive of worse outcome in infection with the novel pandemic strain of influenza a/h1n1 [34] .the primary goal was to improve estimates of clinical prognosis, although observations about prognosis could later be used to generate causal hypotheses for further testing. similarly, observations of disparate rates of severe outcomes by geography within new york city did not initially involve causal judgments about why certain areas had worse outcomes, although they could be used to guide enhancement of services in areas with worse outcomes [35] . even for a well-understood disease like polio, it may be necessary to identify unusual demographic patterns of mortality in order to understand and respond effectively to an outbreak [36] . prognostic exercises such as these cannot suffer from confounding bias because no causal interpretation is attached to the conclusions. they can, however, suffer from selection bias. returning to the ebola context, one might wish to know whether pregnant women infected with ebola are at greater risk of death from ebola infection than other cases [37] , for example, in order to give them greater supportive care. if the probability of entering the database depends on whether an ebola patient is pregnant and on whether she ultimately dies of the infection, then the probability of death given pregnancy will likely differ in the database from the value in the population of direct interest for a clinical or public health decision maker. if the goal of analysis is to inform public health decision makers on the value of efforts to prevent infection in pregnant women, then the population-wide cfr among pregnant women is the value of direct interest. if, on the other hand, the goal of analysis is to inform health care providers at a treatment center to make a better clinical decision based on an accurate prognosis of the patient presenting to them, the quantity of direct interest is the probability of death among pregnant women in the population they encounter-those admitted to the treatment center. table 5 . effect of selection bias on estimates of relative cfr on the risk ratio (rr) odds ratio (or) and risk difference (rd) scales. joint frequencies of hospitalization and death in the whole population among those alive at day 8 of symptoms this value, again, will differ from that in the database, which may (in our running example) have been enriched for individuals entered in the database because they died of the infection. it will also differ from that in the overall population. the general point is that selection bias can be operative if the population on which analysis is performed is not a representative sample of the population for which the value of the cfr is sought, and selection bias of this form can lead to spurious conclusions in prognostic estimates as well as in causal ones. as in the case of causal inference, prognostic estimates will avoid selection bias to the extent they can be performed on a randomly chosen cohort of cases, identified via tracing of healthy contacts, for example. to determine the appropriate scope and magnitude of public health response to an infectious disease outbreak, it is important to estimate the cfr and the determinants of its variation [1, 5] . for example, in the 2009 influenza pandemic, early point estimates of the cfr ranged over orders of magnitude, from a value below that of seasonal influenza, which would have justified a modest response, to values around 1%, approximately half that of the 1918 pandemic, which would have indicated the need for massive interventions to protect public health [12, [15] [16] [17] 21] . to a large degree, this variation reflected judgments that one or the other of the biases in table 1 was more important, judgments that were difficult to make accurately and confidently on the rapid timescale required for decision making [26] . in other situations, accurate assessment of the cfr is not as crucial for decisions about the scale of response required; for example, in the ongoing 2014 ebola epidemic in west africa, the uncertainty about the cfr is limited to a range between high values and very high values, and it is not clear that any greater response would be indicated by a 90% cfr than a 60% cfr [22] . either way, a rapid and massive response is warranted. even when the overall cfr is not a key input to decision making, there is obvious value to inferences about which conditions lead to a lower cfr, whether these be specific treatment, particular types of supportive care, or hospitalization in general. moreover, treatment facilities might be evaluated by the proportion of their patients who survive; here the relative cfr calculated would be for treatment in one facility versus treatment in another. there will be a temptation to conclude that treatment facilities with higher cfr are doing a worse job-that is, to apply a causal interpretation to observed differences in the cfr. even in settings with more resources to measure covariates, methods of risk-adjustment of comparative outcomes to account for the mix of patients seen are complex and controversial [38] . in an emergency setting, with few covariates available to characterize the "case mix" of a health care provider, causal interpretation of differences in cfr would be particularly prone to error, potentially producing conclusions that mislead and thereby damage control efforts. for instance, if through confounding, larger referral treatment centers primarily receive patients who have survived infection for some time and are therefore less likely to die, independently of treatment, this may be erroneously interpreted as more effective treatment in these centers. similarly, if certain treatment centers preferentially admit the most symptomatic patients, they may falsely appear to be less effective or even harmful to patient outcome. with at least five separate sources of bias in cfr or relative cfr estimates, and only imperfect solutions typically available for most due to lack of data, separating causal from non-causal factors in relative cfr estimation seems extremely risky. this is not to deny that data should be gathered or analyzed; on the contrary, the biases here suggest that more thorough data gathering is necessary before analyses of such quantities as relative cfr are relied upon for any decision. there has been much debate, particularly in the area of ebola treatments, about whether randomized studies comparing a treatment to a placebo are ethical [3, 39] . whatever one's view on this debate, it seems likely that some observational (non-randomized) studies of the effectiveness of particular therapies, or the comparative effectiveness of two or more therapeutic approaches will occur, whether for ethical reasons, logistical reasons, or both. such studies-in which a key endpoint will be mortality-will be vulnerable to the sorts of biases described in this article, particularly in cases in which the true effect size of the treatment is limited. the biases described here should be kept in mind when evaluating the conclusions of such studies, and wherever possible, studies should be designed to minimize them. small studies conducted using systematic approaches to enrollment and follow-up of patients may be more precise and less biased than studies with larger sample sizes that use databases collected for other reasons. similarly, there may be situations in which efforts are made to administer scarce therapeutic agents to those most likely to benefit from them. such efforts rely on estimates, formal or informal, of the prognosis of patients with and without the treatment, depending on variables such as the time since they became symptomatic. these estimates, too, may be affected by the biases discussed. in the current ebola outbreak in west africa, such data gathering has not routinely occurred, for a number of reasons, including lack of health system infrastructure [40] and prioritization of crisis response and other directly lifesaving activities. in future outbreaks of other diseases, as in the past with pandemic influenza, setting up systematic approaches to gather data useful for such assessments should be a priority [1, 5] . meanwhile, emphasis on recording for each patient in a database the time, place, and circumstances (e.g., hospital, clinic, funeral, contact tracing) under which the information is being gathered can substantially improve our ability to account for biases induced by a database with unplanned entry criteria. to reduce the impact of the biases identified on causal and (where applicable) prognostic inference, it appears desirable when possible to limit analysis to a subset of cases who have been followed prospectively since they became cases. these individuals might most likely be identified by forward contact tracing, in which cases are asked to name healthy individuals with whom they have had contact, and those individuals are followed to identify further infections. it has previously been noted that cases identified by contact tracing are more representative of cases in the general infected population than those identified because of symptoms, medical need, or death [20, 41] . use of such a sample does not guarantee to eliminate biases, as there may be residual confounding not adequately controlled in the analysis or subtler forms of selection bias (e.g., differential loss to follow up within the sample) [32] , but should significantly reduce them. we have emphasized the relevance of several biases to interpretation of datasets gathered in an emergency, such as the early phases of an emerging infection. while the downward bias in estimation of the cfr due to delayed reporting of deaths is most acute in rapidly growing epidemics, the other biases described may apply regardless of the overall trajectory of an epidemic, and thus may apply to endemic diseases as well as emerging ones. nonetheless, due to the sense of urgency to gather data and scale-up a response simultaneously, datasets assembled during infectious disease outbreak or emergency settings are especially prone to include unplanned mixes of cases who enter the dataset for various reasons. biases of the sorts described here should be systematically considered whenever one attempts to extract causal inferences from such observational data, and alternative, more systematic data collection should be considered when possible. • datasets available at the onset of new epidemics of infectious diseases are often collected for reasons other than epidemiologic analysis of absolute and comparative casefatality risks (cfr), and estimates of such quantities based on these data may be subject to biases, the relative magnitudes of which are difficult to ascertain and vary by situation. • major sources of bias affecting the estimation of absolute cfr are differences in severity between all cases and the subset of cases who enter the dataset, typically leading to inflated estimates of cfr, and more rapid reporting (less delay) in reporting cases than in reporting the deaths of those cases, typically leading to underestimates of cfr. • biases affecting the causal interpretation of relative cfr (causal attribution of different cfr in different groups to a particular intervention in one group, e.g., hospitalization) may arise from survivorship bias, in which individuals who survive longer may be more likely to receive the intervention; from confounding, in which a common factor (e.g., disease severity) affects the probability of both the intervention and mortality; and from selection bias, in which individuals are more or less likely to enter the dataset as a function of whether they receive the intervention and whether they have the outcome. • these biases may be severe enough to lead to qualitatively mistaken inferences about the severity of the infection or about the impact of interventions (such as hospitalization) on mortality, and may be particularly misleading when comparing, for example, the effect of hospitalization at different centers, given that cases hospitalized at different centers may enter the dataset for different reasons. • methods exist to identify and reduce these biases. in particular, the use of small but carefully defined cohorts of individuals who are followed from the time of infection or symptom onset (perhaps those identified via contact tracing) may ameliorate many of these biases. epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong basic methods for sensitivity analysis of biases assessing the severity of the novel influenza a/h1n1 pandemic a structural approach to selection bias h1n1 surveillance group. improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 studies needed to address public health challenges of the 2009 h1n1 influenza pandemic: insights from modeling interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early targeted layered use of nonpharmaceutical interventions. department of health and human services randomised controlled trials for ebola: practical and ethical issues middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 human infection with avian influenza a h7n9 virus: an assessment of clinical severity seroevidence for h5n1 influenza infections in humans: metaanalysis comment on "seroevidence for h5n1 influenza infections in humans: meta-analysis epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the severity of pandemic h1n1 influenza in the united states changes in severity of 2009 pandemic a/h1n1 influenza in england: a bayesian evidence synthesis pandemic influenza a(h1n1)v in new zealand: the experience from under-reporting and case fatality estimates for emerging epidemics notes from the field: outbreak of 2009 pandemic influenza a (h1n1) virus at a large public university in delaware pandemic potential of a strain of influenza a (h1n1): early findings use of cumulative incidence of novel influenza a/h1n1 in foreign travelers to estimate lower bounds on cumulative incidence in mexico optimizing the precision of case fatality ratio estimates under the surveillance pyramid approach estimating infection attack rates and severity in real time during an influenza pandemic: analysis of serial cross-sectional serologic surveillance data the infection attack rate and severity of 2009 pandemic h1n1 influenza in hong kong transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart assessing the severity of the novel influenza a/h1n1 pandemic ebola virus disease in west africa-the first 9 months of the epidemic and forward projections case fatality rate for ebola virus disease in west africa methods for estimating the case fatality ratio for a novel, emerging infectious disease non-parametric estimation of the case fatality ratio with competing risks data: an application to severe acute respiratory syndrome (sars) managing and reducing uncertainty in an emerging influenza pandemic assessment and control for confounding by indication in observational studies effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data effectiveness of antiviral treatment in human influenza a(h5n1) infections: analysis of a global patient registry strengthening observational evidence for antiviral effectiveness in influenza a (h5n1) determinants of antiviral effectiveness in influenza virus a subtype h5n1 a structural approach to selection bias basic methods for sensitivity analysis of biases risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis pandemic (h1n1) 2009 surveillance for severe illness and response investigation of elevated case-fatality rate in poliomyelitis outbreak in pointe noire ebola hemorrhagic fever and pregnancy use of risk adjustment in setting budgets and measuring performance in primary care ii: advantages, disadvantages, and practicalities evaluating novel therapies during the ebola epidemic ebola: the teaching and learning moment household transmission of 2009 pandemic influenza a (h1n1) virus in the united states we thank lina nerlander for helpful suggestions on an earlier draft. key: cord-006924-1i3kf01j authors: nan title: abstracts from uscap 2020: pulmonary, mediastinum, pleura, and peritoneum pathology (1869-1980) date: 2020-03-05 journal: lab invest doi: 10.1038/s41374-020-0400-0 sha: doc_id: 6924 cord_uid: 1i3kf01j nan conclusions: a panel of immunostains including bap1, mtap, cd5, cd117 and tdt can be useful in the distinction between thymomas and thymic carcinomas with only a minority of cases being inconclusive. feasible markers of cellular senescence on paraffin but the expression of p16 protein and a low ki67 are surrogate markers of that cellular stage. sclerosing pneumocytoma (sp) is a benign tumor with a combination of patterns and a characteristic dual population of surface cells and round cells. the first express ttf1 and ck and the last only ttf1. no driver mutations have been described in this lesion. we reviewed the histological characteristics and immunohistochemical findings of 4 ba from 3 patients and compared it to 1 sp and 1 adk (from the patient with 2 ba). the antibodies used were: ttf1 (ventana, clone 8g7g3), ki67 (ventana, , p16 (cintec, e6h4) and p40 (ventana, bc28) . we also performed next-generation sequencing (ngs) (thermofisher, panel oncomine solid tumor) for all the ba and the adk. results: all 4 ba displays high expression of p16 protein in over 80% cells and a proliferative index below 3%. one of the ba contained round cells ck-and ttf1+, similar to round cells in sp. sp, sp-like cells and adk did not show a significant p16 expression. see we have identified a sp-like component that has not been described in classic ba. ba may represent another kind of benign tumor with senescent features and oncogenic alterations, similarly to nevus or pylocitic astrocytomas. these findings can be relevant in the differential diagnosis with adenocarcinomas as well as in the understanding of the malignant transformation in lung tumors. further research in a larger number of cases is needed. conclusions: in summary this study of selected images suggests that the distinction between stas and artifacts is achievable and reproducible. further work on reproducibility is needed using glass slides and frozen sections. these data suggest that these originally proposed criteria to make the distinction between artifacts and stas are readily applicable. this likely has helped the many independent investigators who have performed studies demonstrating the clinical significance of stas. design: a retrospective multi-center retrieve was performed for a ten-year period. ninety cases of mm, 50 cases of non-small cell lung adenocarcinoma (nscla) and 50 cases of breast carcinoma (bc) were retrieved from the pathology archives. all slides were reviewed and reclassified by expert pathologists. a tissue microarray (tma) was built from three sections of each case, resulting in 570 cores. immunofluorescence (if) was performed with a col (v) in-house clone (described by atayde et al, plos 2018) . images were captured through a microscope camera and the percentage of the positive area was accessed by threshold properties in the imagej software. patterns of deposition were defined as fibrillar (a linear pattern in fine bundles of intercellular deposition), surrounding (a membrane pattern, surrounding and isolating each cell) and mixed. for the mm tma, a double col (v) d2-40 stain was also performed to enhance mesothelial cell specificity. statistical analysis was performed using spss 25 by anova followed by spearman's test. a p-value of less than 0.05 was considered significant. the analysis was possible in 419 spots (74% of total):126 nscla (84%), 102 bc (68%), 229 mm (70%). the fibrillar type was expressed in 117 (93%) of nscla, 100 (98%) of bc and 9 (0.05%) of all spots of mm. the surrounding pattern was expressed in 182 (95%) of mm spots (p-value <0.05). the mixed pattern was expressed in 9 (7.7%) and 2 (1.2%) of nscla and bc spots, and when present, fibrillar dominated. average col (v) expression was: 3.58% (nscla), 12.1% (bc), and 10.65% (mm). lung expressed significantly less than the other groups (p<0.05). than that of patients with zeb1-negative tumors (781 days and 1798 days, respectively; p = 0.008). among epithelioid tumors, median survival of patients with zeb1-high tumors was significantly shorter than that of patients with zeb1-low tumors (476 days and 1798 days, respectively; p = 0.001). conclusions: as expected, biphasic morphology was associated with zeb1 staining and poorer survival. zeb1-positive epithelioid tumors had shorter median survival. furthermore, strong and diffuse positive staining in epithelioid mesotheliomas was associated with worse prognosis. use of this immunohistochemical stain may help to risk-stratify patients with epithelioid mesotheliomas, even in the absence of sarcomatoid morphology. results: using the above criteria, we identified 19 cases, of which 11 were diagnosed as lam on histology. all cases were females, ranging from 25-62 years, with an average age of 44 years (table 1 ). 1. the vegf-d levels ranged from 85 to 794 pg/dl. no case was above the present diagnostic cut-off of 800 pg/ml. all 7 cases with vegf-d levels >400pg/ml were diagnosed as lam on histology. 7 out of 9 cases with vegf-d levels <400 pg/ml did not have lam. (ppv=100%, npv=77.7%) 2. all positive cases had a typical radiological findings of multiple, diffuse, bilateral small cysts, usually <1cm. radiological findings in cases not diagnosed as lam were variable, including predilection for lower lobes, coexisting nodular lesion, varying size cysts, etc. 3. cases where the radiology was characteristic but histologically not proven to be lam, were noted to have vegf-d levels of <300pg/ml age sex vegf-d (pg/ml) radiological findings histopathology it is important to correlate radiological findings and vegf-d levels when considering the need for a surgical lung biopsy, as those cases with non-characteristic radiological findings and vegf-d <400pg/ml were unlikely to be diagnosed as lam on histology. considering that all patients with biopsy proven lam had a vegf-d levels of greater than 400 pg/ml, we speculate that this level might be used as new cut-off for triaging patients with cystic lung disease for a biopsy, in an appropriate clinical setting. lorelle brownlee 1 , robert bentham 2 , nicholas mcgranahan 3 , charles swanton 4 , david moore 2 , mariam jamal-hanjani 2 , tracerx consortium 5 results: 45 patients were identified. on fu (mean 31 months) 6 cases (13.3%) had a final diagnosis of malignancy (2 adenocarcinomas, 1 squamous cell carcinoma, 1 malt lymphoma, 1 dlbcl, 1 nsclc). of these, 3 were diagnosed on repeat cnb, 1 on wedge resection after repeat benign cnb and 2 on fu fna. other 11 cases (24.4%) with repeat biopsy and 2 cases (4.4%) with fu resection were benign (1 wedge resection and 1 lobectomy, both nodular lymphoid hyperplasia). all nodules were solid (86.7%) or part-solid on ct. most benign and all malignant cases had spiculated (24.4%) or irregular margins (55.1%). initial biopsies in malignant cases had either inflammatory (5/6) or lymphoma-like pattern (1/6). all cases with scar-like pattern (7/45, 15.5%) were benign. shorter solid lesional component on cnb correlated with final benign diagnosis (p=0.01; solid component of all malignant cases measuring ≤10 mm). cases with final malignant diagnosis also had cnb with shorter lesional tissue and higher suvmax, which approached statistical significance despite low number. conclusions: combined radiological-pathological approach may improve the diagnostic accuracy of patients with focal lung lesions and non-specific inflammation on initial cnb. good representation of the solid component of the lesions is associated with final benign diagnosis and should be considered when sampling and making management decisions. matthew cecchini 1 , tara tarmey background: many forms of interstitial lung disease (ild) are diseases of aging secondary to repetitive injury or exposures that causes an abnormal cellular senescence. a subset of ild occurs in patients with short telomeres who can also have premature greying of hair, liver fibrosis and bone marrow failure. cells with critically shortened telomeres can enter into a cellular senescence mediated in part by upregulation of the cdk inhibitor p16. despite our increasing understanding of the underlying pathogenetic mechanisms, histopathologic and radiologic features of ilds in patients with confirmed short telomeres have not been well characterized. design: cases diagnosed as positive for short telomeres (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) (2019) were identified by testing of peripheral blood granulocytes and/or lymphocytes with 10 th percentile of telomeres or less. lung explants or wedge biopsies from the patients with short telomeres were reviewed independently by 2 pathologists using defined morphologic parameters. discordant cases were reviewed by a third pathologist. high resolution ct scans were reviewed by 2 radiologists independently. immunohistochemistry for p16 (clone e6h4) was performed on a section of each case showing the most areas of active fibrosis with abundant interfaces between fibrotic and architecturally preserved lung parenchyma. all foci showing p16 positivity in both epithelial cells and associated fibroblasts were counted. cases without short telomeres were used as controls. the morphologic and radiologic features of cases with short telomeres (n=15) as compared to those without short telomeres (n=5) are outlined in table 1. among 8 cases with short telomeres that were tested for mutation, 5 (62.5%) cases had a germline mutation in a gene related to telomere maintenance (naf1, tert, rtel1, terc). the cases in the short telomere group often demonstrated features atypical for usual interstitial pneumonia (uip) on both histopathologic and radiologic examination. nine of 15 (60%) cases with short telomeres were classified as non-uip, while 3 of 5 (60%) without short telomeres were diagnosed as uip. the average number of p16-positive foci was higher in the cases with short telomeres though not statistically significant (p=0.4) ( table 1) . the majority of ilds in patients with short telomeres showed morphologic and radiologic features atypical for uip and were often diagnosed as chp or unclassifiable ild. both groups with and without short telomeres demonstrated p16-positive foci. we have recently reported on the utility of comprehensive next-generation sequencing (ngs) for distinguishing separate primary lung carcinomas (splc) from intrapulmonary metastases (ipm) in clinical practice. here, we report on detailed review of histologic challenges in determining the relationships between multifocal adenocarcinomas using ngs as a gold standard. design: a total of 70 surgically-resected adenocarcinoma pairs underwent molecular profiling using 341-468 gene hybridization-capture based ngs assay. comparative genomic profiles were used to stratify tumor pairs into clonally-unrelated (splc) and clonally-related (ipm). relationship of tumor pairs predicted by prospective histopathologic assessment was compared with ngs-based classification. histopathologic features contributing to challenges in distinguishing tumor relationships were assessed retrospectively. of 70 adenocarcinoma pairs, ngs classified 47 as splcs and 23 as ipms. prospective histologic prediction was discordant with ngs in 16 cases (23%), with significantly higher histologic misclassification rate for ngs-confirmed ipms than splcs (43% vs 13%, p=0.0003). the discordance rate was significantly higher when histologic prediction was regarded as potentially equivocal by a pathologist and confirmation by ngs was suggested (p=0.02). retrospective review highlighted several specific factors contributing to misinterpretation of ngs-defined ipms as morphologically unrelated tumors, including 1) morphologic progression leading to higher proportion of solid and micropapillary patterns in secondary tumors (n=6), and 2) presence of significant amounts of non-predominant lepidic pattern in both primary and secondary tumors (n=4). ngs-defined splcs that were initially misinterpreted as morphologically related tumors (n=6) showed closely overlapping architectural or cytologic features. comprehensive histopathologic assessment is adequate for distinguishing splcs from ipms in most cases, but has notable limitations in the recognition of a subset of cases, particularly ipms. our results support the adoption of molecular testing to supplement histologic assessment for robust discrimination of clonal relationships of multifocal adenocarcinomas in clinical practice, and we propose an algorithm incorporating specific histopathologic scenarios where molecular profiling may be most helpful. background: malignant mesothelioma (mm) is rare but lethal; some affected individuals develop mm in the setting of predisposing inherited mutations. our group found a 12% prevalence of pathogenic or likely pathogenic variants in prospectively tested mm patients. these patients showed longer survival after platinum therapy, suggesting distinct biology. germline mutated bap1 mm, the most prevalent mutated gene in mm, appears to be more indolent than wild-type. this study aims to compare the morphology of epithelioid (e-) mms with germline mutations to those without. design: eligible patients with pathologically confirmed mm underwent panel-based hereditary cancer susceptibility germline genetic testing. there were two control groups with no germline mutation matched by mm histologic subtype, age at diagnosis, and sex; 22 patients had retained bap1 and 9 had loss of bap1 on ihc. in e-mm, nuclear grade (3-tiers comprised of mitotic count and nuclear grade), presence of necrosis (yes/no), and patterns were compared using graphpad prism via fisher's exact tests. results: of 265 mms tested, 25 had germline mutations; an additional 5 cases were from other institutions (n=30). pathology was unavailable in 7. of the 23 left (table 1) , there were 22 e-mm and 1 biphasic mm with 10 gene variants. bap1 was most common (n=9). among all mms with a nuclear grade of 1 (n=79), 15 (19%) had a germline mutation. the e-mms had a lower nuclear grade than those without (p=0.01). the mitotic count was a significant contributor to lower grade (p=0.03). by site, there was a trend in lower grade for peritoneal mms (n=13) between germline mutated and non-mutated cases (p=0.06); that difference was not observed for pleural mms (n=9, p=0.24). when focusing on bap1, lower nuclear grade was also present when comparing bap1 mutated mms to sporadic bap1 ihc loss mms (p=0.05) and bap1 ihc retained mms (p=0.05). there was a trend of lower mitotic count in the bap1 germline mutated cases compared to the bap1 ihc loss and the retained cases (p=0.16, p=0.13). there was a trend of less necrosis in bap1 germline mutated mms vs mms with retained bap1 (p=0.11). solid and trabecular patterns were most frequently observed in both study cases and controls. background: lung cancer has been the most prevalent and the most deadly malignant cancer globally, which is still incurable disease in essence. immunotherapy targeting pd-1/pd-l1 represents a breakthrough in the treatment of lung cancer. nevertheless, the response rate of anti-pd-1/pd-l1 immunotherapy remains unsatisfactory, due to tumor resistance and complexity of immune microenvironment. to enable more patients to benefit from immunotherapy, a thorough understanding of the regulatory mechanisms of pd-l1 expression will be pivotal for novel combinational immunotherapies. pyruvate kinase m2 (pkm2) is a critical player of glycolysis, conducing to tumor progression and immune response. however, the correlation and clinical significance of pkm2 and pd-l1 expression in human lung adenocarcinoma (luad) remain not entirely explored. design: expression of pkm2 and pd-l1 were detected by immunohistochemistry in 74 cases of luad and the corresponding noncancerous tissues. simultaneously, multiplex immunofluorescence was used to detect pkm2, pd-l1, cd3, cd68, cd163 and pan-ck by using the opal 7-color ihc kit, combined with multi-spectral imaging system and inform software. we measured expression patterns and co-localization of these targets, evaluating their correlation with clinicopathological features and overall survival. validation of findings was conducted using mrna expression data from the cancer genome atlas (tcga) of 515 lung adenocarcinoma cases. results: co-expression of pkm2 and ck, cd3, cd68 were found. pd-l1 protein expression was detected in the tumor cells and immune cells including t cells and tumor associated macrophages (cd68+cd163+). high expression of pkm2 in tumor cells (tcs) was significantly related to lymph node metastasis and tnm stage. moreover, pkm2 expression in tcs was positively correlated with pd-l1 expression in tcs. high expression of pkm2, pd-l1, as well as both pkm2 and pd-l1 in tcs and immune cells predicted high mortality rate and worse survival, respectively. additionally, multivariate cox regression models indicated that high expression of pkm2 in tcs was an independent prognostic factor. based on tcga genomic data, high pkm2 mrna expression was significantly associated with poorer survival. background: immunotherapeutic agents have revolutionized the standard of care in patients with advanced non-small cell lung cancer (nsclc) and have emerged as novel treatment strategies as they have demonstrated promising treatment advantage over traditional chemotherapeutic agents. immunohistochemical (ihc) detection of pd-l1 expression in nsclc cases is considered to be a clinical decision-making tool to support the use of checkpoint inhibitors in nsclc patients. fda-approved assays and other antibodies are commercially available. this validation study is designed to compare a commercially available assay (28-8) with another clone (cal10), and to also determine the distribution patterns of expression of pd-l1 and pd1 in the tumor and tumor-associated environment. design: 134 consecutively diagnosed nsclcs with available whole tissue sections were evaluated for the study, which includes testing with the fda-approved pd-l1 assay 28-8 (clone 28-8; agilent/dako), pd-l1 clone cal10 (biocare medical) and pd1 clone (nat105). whole sections were chosen over tissue microarrays to adequately assess intra-tumor heterogeneity. deparaffinized tissue sections were pretreated using either online (for the two non-fda approved tests) or offline (for 28-8), followed by antibody incubation, polymer detection and dab visualization. for the 2 pd-l1 assays, the 2019-revised fda tps scoring criterion of ≥1% was employed. for the pd1 assay, immunoe cell score was employed. results: 50% of tumors (n = 67) showed tps positive expression of ≥1% using 28-8 as a gold standard assay for pd-l1. agreement between 28-8 and cal10 was 100%, with a consistently higher tps score observed with cal10 compared to 28-8. immune cell score of nat105 anti-pd1 antibody was positive in 8% of tumors (n = 12). when comparing the distribution of pd-l1 and pd1 expression on the same tumor, out of the 12 pd1 positive cases, 4 showed positive pd-l1 expression, and 8 showed a mutually exclusive expression with only pd1 positive expression. the validation study shows that pd1 and pd-l1 expression on the same tumors in nsclc is uncommon, a phenomenon that can be exploited to expand targeted therapy treatment strategies in this setting, and could possibly be expanded into other oncology settings. it also demonstrates the slightly higher sensitivity of detection achieved by clone cal10 compared to 28-8 using the tumor proportion score, which warrants an expanded evaluation. swi/snf complex, sensitizes smarca4-mutant lung tumors to chemotherapy (fillmore, nature 2015). however, the expression of ezh2 in smarca4-mutant lung tumors is unknown. we reviewed a cohort of 282 thoracic specimens sampled between 2014-19 that previously underwent next generation sequencing (ngs) of over 320 cancer-associated genes as part of routine clinical care. smarca4 frameshift, nonsense and splice site mutations were defined as loss of function (lof). ezh2 immunohistochemistry (ihc) was performed and expression was quantitatively assessed on tumor cells based on the extent of staining as follows: loss (0), weak (1+), moderate (2+) and strong (3+). a cohort of 5 lung carcinomas without smarca4-mutations were also stained as controls. we identified 13 patients (mean age 62, range 42 to 81 years, 64% male) with smarca4-mutant lung cancers (incidence 4.6%). lof mutations were present in 8 (62%) cases and included 3 nonsense, 3 frame shift and 2 splice site variants. two additional cases had smarca4 duplications. all but two cases (92%) were lung adenocarcinomas, with 1 poorly differentiated non-small cell carcinoma and 1 sarcomatoid carcinoma. all cases with lof mutations exhibited a solid growth pattern and 40% presented as distant metastatic disease. tumor tissue was available for 8 cases; ezh2 was expressed in 100% of cases with confirmed smarca4-mutations (mean 2.5 intensity). of 5 smarca4 non-mutated tumors, only 1 tumor demonstrated ezh2 expression (mean 0.2 intensity, p<0.01) conclusions: ezh2 is overexpressed in smarca4-mutant lung cancers and may serve as a potential prognostic and predictive factor for those treated with epigenetic inhibitor therapy. this is the first study to assess ezh2 expression in this subset of tumors that are known to be resistant to conventional chemotherapy. results: oncogenic rearrangements were found in 16/58 samples (28%) with contributive analyses. the most frequent event was a kif5b-ret translocation (6 patients). we also found 4 ros1 cases (cd74 and ezr), 2 nrg1 (slc3a2 and flywh1), 2 egfr (vopp1), 1 alk (eml4) and 1 ntrk1 (egfr). moreover, 4 patients had met exon14 skipping confirmed by the identification of a splice mutation. all but egfr-vopp1 fusions were targetable (18/20, 88%). ret fish was negative or equivocal in 3/6 cases, ros1 fish was negative in 1/4 positive case. alk (5a4) and ntrk1 ihc were negative in both positive cases. patients with fusions were younger than usually described and the sex ratio was close to 1. the proportion of smockers was higher in the group without oncogenic events (p = 0.045). patients without an oncogenic event were more likely to express pd-l1 (p < 0.05). five patients were treated, with partial response. the clinicoradiologic presentation of these four cases are similar to those recently reported in the literature. our small series suggest that vapi is characterized by organizing acute lung injury and should be considered in the differential diagnosis of diffuse alveolar damage of unknown etiology, particularly in younger patients. while foamy macrophages were found in 3 cases, the characteristic features of lipoid pneumonia were not evident on histologic examination. as such, the significance of lipid-laden macrophages on bal requires further investigation. background: congenital pulmonary airway malformations (cpams) are rare developmental malformations of airway branching in the lung, typically diagnosed antenatally. they are classified into 4 subtypes (type 1, type 2, type 3, and type 4) based on their histologic appearance. their origin, natural history, and genetics remain poorly understood as does their malignant potential. a subset of type 1 cpams are thought to possess malignant potential and eventually go on to develop into invasive mucinous adenocarcinoma. these lesions are characterized by areas of mucinous epithelium that can often represent a very small fraction of the total type 1 cpam. it remains to be determined if type 1 cpam lesions with mucinous epithelium are molecularly distinct from type 1 cpams that lack mucinous epithelium. design: a total of 10 type 1 cpams and 10 type 2 cpams with available slides and tissue blocks were identified. each type 1 cpam was assessed for the presence or absence of mucinous epithelium. four cases of type 1 cpam had microscopic evidence of mucinous epithelium and these areas were separately micro-dissected from areas without mucinous epithelium. genomic dna was extracted and used for molecular testing. capture-based next generation sequencing was performed at the ucsf clinical cancer genomics laboratory, using an assay (ucsf500 panel) that targets the coding regions of 480 cancer-related genes, select introns from approximately 40 genes, and the tert promoter with a total sequencing footprint of 2.8 mb results: among the four type 1 cpams that contained mucinous epithelium, a pathogenic hotspot mutation in kras codon12 was detected in all four cases. this pathogenic mutation was detected in both the mucinous and non-mucinous epithelium at roughly similar mutant allele frequency. none of the remaining 6 cases of type 1 cpams that lacked mucinous epithelium or the 10 type 2 cpams contained a kras mutation. results: biopsies with dsa had a statistically significant difference versus nabs with regards to ali (p=0.0421), presence of capillary neutrophilic inflammation (p= < 0.00001), and the presence of asw (p=0.0007). similar to earlier reports, we found a positive correlation between the capillary neutrophils, ali, and dsas. frequent asw was noted in our patients with dsas and was an easily recognized feature at low-power magnification. endothelialitis was not appreciated in any of our cases, likely due to a small sample size. complement 4d (c4d) was available in 16.6 % of specimens and showed only focal staining (<50%). c4d, acute cellular rejection, and airway inflammation did not reveal any significant statistical association with dsa status. conclusions: ali, capillary neutrophilic inflammation, and asw were morphological features found to be statistically significant in this small sample size study. these are not pathognomonic, and currently, the triple test (clinical allograft dysfunction, dsas, pathologic findings) is the best approach for the diagnosis of amr. further research investigating asw, exploring its likelihood to be included among conventional histopathologic patterns prompting clinical and serologic evaluation is needed. figure) , storiform pattern, myofibroblastic proliferation, granulation tissue, maximum nuclear length (defined in relation to lymphocyte size, right figure), nuclear atypia (grades 1-3), mitotic score of 1(0-1/2mm 2 ), 2(2-4/2 mm 2 ) or 3(≥5/2 mm 2 ) and infiltrating inflammatory cells (%). overall survival (os) was determined by kaplan-meier method and compared between groups using log-rank test. results: invasion, bland necrosis, proliferation nodules, nuclear atypia (grade >2), mitotic score (>2), nodular growth and storiform pattern showed 100% specificity (all p<0.001) but variable sensitivity (75%, 67%, 69%, 81%, 88%, 89% and 96%, respectively) for mm. nuclear length was significantly greater in mm than in cfp (median 4.0 vs 3.0x size of lymphocyte, respectively; p<0.0001). myofibroblastic proliferation and granulation tissue were more frequently observed in cfp (69% and 77%, respectively) than in mm (both 19%) (p=0.001 and p<0.001, respectively). percentage of infiltrating inflammatory cells was not significantly different (p=0.3) between mm and cfp (median 10% and 15%, respectively). there were no significant differences in gender (p=0.4), age (p=0.6), stage (p=1) and os (p=0.13) between patients with sarcomatoid mm (n=26) and dmm (n=28, defined as >50% desmoplastic component by who). conclusions: in the distinction from cfp, several features appear specific for mm, among which nuclear atypia, mitotic score, nodular growth and storiform pattern are most sensitive. myofibroblastic proliferation and granulation tissue, while not exclusive to cfp, may be a reliable indicator of benignity in absence of features specific for mm. based on this limited cohort, there appear to be no significant survival differences in patients whose tumors meet who criteria for dmm compared to those with mm showing <50% desmoplastic component. mona farahi 1 , alain borczuk 2 , kartik viswanathan 3 , michael kluger 4 , hanina hibshoosh 5 background: well-differentiated papillary mesothelioma (wdpm) is a rare indolent mesothelial tumor that is usually found incidentally in the peritoneal cavity. there is controversy about the prognosis of the disease with few reported cases of invasive and malignant behaving wdpm. thus, some consider it a tumor of uncertain malignant potential. in contrast, diffuse malignant peritoneal mesothelioma (dmpm) is an aggressive tumor associated with asbestos exposure with a poor prognosis. the distinction between these two entities relies heavily on histopathological findings. prior studies identified traf7 and cdc42 (mutually exclusive) somatic mutations in wdpm. l1 cell adhesion molecule (l1cam) an nf-kb activated gene was overexpressed in wdpm. the goal of the present study is to assess the diagnostic utility of l1cam in a larger cohort of peritoneal mesotheliomas. design: immunohistochemistry utilizing mouse monoclonal anti-l1cam antibody (clone uj127.11, sigma, l4543, 1:1000 dilution) was performed on formalin-fixed, paraffin-embedded tissue sections in 197 total cases consisting of: 41 cases of biphasic/sarcomatoid dmpm, 16 cases of wdpm, 8 cases of benign multicystic mesothelioma (bcm), and 132 cases of epithelioid dmpm. the staining was scored as either positive or negative but since 15% was the minimum percentage to show complete and intense membranous staining among the wdpm, this was chosen as the true positive cutoff. background: adenosquamous lung carcinoma (asc) is a rare yet aggressive malignancy thought to potentially arise from a bipotential undifferentiated precursor cell. a topic of controversy until coined a distinct entity in the 2015 5 th edition of the who classification of lung cancer, asc is defined as a tumor with two separate malignant populations (>10% glandular and squamous components). the coexistence of glandular and squamous differentiation within the same tumor cells has also previously been reported (pelosi, journal of thoracic oncology 2015) and termed "asc immunophenotype" due to its amphicrine phenotypic nature. the single case report showed dual ttf-1 and p40 immunohistochemical (ihc) expression, along with specific electron microscopy and molecular findings. we report here for the first time a case series with dual differentiation and further characterize the findings with molecular analysis. design: from 58 primary lung ascs diagnosed between 2017 and 2019 at our institution, we identified 7 patients (mean age 72.1 years) with dual glandular and squamous differentiation within the same population of malignant tumor cells. ihc stains for ck5/6, ck7, ttf-1, p40, and p16 were performed. the ion ampliseq tm cancer hotspot panel v2 (chp2) and variantplex myeloid kit (archerdx) next generation sequencing (ngs) assays targeting 125 oncogenes were performed on two cases. fluorescence in-situ hybridization (fish) was also performed on the two sequenced cases. ascs with dual differentiation occurred predominantly in women (85.7%, age range 60 to 85 years) with variable smoking history. all cases were poorly differentiated with a solid growth pattern and 43% (3/7) presented with distant metastasis. the following ihc stains were positive in the tumor population: ck5/6 (4/4, 100%), ck7 (3/3, 100%), ttf-1 (7/7, 100%), p40 (7/7, 100%), and p16 (7/7, 100%). ngs revealed cdkn2a and tp53 mutations in both cases; one cdkn2a variant is known to disrupt p16ink4a-and p14arf-dependent regulation of cdk4/6 and p53. loss of function atrx, bcorl1 and notch1 mutations were also identified. fish revealed cdkn2a mutations and relative loss of 9p21 in one case, and chromosome 9 monosomy in the other. we report for the first time a case series of these rare lung ascs with dual differentiation and show an association with cdkna2 mutations and corresponding p16 overexpression. background: there is considerable interobserver variability in the pathological diagnosis of uip and other ild. the variability in finding "ground truth" presents a difficult problem during the development of deep learning platforms designed to diagnose ild using ai. we describe the use of a smartphone application that allows for the collection of multiple opinions from sixteen pulmonary pathologists from nine countries as a novel method for finding "ground truth" for uip diagnoses. design: whole slide images from 14 consecutive interstitial lung disease cases were scanned by aperio cs2 at 20x objective and sliced into multiple individual 7x7mm images, resulting in 355 jpeg image patches. these individual images were randomized and shown over the internet to 16 expert pathologists from 9 countries using the novel smartphone application, bonbon system. expert lung pathologists were asked to classify each image into one of 8 categories: uip/ipf; ctd/uip; chp/uip; uip/other cause; non-uip; "not sure"; "normal"; and "exclude". images classified as "exclude" by any of the experts were deleted from analysis. all individual diagnostic classes selected for individual images were grouped by case, and the predominant class for each case, was used as "diagnosis". all diagnoses were analyzed using clustering analysis and kaplan meier statistics using jmp. interobserver agreement was calculated by fleiss kappa coefficient using r. the diagnosis of each of the 14 cases by each of the 16 pathologists showed that interobserver agreement using the 7 categories was poor (k=0. 19) . none of the cases were classified as chp-uip or uip/others by a majority of pathologists. in order to simplify the diagnoses into more clinically relevant classes, the categories uip/ipf, chp-uip, and uip/others were grouped into uip and non-uip and ctd-uip into non-uip as shown in figure 1 . clustering analysis stratified the diagnoses of each case into 3 clusters as shown in figure 2a (k= 0.77, 0.63, and 0.03, respectively), validating cluster a and b were meaningful. log rank test showed significant survival difference between uip and non-uip groups only for cases in cluster a (p=0.017) ( fig 2b) but not for cases in cluster b (fig 2 c) . eventually, 269 h&e images with high agreement of uip and non-uip group were selected as "ground truth" for ai training. conclusions: classification of image patches from wsi over app offers a useful method to standardize uip diagnosis and to develop diagnostic ai. results: precision, reproducibilty and lod were assessed using hd300 in triplicate by 2 operators on 2 instruments. the 27 ffpe and hd300 were used for accuracy. idylla™ results were in complete agreement with those obtained by ngs for egfr mutations targeted by idylla™ except for one sample. these include l858r (1), g719a (3), g719c (2), l861q (1), s768i (2), l861q (1), t790m (3), exon 19 deletions (16) and exon 20 insertion (1). the discrepant sample had an exon 19 deletion identified by ngs but was wild-type by idylla™. upon review of ngs data, an egfr synonymous snv was present downstream of the deletion which might impair the probe binding, leading to false negative results. lod of 5% vaf and tumor content of 10% were confirmed. no egfr mutations were detected by idylla tm in the samples determined by ngs as having wild-type egfr. the fully automated idylla™ system offers rapid (turnaround time of approximately 2.5 hours) and reliable testing of clinically actionable mutations in egfr directly from ffpe tissue sections. in our center, it will complement ngs testing by providing rapid egfr results within 1-2 days of diagnosis. background: identification of patients with synchronous lung adenocarcinomas has crucial implications for their staging and clinical management. these tumors are thought to represent independent primary neoplasms, but their oncogenesis is still poorly characterized. a few studies based on the analysis of limited panel of genes suggest that synchronous tumors have a high level of genetic heterogeneity. we sought to investigate the genetic mutations in a well-defined cohort of synchronous adenocarcinomas using whole-exome sequencing (wes). design: a retrospective cohort of 34 caucasian patients that underwent surgical resections for synchronous lung adenocarcinomas between 2013 and 2016 in our center was selected. all slides were reviewed by a thoracic pathologist to confirm the diagnosis based on a comprehensive histologic assessment. clinical characteristics and outcome were extracted from the electronic medical files. for each patient, three formalin-fixed paraffin embedded blocks were selected: one from two synchronous tumors and one from normal lung tissue. dna was extracted and samples were submitted to wes on an illumina hiseq platform (agilent sureselect xt). data processing was made according to gatk best practices and somatic variants (single nucleotide variants and insertion-deletions) were called using strelka and mutect. the cohort comprised 44.1% men with a mean age of 65.0 years, 91.2% had a stage i or ii disease and 94.1% were smokers or ex-smokers. mean follow-up was of 38.4 months with an 82.4% overall and 67.7% progression-free survival rates. wes showed an average of 317 called somatic variants per tumor. variants were identified in known driver genes at the following prevalences: kras 35.3%, egfr 8.8%, braf 8.8%, ros1 5.9%, alk 1.5%, met 4.4%, ret 1.5%. synchronous tumors from the same patients showed a high level of heterogeneity, as pairs shared 0 to 12 (0% to 3.3%) variants. all shared variants were likely passenger mutations, but one pair of tumors shared three variants including one in a driver gene, a kras p.g12c mutation. we showed a high level of genomic heterogeneity between two synchronous adenocarcinomas from the same patient using wes, supporting independent primary tumors. one pair of tumors had an identical kras mutation with a high level of genomic heterogeneity, emphasizing the fact that synchronous tumors can share a mutation in a frequent driver gene randomly. andréanne gagné to improve their recognition, the international association for the study of lung cancer (iaslc) recently proposed a classification based on clinicopathologic criteria. it divides msla in three categories: second primary, multiple ground glass opacities (ggo) and pneumonic type. however, the prevalence of these three subgroups remains poorly described and their clinical characteristics have been mostly described in asian cohorts. we aimed to establish the prevalence, clinicopathologic characteristics and prognosis of patients with msla in a caucasian population according to the iaslc criteria. we selected a retrospective cohort of 1012 consecutive patients, including 432 surgical patients, with a diagnosis of lung adenocarcinoma in our center between 2011 and 2014. the cohort was divided according to the iaslc classification: a group of sporadic tumors, comprising patients with one tumor and those with intrapulmonary metastasis (ipm), and a msla group further divided in second primary, multiple ggo and pneumonic type. prevalence of each group was calculated in the whole cohort. chi-square and t-tests were used to evaluate the associations between clinicopathological characteristics and the iaslc groups in surgical patients. overall survival of surgical patients was compared using a cox proportional model. : 143 patients (14.1%) with msla were identified, including 52 (5.1%) second primary, 84 (8.3%) multiple ggo and 7 (0.7%) pneumonic type. age (p=0.74), gender (p=0.38) and smoking status (p=0.77) were not associated with msla. msla patients had significantly more metachronous lung tumors (p=0.006), atypical adenomatous hyperplasia foci (p<0.001) and tumors with lepidic and acinary predominant patterns (p=0.03). there were no significant differences between the msla groups. compared with patients with a single tumor, the multiple ggo group tended to have the best prognosis (hr=0.75, p=0.41) and the second primary (hr=1.97, p=0.02) and pneumonic type (hr=2.33, p=0.41) had worse survival when using a multivariate model. to our knowledge, this is the first report to assess msla prevalence using the ialsc criteria to define patients with multiple adenocarcinomas. even though there were no differences in the clinicopathological variables between the msla groups, their survival disparity supports the iaslc classification. design: histologic slides of pulmonary lobectomies for high grade necs from the 2007-2018 period were reviewed. high grade was characterized by a mitotic count >10 per 2 mm². necrosis was subtyped as massive or punctuate. carcinoid morphology was identified according to the who morphological criteria. tumors with diffuse architecture, nuclear polymorphism and marked nucleoli were classified as "classic" lcnec. proliferation was also assessed with immunodetection of ki67. results: ten cases were available, 4 were classic lcnec, 6 had carcinoid-like features. clinical and pathological data are listed in table 1 . there were no differences between the 2 groups for clinical data. carcinoid-like subgroup had lower mitotic count (mean 28 vs 70), ki67 index (mean 54% vs 84%) and better survival (61 vs 18 months) than classic lcnec respectively. no reliable statistical analysis was allowed due to the small size of our cohort. classic lcnec (n = 4) carcinoid-like lcnec (n = 6) age mean ( conclusions: lcnecs with carcinoid features form a homogeneous subtype with precise morphological criterias. it is rare (0.3% of resected lung tumors in our center), nevertheless its real incidence is probably underestimated. indeed, for sampling reasons, we chose to report a series including only surgical specimens but we have already identified this tumor-type on biopsies from on resectable mediastinal lesions. proliferation markers are lower in carcinoid-like lcnecs than in classic lcnecs. nevertheless, results differ depending on the method of assessment. there is no overlap between the mitotic count values in both subgroups whereas ki 67 index overlaps with no upper limit value in carcinoid-like group. these results rise two questions : 1/ the choice of the method of evaluation of proliferation between mitotic count or ki67, 2/ the place of carcinoid-like lcnec within pulmonary nets classification: an intermediate group between atypical carcinoids and classic lcnecs or a high grade well differentiated net equivalent to that described in the digestive tract. however, the interests of individualizing the carcinoid-like lcnec subgroup are : diagnostic (must be distinguished from atypical carcinoid), prognostic (better than classic lcnec) and probably therapeutic (therapeutic response) jonathon gralewski background: lung cancer is a leading cause of cancer death worldwide. however, the introduction of targeted therapies in recent years has led to improved overall prognosis and survival. current guidelines for lung adenocarcinoma require epidermal growth factor receptor (egfr) exons 18-21 mutational testing. furthermore, it outlines molecular methods, such as next generation sequencing (ngs) with an acceptable turnaround time (tat) up to two weeks. this prolonged tat delays treatment decisions and increases healthcare costs. recently, a fully-automated, cartridge-based platform has been introduced with an ultra-rapid (i.e. <3 hours) sample-to-result tat that does not require traditional sample preparation, such as dna extraction, library preparation, and pcr amplification. design: twenty-one archived formalin-fixed paraffin embedded lung adenocarcinoma including 5 cytology and 16 surgical samples with previously characterized exon 18-21 egfr mutations were selected for this study. a single unstained section of the selected paraffin blocks was macrodissected, placed between filter papers, and subsequently placed into the cartridge and run for 51 egfr variant mutational analysis as per the manufacturer instructions. results were generated in <3 hours without the need for complex bioinformatics analysis and interpretation. results: of 23 previously characterized egfr mutations in 21 analyzed samples, 22 egfr variants were successfully detected by fully integrated ultra-rapid platform (96% concordance). the automated platform failed to identify an egfr g719c variant with allelic frequency of 6% that had been detected using conventional ngs platform. an additional egfr l858r variant was only seen by the cartridge based pcr platform. this variant was re-interrogated on the ngs-based platform and deemed likely a false positive result. conclusions: the cartridge-based fully integrated platform with ultra-rapid tat demonstrated high concordance with the conventional ngs-based platform. the fully-automated and integrated platform has minimized the need for significant molecular expertise and laboratory infrastructure. however, this platform does have its limitations, such as only detecting the most common egfr mutations. overall, this fully automated platform provides an ultra-rapid, and reliable cost-effective method in detecting the most common egfr variants, as outlined in the current guidelines with a detection sensitivity comparable with the conventional ngs platform. nancy greenland background: lung transplant recipients undergo bronchoalveolar lavage (bal) and biopsies to detect rejection and infection that may be antecedents of chronic lung allograft dysfunction (clad), the major limitation to long term survival. bal cytology is routinely performed but recently some centers have advocated abandoning this practice because of the low diagnostic yield. we hypothesized that inflammation observed on bal cytology would predict clad risk. we grouped diagnostic findings on bal cytology between 2012 and 2019. bronchoscopy indication, infection treatment, bal and biopsy results, and clad-free survival were abstracted from medical records. cytology associations with clinical characteristics were compared using generalized-estimating equation-adjusted logistic regression. the association between bal inflammation and clad or death were determined using time-dependent cox proportional hazards models adjusted for age, gender, diagnosis, lung allocation score, and transplant type. we evaluated 3,578 cytology reports from 491 subjects. inflammation was the most common finding (6.4%), followed by fungi (5.2% of which 0.8% were likely pathogenic). there were 5 cases of malignancy and 2 cases of cmv. inflammation on bal cytology was more common in procedures performed for symptoms (11%) versus surveillance (3%, p<0.001), associated with antimicrobial initiation (a proxy for clinically significant infection, 9% vs 5%, p<0.001), associated with acute cellular rejection (p=0.01), and linked to increased bal neutrophil and lymphocyte concentrations (p<0.001). inflammation on bal cytology was present for 32% of subjects on at least one sample, was more frequent around the time of clad onset, and was associated a 2.6-fold hazard ratio (ci 1.2-5.3) for clad or death ( figure 1 ). however, this association was not significant after adjusting for bal cell counts and acute cellular rejection (p=0.22). the presence of inflammation on bal cytology specimens is clinically significant, suggesting acute rejection or infection and increased risk of clad or death. however, other indicators of allograft inflammation can substitute for some of the information provided by bal cytology. hongxing gui background: primary pulmonary myxoid sarcoma (ppms) is an exceedingly rare low-grade lung neoplasm characterized by reticular/lacelike growth of spindle to epithelioid cells embedded in an abundant myxoid matrix. it overlaps with myxoid variant of angiomatoid fibrous histiocytoma (afh) morphologically. in terms of molecular genetics, they both have ewsr1 gene rearrangements, with ewsr1-creb1 fusion in ppms and either ewsr1-atf1 or ewsr1-creb1 fusion in afh. it is unclear whether they are distinctive entities or the same entity with a spectrum of histomorphologies. we evaluated two cases of low-grade myxoid spindle cell tumor of the lung by histomorphology, immunohistochemistry and fish studies. results: case 1 was a 49-year-old man with a 3.8 cm right lower lobe mass extending into bronchus. sections of the mass revealed proliferation of spindle cells in cords, strands and reticular patterns within an abundant myxoid stroma ( figure 1 ). the tumor cells were positive for ema and negative for desmin, ae1/3, cam5.2, ck7, ck20, ttf-1, c-kit, vimentin, cd1a, p63, and s100. case 2 was a 44year-old man with left main bronchial mass. the periphery of the mass was partially encapsulated with a lymphoid cuff. the central portion contained juxtaposed myxoid and nodular components. the former was composed of spindle cells in myxoid matrix and the latter consisted of histiocytic and spindle cells in whorled and storiform patterns ( figure 2 ). the tumor cells from both areas were diffusely positive for desmin and negative for ema, panck, ae1/3, s100, sma, cd34 and alk1. fish analysis demonstrated positive ewsr1 gene rearrangements in both cases, showing ewsr1-atf1 fusion gene in case 1 and ewsr1-creb1 fusion in case 2. we reported for the first time a case of ppms with a novel ewsr1-atf1 translocation, which is usually common in afh. the second case represented a hybrid of jaxtaposed ppms and afh components. these findings provide new evidence supporting that ppms and myxoid afh may represent a continuum with overlapping histologic, immunohistochemical and genetic features. sarika gupta 1 , sagar vishal 2 , anthony snow 3 , andrew bellizzi 1 1 university of iowa hospitals and clinics, iowa city, ia, 2 coralville, ia, 3 north liberty, ia disclosures: sarika gupta: none; sagar vishal: none; anthony snow: none; andrew bellizzi: none background: the differential diagnosis for epithelial tumors in the mediastinum includes carcinomas (ca) of the lung and thymus and thymoma. cd5 and kit are often used to distinguish thymic (+) from lung (-) ca, but these markers are rarely positive in thymoma. prior studies demonstrated frequent polyclonal pax8-positivity in thymoma (≥90%) and thymic ca (67%). we recently switched to monoclonal pax8, which has been shown to be negative in thymic tumors. given prior experience, we hypothesized that polyclonal pax8-positivity in the thymus represents cross-reactivity with another pax-family transcription factor. pax1 is normally expressed during thymic development, and a high-quality monoclonal antibody recently became commercially available. design: pax1 immunohistochemistry (ihc) (clone 5a2) was performed on tissue microarrays of 79 thymomas, 10 thymic cas, and 396 other cas with an emphasis on differential considerations and pax8-positive tumors: 175 squamous cell cas (25 lung); 41 urothelial cas; 36 lung adenocas; 28 renal cell, 25 serous, 22 endometrioid, 22 and 22 papillary and follicular thyroid, 10 breast, 7 colon, 5 esophagus, 2 poorly differentiated neuroendocrine, and 1 prostate ca. intensity (0-3+) and extent (0-100%) of expression was evaluated, and an h-score was calculated. fisher's exact and mann whitney tests were used with p<0.05 considered significant. results: pax1 was expressed by 91% of thymic tumors, including 94% of thymomas (mean/median h-score 198/210) and 70% of thymic cas (mean/median h-score 144/145). the differences in frequency (p=0.08) and h-score (p=0. 19) were not significant. pax1-positivity was noted in only 3.5% of non-thymic cas at a mean (median) h-score of 15 (5) (both p<0.0001 compared to thymic tumors); these included 6 thyroid cas (14%), 4 sccs (2%) [including 2 lung (8%)], 3 urothelial cas (7%), and 1 (10%) breast ca. two of the pax1negative thymic cas were kit-positive; all 3 were cd5-negative. detailed pax1 expression data by thymoma type are presented in the table; pax1 was very frequently, strongly expressed across types. conclusions: pax1 ihc using a novel monoclonal antibody is sensitive and specific for thymic epithelial neoplasms. occasional weak positivity in thyroid tumors may represent low-level cross-reactivity with pax8. this study exemplifies "next-generation ihc," which seeks to apply knowledge from developmental biology and molecular genetics to "intelligently design" novel ihc markers. background: tumor spread through the air space (stas) is an invasive pattern of lung cancer recently described. but there are some debates on its definition, quantification and clinical impact. in this study, we investigated the association between stas grade and clinicopathological characteristics, as well prognostic impact in resected lung cancers. design: stas has been prospectively described from 2008 and it was graded according to the distance from the edge of tumor margin as 2-tier system, i or ii from 2011. correlations between stas grade and clinicopathologic characteristics and prognostic significance were analyzed in 2000 surgically resected lung cancers. conclusions: stas was more frequently found in nets and mp-predominant adcs. it was associated with well-known aggressive features, and stas-gr ii tumors more frequently showed these features than gr i tumors in adcs. in stage ia non-mucinous adc, multivariate analysis revealed that stas grade was independent prognostic factor for rfs regardless of the extent of surgery. moreover, comparable rfs rates were observed in patients with stage ia/stas-gr ii and those with stage ib. 50% (19) 24% (10) 28% (17) tps=tumor proportion score. note that pdl1 high cases are also counted in the pdl1 positive category. *tumors with one of the following: alk fusion, nrg1 fusion, met exon 14 skipping, egfr mutation, or mutation in erbb2, braf, ret, pik3ca, or idh1/2 our data indicate that at least half of ras-driven nsclc have high pdl1 expression, an important consideration for frontline therapy selection. the majority of genomically-actionable tumors have at least low-positive expression of pdl1, highlighting the potential importance of immune checkpoint therapy in the resistant-progression setting. less than one third of genomically-negative cases have high pdl1 expression, and a significant percent are completely negative; additional innovations are needed to better tailor therapy for this patient subset. background: identification of ros1 rearrangements in advanced lung cancer carries therapeutic implications, given the available ros1targeted therapy, but can be technically challenging. immunohistochemistry (ihc) for ros1 show expression in ros1-rearranged tumors, but staining may be weak in some cases and seen in reactive pneumocytes. fluorescence in situ hybridization (fish) using break-apart probes may be difficult to interpret in cases with subtle intrachromosomal rearrangements. next-generation sequencing (ngs) can be useful but requires sufficient tissue and a turnaround time of at least 1-2 weeks. given that these current methods may have drawbacks, this study explores the utility of rna in situ hybridization (rna-ish) in detecting ros1 rearrangements in lung adenocarcinomas. results: using the ros1 rna-ish, all seven (100%) genetically-confirmed ros1-rearranged lung adenocarcinomas showed positivity; whereas none of the controls (0%) was positive. the fraction of cells in ros1-rearranged lung adenocarcinoma showing positivity ranged 60-90% (median 80%). the average number of ish signal dots was far higher in confirmed ros1-rearranged lung adenocarcinomas than controls (average: 29.6 per cell vs. 0.058 per cell). background lung showed minimal ish signal (average: 0.71 per cell). also, the positive ish signals were easily observed and could be seen using 10x objective in all cases or with a 4x objective in 6 of 7 cases. the automated ros1 rna-ish assay appears sensitive and specific in identifying ros1 rearrangements in lung adenocarcinoma, with ease of use for minimally-trained eyes. ros1 rna-ish may be a quick orthogonal tool in confirming ros1 rearrangements in clinically problematic cases. nonetheless, systematic comparison on its performance with that of ros1 ihc and fish may be warranted. on univariate analysis of all mpems, shorter os was significantly associated with year of diagnosis, asbestos exposure, poorer performance status, lymph node metastasis, higher peritoneal disease burden, absence of cytoreduction and hyperthermic intraperitoneal chemotherapy, biphasic or sarcomatoid histotype, and tumor necrosis. on univariate analysis of epithelioid mpem only, shorter os was additionally associated with nuclear pleomorphism, higher mitotic rate, higher composite nuclear grade, and non-tubulopapillary architecture. on multivariate analysis, sarcomatoid and biphasic histotypes predicted shorter os when adjusted for sex, asbestos exposure, and year of diagnosis. on multivariate analysis of epithelioid mpem only, nuclear grade and non-tubulopapillary growth were independently predictive of shorter os when adjusted for sex, year of diagnosis, and tumor necrosis. among patients with epithelioid mpem, shorter pfs after cytoreduction was associated with lymph node metastasis, tumor necrosis, nuclear pleomorphism, higher mitotic rate, higher composite nuclear grade, and solid growth. on multivariate analysis, nuclear grade and solid growth were independently predictive of shorter pfs when adjusted for year of diagnosis, sex, and necrosis. background: there is emerging evidence that vaping can result in significant lung injury, the severity of which can be variable with few cases resulting in the patient's death also been reported. there is limited data on the pathologic findings in vaping-induced lung injury. here, we report histologic and cytologic findings of four patients who developed lung disease following vaping. design: review of pathologic material and clinical information of four patients with a history of vaping (6 specimens, 3 male and 1 female patient, age 16-37 years old). we have identified 4 patients with a history of vaping who presented to the hospital with severe respiratory dysfunction. three of the patients are teenagers, while one is a younger adult and all of them required admission to the icu. the three younger patients had bronchoscopy with bronchoalveolar lavage (bal) with transbronchial biopsy in two. bal showed lipid laden macrophages in all three with acute inflammation in one. transbronchial biopsy showed intraalveolar fibrin with acute inflammation and organization in one of the biopsies. the adult patient is a known chronic alcohol and drug abuser who presented to the ed with dry cough and had subsequent wedge resection showing organizing pneumonia and chronic interstitial changes with the organizing pneumonia attributed to vaping. background: lymphangioleiyomomatosis (lam) is a rare low-grade neoplasm associated with widespread interstitial infiltration of spindle cells and subsequent cystic changes of the lesions. lam is also uniformly distributed in the lungs. target therapies such as mtor inhibitor have been to manage lam but there are no curative therapies at this juncture. lam cells are known to produce vegf-c and vegf-d with their receptor, vegfr3, which promote proliferation of lam cells also present in tumor cells. however, roles of other angiogenic factors have remained unclear. therefore, in this study, we examined the expression of angiogenic factors such as vegfr family and vasohibin (vash) and examined the correlations between these angiogenic factors and histological and clinical findings. design: 36 lam cases were obtained from 32 patients who underwent lung transplantation in tohoku university hospital from 2006 to 2018. we performed hierarchical clustering analysis to classify the cases based on the results of vegfr1, vegfr2, vegfr3, vash-1, and vash-2 immunoreactivity in lam cells. we also immunolocalized vash-1/2, cd31, and d2-40 in microvessels of the lesions. one of these clusters harbored higher vegfr1/3 and lower vegfr2 and vash-1/2, and the patients in this cluster clinically manifested symptom much older and higher p/f ratio was detected at the time of lung transplantation than those in the cluster with higher expression of all of the factors above. the cluster with higher vegfr1/2/3 expression and lower vash-1/-2 expression also demonstrated significantly higher vash-1/cd31 and vash-2/cd31 ratios in microvessels than the cluster with lower expression of all these factors above. however, there were no significant differences of lymphatic vessel strength or other histological characteristics detected in our present clustering analysis. conclusions: angiogenic factors such as vegfr2 and vash-1/-2 influenced on an early onset and progression of the clinical symptoms of lam. in addition, lam cells expressing vegfr1/2/3 promoted angiogenesis. therefore, not only vegf-c/d-vegfr3 axis but also other angiogenic factors may also enhance lam progression. background: micro-computed tomography (micro-ct), is a non-invasive method which allows 3-dimensional morphometric analysis of tissues in formalin fixed paraffin-embedded (ffpe) tissue blocks without any sectioning or loss of sample. lung adenocarcinoma has a major five tissue patterns according to the 2015 who classification (lepidic, acinar, papillary, solid and micropapillary). each tissue pattern has different prognostic indicators and outcomes. in practice, pathologists have to diagnose a predominant tissue pattern and measure each percentage of tissue pattern. the aim of this study is to analyze the structure of lung adenocarcinoma tissue patterns using micro-ct images from ffpe tissue blocks. the ffpe blocks were then sectioned, stained with hematoxylin-eosin (h&e) and scanned to create whole slide images for comparison. design: ffpe tissue blocks from five lung adenocarcinoma cases were scanned using a custom-built micro-ct scanner (nikon metrology) and digitally re-constructed for visualization and analysis using a digital image system. all h&e slides were scanned with 0.5um/pixel by nanozoomer s60 (hamamatsu photonics, japan) whole slide imaging scanner. we then investigated features of each tissue pattern and correlate between micro-ct images and whole slide images as well as histology 3d. results: micro-ct of ffpe blocks highlighted the structure of lung adenocarcinoma ( figure. 1) and normal lung tissue ( figure. 2) in 3d images. we can detect lung adenocarcinoma in a micro-ct image, and detect the tissue pattern, such as lepidic, acinar, papillary, solid, and micropapillary pattern as well as spread through air spaces (stas) (figure 1 ). we could detect the co-relation of normal lung tissue (bronchus, alveolar wall, pulmonary vessels, and visceral pleura) on h&e slides and micro-ct images ( figure 2 ). conclusions: whole block imaging by micro-ct allows for the identification of lung adenocarcinoma tissue patterns in ffpe blocks in a non-invasive and non-destructive manner. correlation between micro-ct images of ffpe blocks and h&e histology images suggests that there is potential for (1) detecting pathologic features without sectioning and staining of the tissue and (2) to measure the volume of each adenocarcinoma tissue pattern including the invasive component in a block accurately. metastasis, respectively, and more frequently associated with recurrence and death (22.6% vs. 1.9%, p<0.001 and 6.5% vs. 1.4%, p=0.05) compared with mp/s-group. survival analysis indicated that mp/s+ and mp/s <5% were associated with shorter recurrence free survival compare with mp/s-(hr=9.1, 95% ci=3.1-26.8, p<0.001; and hr=10.1, 95% ci=2.9-35.5, p<0.001, respectively). mp/s+ and mp/s<5% were more powerful predictor of recurrence than t or n stage in multivariate analyses. even very small proportion of mp/s subtype component was a significant predictive factor for recurrence in surgically resected lung adenocarcinoma. further investigation on the underlying biological mechanism of poor prognostic effect of mp/s subtype is warranted. results: patients' characteristics of 77 cases were as following; median age was 60 years old (range 33-77); 67 male and ten female; 16/16/41/4 of clinical stage i/ii/ii/vi; 21 chemotherapy, 52 chemoradiation and 4 radiotherapy; 52 adenocarcinomas, 18 squamous cell carcinomas and seven other types of histology. mpr was observed in 42 (55%), and pcr in 8(10%). the concordance rate of mpr and pcr assessment among two pathologists was high (96% and 96%). inter-observer agreement was high in mpr (kappa 0.928, p<0.001) and pcr (kappa 0.825, p<0.001). the discrepancy of mpr/pcr was due to the different judgment of tumor bed area and atypical cells whether they are benign or malignant. pathological findings of discrepancy cases had temporal and spatially heterogeneity of fibrosis, active inflammation with reactive stromal and epithelial cell changes. survival analysis will be updated at the time of presentation. our results revealed high reproducibility of mpr, and higher incidence compared to pcr, indicating mpr as a useful method for pathological therapeutic response. background: interstitial lung disease (ild) encompasses a spectrum of conditions with distinct clinical and pathologic features. a subset of ild has been linked to abnormal cellular senescence which can induce a pro-fibrotic senescence associated secretory phenotype that results in progressive pulmonary fibrosis. the senescence is mediated in part by activation of the cdk inhibitor p16 which can arrest the cell cycle and can be used to mark senescent cells. we aim to demonstrate that a distinct subset of ild associated with a senescent phenotype can be identified by expression of p16. design: 70 cases of ild diagnosed with surgical lung biopsy were identified between 2003 and 2018 at a large tertiary-care level hospital ( figure 1 ). additional p16 staining (clone e6h4, roche) was performed on representative sections with the most active fibrosis. p16 positive senescent foci were defined as a loose collection of p16-positive fibroblasts with an overlying p16-positive epithelium and scored as p16-low (0-2 foci/slide) or p16-high (≥3 foci/slide). the diagnosis was verified with the original pathology report and outcome data by time of biopsy to time of death or lung transplant. the presence of any p16-positive senescent foci was highly specific (92%) for the diagnosis of usual interstitial pneumonia (uip). in the uip group there was variable expression of p16 with a range of senescent foci between 0 and 23 per slide with 30 (67%) cases expressing some level of p16 and 20 (44%) cases expressing high levels of p16. comparing cases with high levels of p16 to cases with low to absent p16, there was a reduced survival (hr 2.26; 95% ci, 1.17 to 4.24; p = 0.016) in the p16 high group that was an independent predictor of lung transplant-free survival (figure 2) . in a sub-group analysis of only cases with a diagnosis of uip, p16 status trended towards significance (hr 2.06 ; ci, 0.99 to 4.24)( table 1) . conclusions: increased levels of p16 positive foci are highly specific for the diagnosis of uip and identify a subset of cases with a significantly worse transplant-free survival. these cases may represent an important subset of ild that may be most effectively treated with emerging classes of drugs that target senescent cells. design: 16 cases of nsclc patients who received ici were identified from departmental surgical archive and response was retrieved from the electronic medical records, including 7 cases of responders and 9 cases of non-responders. responders were defined as achieving a progression free response for more than 4 months. immunohistochemistry (ihc) studies for pd-l1, cd3, cd4, cd8, cd21, cd34, cd56, cd163, foxp3, and mmr proteins (mmrp mlh1/msh2, pms2/pms6) were performed. for cd3/4/8 ihc, the lymphocytic density (%) as well as the location (peripheral vs infiltrating) of positive t-cells were evaluated. results: all responders displayed a high pd-l1 expression (avg. of 68.8%, range 5% to 100%). the cd3/4/8 positive t-cells heavily infiltrated the tumor (avg. of 46.6%, range 30% to 80%). for non-responders, only 4 out of 9 cases displayed a lesser pd-l1 expression (avg. of 35.25%, range 1% to 70%). the cd3/4/8 positive t-cells were much less dense and infiltrative (<5%), and tended to concentrate at the periphery. the ihc results for cd56, foxp3, cd21 were negative. cd163 and cd34 had a similar pattern in both responders and non-responders. mmrp showed intact mlh1, msh2, pms2, and pms6 except for one non-responder. in addition to high pd-l1 expression, the percentage and pattern of cd3/4/8 positive t-cells appear to be more predictive of ici response in nsclc than pd-l1 alone and may easily be implemented in routine testing. the tumor microenvironment may also play an important role in facilitating immunotherapy response. however, nk-cells (cd56), tumor-associated macrophages (cd163), regulatory tcells (foxp3), and mmrp were not predictive of ici response in our pilot study. additional studies are needed to further investigate our preliminary findings. background: high-grade fetal adenocarcinoma (hfa) and enteric adenocarcinoma (ea) are both rare histopathological subtypes of lung adenocarcinoma. hfa and ea are occasionally combined with conventional-type lung adenocarcinoma, but pure types of both malignancies exist. the fact that the lungs and colon develop from the primitive striatum led us to speculate that these subtypes might share a common pathogenesis. design: among the 2253 cases of primary lung adenocarcinomas reported in our hospital, we identified 4 and 5 pure (p) hfas (0.18%) and eas (0.22%), respectively. all phfa tumors were high-grade adenocarcinomas with fetal lung morphology, necrosis, and immunopositivity for at least one of the following markers in addition to lacking morule formation: α-fetoprotein, sall-4, or glypican-3. all pea cases involved tumor cells that resembled colonic adenocarcinoma with no history of colorectal cancer. we evaluated the clinicopathological and molecular characteristics of these phfa and pea tumors. next-generation sequencing was performed using the ion-torrent personal genome machine platform and the ion ampliseq cancer hotspot panel v2. results: both phfa and pea were associated with several characteristic clinicopathological features such as smoking exposure, high incidence of lymphovascular invasion, and frequent expression of cdx2 and hnf4α. furthermore, nuclear accumulation of β-catenin was observed in 2 cases of phfa (50%) and 3 cases of pea (60%), which indicate activation of wnt signaling. the most frequently mutated gene was tp53 (3 peas), and other mutated genes that lead to the activation of wnt signaling were ctnnb1 (1 phfa) and apc (1 pea). an analysis of copy number variations revealed that smad4 deletion was the most frequently detected mutation, regardless of wnt activation (2 phfas; 2 peas). additionally, amplifications of fgfr3 were detected in 3 cases (1 ea; 2 phfas), which tend to be mutually exclusive with wnt activation. common mitogenic mutations in lung adenocarcinoma, such as egfr and kras, were not detected. conclusions: phfa and pea have similar clinicopathological features and oncogenic alterations which included frequent association with the activation of wnt signaling, amplifications of fgfr3, and smad4 deletion. recognition of these genetic subsets may help distinguish between lung adenocarcinoma with fetal and enteric morphology. background: gnas hotspot mutations have been described in indolent and slow-growing mucinous epithelial neoplasms in several organs, such as the pancreas and appendix. large genomic databases show that a subset of mucinous and non-mucinous lung adenocarcinomas harbor gnas mutations. however, the clinicopathological impact of gnas mutations on invasive mucinous adenocarcinoma of the lungs (ima) is not fully determined. we evaluated the clinicopathological and molecular characteristics of imas with gnas mutations in comparison with gnas wildtype cases. we examined egfr, kras, gnas, and tp53 mutations by pcr-direct sequencing in 80 imas. subsequently, a nanostringbased screen for 90 tyrosine kinase fusions was performed for all imas with wild type egfr and kras. next-generation sequencing using the ion-torrent personal genome machine platform and ion ampliseq cancer hotspot panel v2 or rna sequence were performed to confirm gnas mutations and tyrosine kinase fusions. mucin core proteins (muc1, muc2, muc4, muc5ac, and muc6) and differentiation transcription factors, particularly differentiating on the basis of the cellular lineage (ttf-1, cdx-2, and hnf4α) were detected by immunohistochemical staining. results: three of 80 imas (3.8%) harbored gnas mutations (2 r201h and 1 r201c). other mitogenic alterations including kras mutations (53 cases, 66.3%), tp53 mutations (11 cases, 13.8%), cd74-nrg1 fusions (2cases, 2.5%), and met exon 14 skipping (1 case, 1.25%) were detected. among 53 cases of kras mutations, g12v was the most frequent (42%), followed by g12d (34%) and g12c (13%). neither egfr mutations nor rearranged alk, ros1, ret, and ntrk were detected. all cases of gnas mutations were found in women who were never or light smokers with wild-type tp53. furthermore, gnas r201h mutations cooccurred with kras g12d mutations in two cases. in comparison with gnas wild-type cases, gnas-mutated cases were significantly associated with the female sex (p<0.05) and immunopositivity for muc4 (p<0.05). however, no significant differences were observed in other clinicopathological and immunohistochemical features, and progression-free and overall survival among both groups. conclusions: gnas-mutated imas are rare, but frequently co-occur with kras g12d mutations and immunopositivity for muc4, and are predominantly found in never-or light-smoking women; however, the prognostic impact of gnas mutations in imas is unclear. background: lung cancer is classified into small cell lung cancer (sclc) and non-small cell lung cancer (nsclc), which mainly contains adenocarcinoma (ac) and squamous cell carcinoma (sq). lung cancer subtyping plays an important role in choosing therapeutic schemes. sputum is a kind of noninvasively accessible biologic fluids containing exfoliated airway epithelial cells. although cytopathological examination of sputum is now available, its positive-rate of malignant cells is very low. it is hard to classify subtype via classic cytology on sputum specimens. we had reported that microrna panels could accurately discriminate between three subtypes of lung cancer in bronchial brushing specimens. the diagnostic value of micrornas in sputum specimens is need to be explored. in this study, 114 sputum specimens (49 ac, 39 sq, and 26 sclc) were investigated. reverse-transcriptase quantitative polymerase chain reaction (rt-qpcr) was performed to evaluate expression of 7 candidate micrornas discovered via microarrays previously. two logistic regression models constructed before was validated in the cohort of 114 sputum specimens. the area under the receiver operating characteristic curve (auc) was used to assess the diagnostic accuracy of microrna panels. the diagnostic performance was compared between microrna panels and cytology. results: panel a, consisting of mir-29a and mir-375, was built to discriminate sclc from nsclc. in the cohort of 114 sputum specimens, the auc value was 0.621 with sensitivity of 81.72% and specificity of 46.15%. similarly, panel b, consisting of mir-34a and mir-205, was used to discriminate sq from ac. in the cohort of 114 sputum specimens, the auc value was 0.678 with sensitivity of 46.94% and specificity of 82.05%. compared with cytology, microrna panels or the combination of microrna panels and cytology were of higher sensitivity and specificity in diagnosis of ac, sq and sclc. conclusions: in sputum specimens, those two microrna panels for lung cancer subtype discrimination could achieve high sensitivity and specificity. moreover, the combination of microrna panels and cytology could improve the diagnostic accuracy in further. these findings could be helpful in therapy of lung cancer. (table) . on image analysis, median total diagnostic area was 1.36 mm 2 and the median size of the largest fragment was 0.29 mm 2 (n=25). the largest fragment size was obtained for a case of diffuse large b-cell lymphoma (total area : 48.40 mm 2 , figure 1 ). there was no significant correlation between lpd diagnostic category and total fragment size (p=0.153) or largest fragment size (p=0.139). preliminary data on surgical specimens obtained by mediastinoscopy show a total diagnostic area ranging from 50.86 mm 2 to 95.30 mm 2 , with largest fragment size varying from 14.61 to 22.53 mm 2 (n=3). we report here a series of lymphoma cases diagnosed by ebus/eus, ranging across several diagnostic categories, based primarily on small size fragments. quantification of endoscopic diagnostic tissue provides insight on tissue yield, on its relationship with lymphoma subtype, and supports ebus/eus as an acceptable procedure for lymphoma diagnosis. background: light chain deposition disease (lcdd) is characterized by amorphous "glassy" deposits of immunoglobulin light chains in organs such as kidneys, heart, and liver. since these amyloid-like deposits lack the secondary structure consisting of beta-pleated sheets, they don't stain salmon-pink with congo red. additionally, a negative congo red may be attributed to suboptimal staining, especially when the deposits primarily involve organs where lcdd is rarely encountered. moreover, congo red stains are compromised when the available sections are 5 micron thick. we noticed in a case of primary lcdd of the lung that the light chain deposits stained bright red with masson trichrome and pink with sulfated alcian blue (sab) stains. in the current study, we tested whether these two stains can distinguish between amyloidosis and lcdd. we reviewed lung cases with eosinophilic deposits and immune infiltrates with negative congo red stain. we assessed trichrome and sab staining on 9 cases of lcdd and 1 control case of amyloidosis involving the lung. results: 9 cases were identified ( table 1 ). the age of the patients ranged from 44 to 75 years and 56% were females. imaging studies of 7 cases showed nodular deposits and the remaining cases showed cystic lesions and/or nodular lesions. underlying hematologic abnormalities were detected in 7 lcdd patients which included malt lymphomas (n = 6) and plasma cell neoplasm (n = 1). systemic involvement was absent in 7 cases with available information. hematoxylin and eosin stained slides of all cases showed "glassy" eosinophilic amyloid-like deposits predominantly around airways and vasculature. congo red staining was negative in all lcdd cases, while it showed salmon-pink staining of amyloid deposits in the control case. for all lcdd cases tested, trichrome stained the deposits as bright red and the sab stained the deposits as pink (figure 1 ). in contrast, amyloid deposits in the control case stained greyish blue with trichrome and bright green with sab stains, as expected. figure 1 -1926 conclusions: when congo red fails to stain "glassy" amorphous eosinophilic material salmon-pink, trichrome and sab stains might indicate the non-amyloid, light chain nature of lcdd deposits when red and pink staining is seen, respectively. these screening studies can inform downstream testing such as immunofluorescence staining for light chains, transmission electron microscopy, and typing of light chains by mass spectrometry. background: pulmonary pleomorphic carcinoma (ppc) is known for its aggressiveness and poor prognosis than other subtypes of nonsmall cell carcinoma. to better understand the molecular characteristics of ppc, we analyzed genetic alterations of ppcs and their metastatic lesions by whole exome sequencing. we included 11 ppc patients who underwent surgical resection for both primary lung cancer and metastatic lesions at seoul national university bundang hospital. carcinomatous and sarcomatous components of each primary ppc along with the metastatic lesions were microdissected. somatic mutation profiles were generated by whole exome sequencing. the majority of ppc patients were male (10 of 11, 90.9%) and smokers (10 of 11, 90.9%). carcinoma components of ppc consisted of adenocarcinoma (8 of 11, 72.7%), squamous cell carcinoma (2 of 11, 18.2%) or adenosquamous carcinoma (1 of 11, 9.1%). tp53 (70%) was the most frequently recurrent genetic alteration, followed by wdfy2 (36%), otog, pxdnl, and slitrk5 (33%). kras mutation was found in 2 cases (18.1%) and egfr mutation in one (9.1%). in addition, mutations discovered to be richer in either carcinomatous, sarcomatous or metastasis included various genes known to be associated with tumor progression and poor prognosis (flnc, nrxn1, csmd2, znf208, auts2). gene alterations associated with somatic hypermutation (pole, pold1) and epithelial-mesenchymal transition (notch1, cdh2, fn1) were also found. four cases (36.4%) had high tumor mutation burden (tmb). of the 43 altered genes investigated in depth in this study, roughly half (n=18) were shared by carcinomatous, sarcomatous and metastasis in at least one case. significant overlap of the mutation profiles between metastasis and either carcinomatous or sarcomatous component was not found. conclusions: this is the first study to analyze the molecular profile of ppc in both metastatic lesions and primary carcinomatous and sarcomatous components. sporadic genetic alterations appeared to occur among carcinomatous, sarcomatous components and metastatic lesions of ppc. various genes associated with tumor progression were altered in both primary and metastatic lesions. christin lepus 1 , julia rotow 2 , pasi janne 2 , lynette sholl 1 background: histologic transformation of egfr-mutant non-small cell lung carcinoma (nsclc) to small cell lung carcinoma (sclc) is a mechanism of acquired resistance to egfr-tyrosine kinase inhibitors (tkis) that occurs in approximately 5% of egfr-mutant nsclcs. however, the natural history/histologic progression of sclc transformation from nsclc is poorly understood. a retrospective analysis was conducted to characterize the morphologic variation during transformation of egfr-mutant nsclc to sclc. we identified 12 patients with egfr-mutant nsclc that transformed to sclc during treatment with egfr-targeted tyrosine kinase inhibitor therapy. histologic evaluation of longitudinal specimens (n=1-5 per patient) from both lung and distant metastases was performed to characterize the morphologic spectrum of lesions obtained from the initial diagnosis of nsclc to sclc transformation. results: all 12 patients had lung adenocarcinoma harboring egfr mutations at initial diagnosis (exon 19 deletion, 91.6%; l858r, 8.4%) and had been treated with at least one egfr tki prior to development of sclc. most patients (10 of 12; 83.3%) were on secondline osimertinib at the time of transformation. when transformation was first documented, four patients (33.3%) were reported to have combined adenocarcinoma and sclc or poorly differentiated carcinoma with mixed adenocarcinoma and sclc morphologic features; the remaining 8 patients (66.6%) were reported to have sclc. retrospective review of this latter group demonstrated focal nsclc-like morphology (mildly increased cytoplasm, variably prominent nucleoli, and rare gland formation) in 3 patients (37.5 %). conversely, 4 of 6 patients who had post-treatment, pre-transformation biopsies reported as adenocarcinoma showed classic adenocarcinoma architecture with superimposed small cell carcinoma-like cytomorphology, including increased nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, and finely granular chromatin. overall, a hybrid/transitional phenotype was captured in 7 of 12 patients (58.3%). among 7 patients who had tumor genotyping upon sclc transformation, all maintained the sensitizing egfr mutation and showed acquired rb loss. recognition of these transitional morphologies may facilitate earlier detection of sclc-driven resistance to egfr-targeted therapy and inform timely selection of alternate treatment regimens. yuan li background: random forest model is a recently developed machine-learning algorithm, and superior to other machine learning and regression models for its classification function and better accuracy. but it is rarely used for predicting causes of death in cancer patients. on the other hand, specific causes of death in lung cancer patients are poorly classified or predicted, largely due to its categorical nature (versus binary death/survival). we therefore tuned and employed a random forest algorithm (stata, version 15) to classify and predict specific causes of death in lung cancer patients, using the surveillance, epidemiology and end results-18 and several clinicopathological factors. the lung cancer diagnosed during 2004 were included for the completeness in their follow-up and death causes. the patients were randomly divided into training and validation sets (1:1 match). we also compared the accuracies of the final random forest and multinomial regression models. we identified and randomly selected 40,000 lung cancers for the analyses, including 20,000 cases for either set. the causes of death were, in descending ranking order, were lung cancer (72.45 %), other causes or alive (14.38%), non-lung cancer (6.87%), cardiovascular disease (5.35%), infection (0.95%), and. we found more 250 iterations and the 10 variables produced the best prediction, whose best accuracy was 69.8% (error-rate 30.2%, figure 1 ). the final random forest model with 300 iteration and 10 variables reached an accuracy higher than that of multinomial regression model (69.72% vs 64.58%). the top-5 most important factors in the random-forest model were sex, chemotherapy status, age, radiotherapy status and nodal status (figure 2 ). we optimized a random forest model of machine learning to predict the specific cause of death in lung cancer patients using a population database. the model also appears more accurate than multinomial regression model. background: primary pulmonary hematolymphoid neoplasms (phlns) are rare, and the incidence is increasing with modern diagnostic advances and treatments. flow cytometry (fc) is a proven powerful tool in the diagnosis of hematolymphoid disease, however its role in phln is not well represented. in the current study, we aim to assess the utility of fc in diagnosis of patients with phlns. we retrospectively reviewed the fc analyses of pulmonary specimens from our institutional database between 2016-2019. the specimens are comprised of pleural fluid, bronchoalveolar lavages, bronchoscopy or ct guided fnas, and vats or surgical biopsies of lung mass. phlns were detected by 10-color panel fc by identification of clonal or immunophenotypical aberrant hematolymphoid populations. primary neoplasms are classified if no extrapulmonary lesions were detected by clinical radiological work up at the time of diagnosis or within three months of diagnosis. lung involvement is also a frequent site in involvement of lymphoproliferative diseases and are referred to as secondary involvement of the lung. we retrieved 471 pulmonary specimens submitted for fc. we identified 111 cases that were positive for phlns. median age was 49 years with m:f ratio=1. 54/111 specimens aided in the diagnosis of a hematolymphoid disease. distribution are as follows: 17 pleural fluids, 33 tissue biopsies, 4 bronchoalveolar lavages. cases were classified as either primary (9/54 (17%)) or secondary (45/54 (84%)), and categorized as myeloid neoplasms, b-cell neoplasm, t-cell neoplasm, primary effusion lymphoma (pel), post-transplant lymphoproliferative disorder (ptld); and plasma cell neoplasms (table 1) . of the phlns, diffuse large b-cell lymphoma (dlbcl) was the most common diagnosis (12/54 (22%)). immunosuppression were notable in 14 patients. the remaining 57/111 cases represent followups of known lymphoproliferative diseases where fc analysis of lung specimen played a role in assessment of disease status. all fc positive cases were confirmed by tissue examination with positive predictive value (ppv) of 100%. conclusions: fc demonstrates a clear utility for immunophenotyping in the diagnosis of phln, with a perfect ppv. appreciating the spectrum of phln is helpful in disease detection. in addition, fc provides the ability to monitor residual disease and response to therapy; especially in immunosuppressed patients where expedited treatment can impact prognosis. background: piwi-interacting rnas (pirnas) are small non-coding rnas (24-35nt) that play an essential role in maintaining genome integrity trough regulation of transposable elements. their expression was tough to be limited to germinal cells and early embryogenesis but recently an implication of pirnas in cancer biology has been reported. the aim of this study was to explore the expression of pir-796 pirna, which was identified by small rnaseq, in resected nonsmall cell lung cancer (nsclc) patients, and to analyze the correlation with the clinic-pathological features. we have analyzed 191 resected nsclc samples from patients who underwent surgery in hospital clínic between 2007 and 2015. pir-796 pirna was quantified using custom taqman non-coding rna assays in tumor and normal tissue. in vitro studies using sirnas to inhibit pirna expression were performed in two lung adenocarcinoma cell lines: hcc44 and a549. results: pir-796 was overexpressed in tumor tissue compared to normal tissue (p<0.001). it also appeared to have higher expression in squamous cell carcinoma compared to adenocarcinoma histological subtype. expression of pir-796 had prognosis impact in the group of early stage (i-ii) adenocarcinoma patients. higher pir-796 levels were associated with shorter disease-free survival (p=0.045). in vitro analysis showed that the silencing of pir-796 was associated with decreased cell migration in a549 cell line (p<0.0001) and increased apoptosis in hcc44 cell line (p=0.03). in conclusion, pir-796 may have an important role in carcinogenesis in nsclc, promoting cell migration and regulating apoptosis and may be a potential new prognostic biomarker for nsclc. lucas massoth lcdd rarely involves the lungs in a nodular or diffuse pattern. we present herein a large series of pulmonary nodular lcdd. in an institutional review from >2000 patients in 2000-2019, we identified 12 specimens from 10 patients with pulmonary nodular lcdd. we reviewed clinicopathologic features and performed electron microscopy in all cases. of 10 patients (6 women, 4 men; age 42-74 [median 68] years), 7 were smokers, 4 had a history of autoimmune disease, but none had a history or evidence of a systemic lymphoproliferative/plasma cell disorder. clinical presentations were most often incidental. of 9 patients with radiology available, 3 showed a solitary nodule each; 6 showed multiple nodules (including 3 with associated cystic changes). each nodule ranged 0.6-2.1 (median 1.2) cm. by light microscopy on hematoxylin-eosin staining, each nodule appeared as amorphous eosinophilic deposits; all cases showed multinucleated giant cells engulfing the deposits and prominent plasma cell infiltrates. while the morphology was reminiscent of amyloidoma, staining for congo red was negative for amyloid in all cases. pas was positive in all 5 cases tested, and trichrome was distinctly bright red in 5 of 6 cases tested. of 8 cases with plasma cell cytoplasmic light chain expression tested by immunohistochemistry and/or in-situ hybridization, 6 were kappa-predominant, 1 lambda-predominant and 1 polytypic. immunofluorescence studies in 2 cases confirmed kappa and lambda light chain restriction in 1 case each. ultrastructurally, all cases showed extracellular electron-dense granular deposits, variably admixed with entrapped collagen fibers, often appearing perivascular, and lacking non-branching fibril formation characteristic of amyloid. of 6 patients with >2 years of follow-up (median 4.3 years), 5 were alive (the single death was unrelated to lcdd). pulmonary nodular lcdd appears clinically indolent, radiologically solitary or multifocal, and mimics amyloidoma by light microscopy. yet, special stains (negative congo red, bright red trichrome) and ultrastructural features (granular deposits with variable entrapped collagen) aid the diagnosis of lcdd and its distinction from amyloidoma. the high rate of autoimmune disease in this cohort may suggest a role of chronic immunologic stimulation in the pathogenesis of pulmonary nodular lcdd. background: molecular targeted therapies against egfr and alk have improved the quality of life of lung adenocarcinoma patients. however, targetable driver mutations are mainly found in ttf-1/nkx2-1-positive terminal respiratory unit (tru) types and rarely in non-tru types. design: to elucidate the molecular characteristics of the major subtypes of non-tru-type adenocarcinomas, we analyzed 19 lung adenocarcinoma cell lines (11 tru types and 8 non-tru types). a characteristic of non-tru-type cell lines was the strong expression of tff-1 (trefoil factor-1), a gastric mucosal protective factor. by immunohistochemistry, we examined tff-1, ttf-1/nkx2-1, hnf4-alpha, and muc5ac expressions using 238 primary lung adenocarcinomas resected at jichi medical university hospital. we also examined the correlation between tff-1 expression and clinico-pathological factors and genetic abnormalities. results: an immunohistochemical analysis of revealed that tff-1 was positive in 31 cases (13%). tff-1 expression was frequently detected in invasive mucinous (14/15(93%)), enteric (2/2(100%)), and colloid (1/1(100%)) adenocarcinomas, less frequent in acinar (5/24(21%)), papillary (7/120(6%)), and solid adenocarcinomas (2/43(5%)), and negative in micropapillary (0/1(0%)), lepidic (0/23(0%)), and microinvasive adenocarcinomas or adenocarcinoma in situ (0/9(0%)). tff-1 expression correlated with the expression of hnf4alpha and muc5ac (p<0.0001, p<0.0001, respectively) and inversely correlated with that of ttf-1/nkx2-1 (p<0.0001). these results indicate that tff-1 is characteristically expressed in non-tru-type adenocarcinomas with gastrointestinal features. tff-1-positive cases harbored kras mutations at a high frequency, but no egfr or alk mutations. tff-1 expression correlated with a poor prognosis in advanced stages. moreover, the knockdown of tff-1 inhibited cell proliferation and induced apoptosis in a tff-1-positive and kras-mutated lung adenocarcinoma cell line. these results indicate that tff-1 is not only a biomarker, but also a potential molecular target for non-tru-type lung adenocarcinomas. background: accurate and timely biomarker results are essential for the modern-day treatment of non-small cell lung cancer (nsclc). nonetheless, many institutions around the world, particularly those without in-house testing capabilities, are faced with prolonged turnaround time. this study investigates the clinical impact of implementing a rapid biomarker testing strategy. design: rapid in-house biomarker testing was implemented utilizing immunohistochemical assays for pd-l1, alk, ros, and braf v600e, as well as qpcr for egfr using the biocartis idylla technology. after a 6-month period, we performed a retrospective chart review of nsclc patients presenting pre-and post-implementation of rapid biomarker testing at our facility. results: 198 patients were included in the study (85 underwent rapid biomarker testing and 113 underwent traditional testing). the median (iqr) turnaround time for biomarker reports decreased from 61.5 (31-100) to 4 (2-7) days (p < 0.001), with turnaround time defined as the total number of days between the diagnosis and biomarker result appearing on the medical record. the mean time to initiation of systemic therapy for advanced-stage patients decreased from 52.5 days to 31.9 days (p < 0.01) the proportion of patients who had a complete biomarker report at the time of their first consult with a medical oncologist increased from 26.8% to 58.0%. similarly, the proportion of patients who had a complete biomarker report at the time of initiation of systemic therapy increased from 56.7% to 86.4%. timely biomarker results are essential for the delivery of targeted therapy and immunotherapy to nsclc patients. many centres experience delays in biomarker results for a number of reasons. in this study, we demonstrate that a small panel of rapidlyreported biomarkers can significantly reduce delays in the initiation of systemic therapy, ultimately leading to superior patient outcomes. mitra mehrad background: primary sarcomas of the thorax (heart, lung, pleura, thymus, mediastinum) are rare and little information is available on predictors of their prognosis. the national cancer institute's seer (surveillance, epidemiology, and end results) database contains abundant information on the natural history of soft tissue sarcomas. therefore, we investigated the clinical outcomes of primary thoracic sarcomas (ts) utilizing the seer database. the seer database was queried for ts entered between 1985 and 2013. ts was compared to sarcomas arising in soft tissues of the extremities and trunk. staging was performed based on the ajcc 8 th edition. multivariable cox regression was used to identify prognostic factors. kaplan-meier curves were plotted to assess cancer-specific survival (css). patients <19 years of age and those without confirmed surgical resection were excluded. results: a total of 1308 ts and 20160 soft tissue sarcomas were available for analysis. most ts arose in the lung (785; 60.0%) followed by mediastinum (224; 17.1%), heart (165; 12.6%), pleura (132; 10.1%) and thymus (2; 0.2%). the 5 most common ts were malignant solitary fibrous tumor (sft) (147; 11.2%), leiomyosarcoma (lms) (139; 10.6%), synovial sarcoma (128; 9.7%), undifferentiated pleomorphic sarcoma (ups) (126; 9.6%), and angiosarcoma (122; 9.3%), whereas in the soft tissue were ups (5477; 27.1%), lms (2357; 11.6%), myxoid liposarcoma (lps) (1544; 7.6%), well-differentiated lps (1413; 7.0%), and myxofibrosarcoma (1331; 6.6%). comparing the two groups by multivariate analysis, the ts were larger and more frequent in males (p<0.0001). they were also of higher histologic grade and stage and were less likely to receive adjuvant therapy (p<0.001). among the ts, tumors of heart origin (p<0.0001), greater than 5 cm, stage iii-iv, and histologic grade 3 had worse css (p<0.01). the more common ts tumors including malignant sft, lms, synovial sarcoma, and angiosarcoma still had worse css compared to their soft tissue counterparts even when adjusting for stage, size and treatment. conclusions: primary thoracic sarcomas show worse clinical outcome compared to soft tissue sarcomas. a thoracic specific sarcoma staging system may be more predictive of css. background: primary pulmonary mucinous adenocarcinoma (pma) may be difficult or impossible to distinguish from colorectal adenocarcinoma (crc) due to considerable morphologic and immunohistochemical (ihc) overlap. since the lung is a common site of metastasis, the need to distinguish pma from crc in the lung is a routine challenge. commonly used lineage-specific ihc markers like cdx2, ttf-1, and napsin a are helpful to distinguish non-mucinous lesions but are either insensitive or nonspecific when applied to mucinous lesions. satb2 is a relatively new ihc marker that distinguishes crc from upper gastrointestinal and pancreaticobiliary tumors. its ability to distinguish crc from pma is not yet completely elucidated. design: three tissue microarrays of lung resections containing primary pulmonary mucinous adenocarcinomas (pmas), metastatic colorectal carcinomas (crcs), and primary pulmonary non-mucinous adenocarcinomas (pnmas) were stained with ck7, ck20, satb2, cdx2, villin, ttf-1, and napsin a. two pathologists evaluated the number of positive neoplastic cells semiquantitatively, regardless of intensity: 0 (no staining), 1+ (<10%), 2+ (10-50%), 3+ (>50%). results: thirty-one pmas, 32 crcs, and 34 pnmas were assessed (table 1) . thirty (97%) of pmas and 34 (100%) of pnmas were positive (3+) for ck7, while all crcs were negative for ck7. twenty-seven (84%) of crcs and 2 (6%) of pmas were positive (3+) for satb2, and 29 (91%) of crcs and 2 (6%) of pmas were positive (3+) for cdx2. both pmas and crcs had high rates of villin positivity, with 23 (74%) and 32 (100%) positive (3+), respectively. only 4 (13%) and 7 (23%) of pmas were positive (3+) for ttf-1 and napsin a, respectively. no crcs were positive for ttf-1 or napsin a. in deciding pma vs. crc, ck7 was 97% sensitive and 100% specific for pma. satb2 was superior to cdx2 and villin but was only 96% sensitive and 94% specific for crc. conclusions: although the lineage-specific markers satb2 and cdx2 were fairly specific for crcs, a few pmas were also positive. in contrast, all crcs were negative for ck7 while almost all of the pmas were positive. lineage-specific markers ttf-1 and napsin a had a low sensitivity for pmas. villin showed a low specificity with the majority of crcs and pmas staining positively. our results suggest that a ck7-positive tumor in the lung, whether mucinous or non-mucinous, is unlikely to be of colorectal origin. lineage-specific markers such as satb2 are of questionable value when evaluating mucinous lesions in the lung. massimo milione 1 , patrick maisonneuve 2 , federica grillo 3 , alessandro mangogna 4 , giovanni centonze 5 , giovanna garzone 6 , laura cattaneo 7 , ketevani kankava 8 , adele busico 6 , paola spaggiari 9 , alessandro del gobbo 10 , luisa bercich 11 , luigi rolli 12 , elisa roca 11 , natalie prinzi 6 , giancarlo pruneri 5 , alfredo berruti 11 , ugo pastorino 12 , carlo capella 13 1 fondazione irccs istituto nazionale tumori milano, milano, italy, 2 ieo, milan, italy, 3 university of genova, genova, italy, 4 university of trieste, trieste, friuli venezia giulia, italy, 5 fondazione irccs istituto nazionale tumori milano, milan, italy, 6 irccs foundation, istituto nazionale dei tumori, milan, italy, 7 irccs foundation, istituto nazionale dei tumori, milano, italy, 8 teaching, scientific and diagnostic pathology laboratory, tbilisi state medical university, tbilisi, georgia, 9 istituti clinic humanitas, rozzano, milano, italy, 10 fondazione irccs ca granda ospedale maggiore policlinico, milan, italy, 11 university of brescia at brescia, italy, 12 irccs foundation national cancer institute, milan, italy, 13 with regards to molecular analyses, 26 cases (54,2 %) of the co-lcnec group were studied (see table 1 ). no single next-generation sequencing marker was statistically associated with os. the identification and morphologic characterization of combined features in lcnecs as well as the application of ki-67 cut off at 55% contribute in predicting clinical outcome of pure-lcnec and co-lnec patients. background: ctnnb1 encodes for β-catenin, which is a member in the wnt signal transduction pathway required for proliferation, survival and differentiation of different epithelial cells. mutation of ctnnb1 causes constitutional changes in the β-catenin protein that impedes its degradation, leading to an uncontrolled proliferation of the mutated cell. ctnnb1 exon 3 hot-spot mutations are described in various tumor types and, for instance, in endometrial cancer, are associated with high risk of disease recurrence. the role of ctnnb1, frequency and type of co-mutations has not been well characterized in non-small cell lung carcinomas (nsclc). design: between 2013-2018, lung cancer samples from 855 patients were sequenced on the ion torrent pgm with the 50 gene ampliseq cancer hotspot panel v2. our in-house sequencing database was searched to identify patients with ctnnb1 mutations; co-mutations in genes commonly altered in nsclc were also recorded. results: thirteen patients (1.5%) with ctnnb1 mutations (p.s37f (5) (one also with p.d32y), p.s37c (3), and p.s33c, p.d32h, p.g34e, p.d32v, p.d32n, in one patient each) were identified. all tumors were adenocarcinoma histology. five patients underwent lobectomy and the predominant histologic patterns were solid (2), papillary (2) and micropapillary (1) . the patients' age ranged from 54-82, at the time of diagnosis, and eight were female (61.5%). all patients were current (2) or former (11) smokers. five patients presented with stage iv disease, two with stage iii, two with stage ii, and four with stage i disease. six are deceased and seven are alive with disease. co-mutations were identified in all but one case and consisted of: braf (1 -v600e), egfr (3 -ex19 deletions), kras (4), pik3ca (3), and tp53 (3). in two patients, egfr, pik3ca and ctnnb1 mutations were co-occurring. we identified ctnnb1 mutations in 1.5% of lung adenocarcinomas, which, in our population, were associated with frequent co-mutations. additional clinicopathologic data will be aggregated from our internal patients and available databases (cbioportal) to better understand the clinical implications of ctnnb1 mutations in nsclc. co-mutation with egfr may be a mechanism of primary resistance to egfr tkis. in addition, ctnnb1 mutations make patients eligible for newer small-molecule tkis (e.g. ttki). mohammad mohammad . samples from normal tissue included pancreas (n=13), rectum/appendix/colon (n=39) and ilium/duodenum/stomach (n=13). nuclear expression for otp was interpreted as negative (<5% tumor cells stained), 1+ (<25%), 2+ (26-50%), 3+ (51-75%), and 4+ (>75%). results: six of 10 (60%) pcs were diffusely positive for otp. one of 33 (3%) pancreatic nets, 2 of 24 (8%) bladder sccs, and 1 metastatic net in the liver (negative for ttf1 and no lung mass identified) were positive for otp. all other tumors and normal tissues were negative. representative cases are shown in figure 1 . conclusions: our data demonstrated that otp expression was only rarely identified in non-pulmonary neuroendocrine tumors/carcinomas, which further validated the previous report of otp to be a highly specific marker for diagnosing pcs. the diagnostic sensitivity for pcs in this study appears to be lower than the previous report, which is probably due to the small number of cases included. caution should be taken because rare pancreatic nets and bladder sccs can be positive for otp. background: quantification of pd-l1/pd-1 expression in non-small cell lung cancer (nsclc) is not always predictive of efficacy of immune checkpoint inhibitor therapy, and response to these agents remains limited to a minority of patients. human leukocyte antigen 1 (hla-1) participates in presentation of aberrant peptide antigens, enabling cytotoxic t cells to recognize and destroy tumor cells. correspondingly, loss of hla-1 expression precludes immune recognition, and this loss is one proposed mechanism of treatment failure of immunotherapy. we hereby examine the expression of pd-l1 and hla-1 in a large cohort of nsclcs to determine the patterns and frequency of expression of these two markers. design: nsclc resection specimens from 2011-2014 classified as adenocarcinoma (ac) or squamous cell carcinoma (scc) were identified. tumor microarrays with 1.0 mm diameter cores were constructed, with 2 sections of neoplastic tissue and 1 section of uninvolved lung for each case. immunohistochemistry for pd-l1 (22c3) and hla-1 was performed. membranous tumoral pd-l1 staining was semi-quantitatively scored as <1%, 1-50%, and >50%. hla-1 was scored as intact, partial (clonal) loss, or complete loss with total absence of staining. conclusions: hla-1 loss is common in nsclc and can readily be assessed by immunohistochemical methods. in this study, there is no association between pd-l1 expression and hla-1 expression. given that hla-1 expression may be a determinant of response to immunotherapeutic agents targeting the pd-1/pd-l1 axis, it may be considered as an adjunct immunohistochemical marker prior to initiation of immune checkpoint therapy or in instances of treatment failure. background: short telomere syndromes (sts) are multisystem accelerated aging syndromes caused by inheritable gene mutations in telomere maintenance genes; frequent manifestations include bone marrow failure and interstitial lung disease (ild). insufficient information is available about histologic findings and patterns of ild in individuals with sts and whether these vary by specific inherited sts gene. our study aims to describe the morphology of lung disease in patients with sts. design: probands enrolled in the inherited hematologic disorders registry at our hospital with a personal or familial history of pulmonary fibrosis underwent genetic testing via targeted genomic capture and next-generation sequencing (ngs) of 13 sts genes. available specimens(n-8), including wedge resections(n-6) and lung explants (n-2), were analyzed for 15 histopathological features (table 1) . eight patients with known sts with ild had available histology (m=7, f=1 average age at diagnosis 56.3 years). pathognomonic findings of usual interstitial pneumonia (uip) were seen in all patients. one patient with two different mutations in the telomere-associated genes showed features of both uip and non-specific interstitial pneumonia (nsip). there was associated lymphoplasmacytic infiltration which was mild in 50% and extensive in the remainder. other histologic findings were variable, e.g. nonnecrotizing granulomas and upper lobe predominance are noted in 50% of the patients. pulmonary hypertension was seen in all of the patients except one who had a very early disease. conclusions: sts associated lung disease frequently shows uip with additional findings including inflammatory (lymphoplasmacytic infiltrate) and hypersensitivity pneumonia-like (granulomas and upper lobe predominance) features. consideration should be given to testing for sts in patients with uip with these histologic findings. it is important to identify this cohort of patients because these patients may have a more rapid progression of their ild and an increased toxicity from immunosuppressive drugs especially in post-transplant setting. if a pathogenic gene mutation is identified, first-degree family members (especially siblings) should be informed about disease monitoring options and knowing the risks of future health issues. background: genetic polymorphisms in key genes encoding enzymes involved in the bio-activation (cytochrome p450 (cyp)) or detoxification (glutathione s-transferase (gst)) of environmental carcinogens including tobacco specific nitrosamines are potential lung cancer risk factors. the frequencies of these variants and consequently their effects vary across ethnicities. the interactive effects with cyp-gst combination along with smoking have not been documented. five single nucleotide variants and two homologous deletion variants associated with cyp and gst genes and their interactive effects with smoking in non-small cell lung cancer (nsclc) were investigated design: the case control study comprised 244 cases of histologically diagnosed nsclc and 224 healthy controls. iec approval and informed consent were obtained. mean age of cases was 56.51±11.21+ 2sd, male: female ratio was 4.19, 91 were non-smokers and 153 smokers subdivided into 20 (47.7%), 20-40 (38.7%) and 40(13.6%) in pack-years. histological subtypes included 180 adenocarcinoma (adc), 57 squamous cell carcinoma (scc) and 09 adeno-squamous cases. majority were diagnosed in late stages with only 5 in stage ii, 78.2% had lymph-node metastasis and 139 cases had distant metastasis. dna was isolated from whole blood and genetic polymorphism analyses were determined by polymerase chain reaction coupled with restriction fragment length polymorphism followed by agarose and poly acrylamide gel electrophoresis for genetic variants. all the statistical analyses were performed with graph pad instat version 3.05 and spss version 16(chicago, usa). significantly high risk of nsclc and subtype adc was associated with variant cyp2a6, gstt1 and gstm1 ( conclusions: functionally relevant polymorphisms in cyp and gst genes with gene-gene and gene-environment interactions play a significant role in modifying the susceptibility to nsclc in population of indian ethnicity. design: thirty consecutive surgically resected lung-nens comprising 10 tc, 14 ac and six lcnec, all with long-term follow-up, were immunohistochemically stained for ki-67 and scanned at 40x (nanozoomer xr, hamamatsu, japan). a tailored algorithm was constructed to recognize all ki-67-stained tumor cells and the obtained patterns were described using spatial statistics, graph modeling, fractality and shannon entropy parameters. a support vector machine classifier with polynomial kernel was then trained, employing the 5 parameters that resulted most informative out of the 620 initially computed, to distinguish dead (true positive) from alive (true negative) patients. over 100 repetitions of 5-fold cross-validation, the model averaged 84.3% diagnostic accuracy in the prediction of ultimate clinical outcome (dead vs alive) in the 30 lung-nen patients under evaluation, which resulted to be independent of who classification. the corresponding values of sensitivity, specificity, ppv and npv were 73.6%, 89.6%, 78.2%, and 87.4%, respectively. the intratumor heterogeneity of ki-67 is a powerful and histology-independent resource to unravel clinical outcome of lung-nens by using machine learning algorithms. roshan raza background: cytologic diagnosis of mm is challenging since atypia in mesothelial cells is not specific for malignancy and there is no architecture to assess for invasion. loss of bap1 expression by icc and homozygous deletion of p16/cdkn2a by fish are specific but not sensitive for mm. co-deletion of mtap occurs in most mm with p16/cdkn2a deletions and can be detected by icc, which has advantages over fish. recently, 5-hmc has been reported to show 92% sensitivity and 100% specificity for distinguishing mm from benign mesothelial proliferations. to our knowledge, these 3 markers have not yet been studied in combination in cytology. herein, we determine the sensitivities and specificities of bap1, mtap and 5-hmc icc for the diagnosis of mm in cytology. design: icc with mtap, 5-hmc and bap1 was performed on all available mm cytology specimens from 2017-present and 20 benign specimens with reactive mesothelial cells on cell block. icc was scored as nuclear loss of bap1 expression, loss or marked reduction of cytoplasmic mtap expression, loss of nuclear expression of 5-hmc in at least 50% of tumor cells or non-contributory (nc) in cases without internal positive control. all available clinical next generation sequencing (ngs) data were collected. results: cytology specimens (9 fluids, 3 fna, 1 touch preparation) from 13 patients with diagnosis of mm confirmed by histology (11 epithelioid, 2 biphasic) contained adequate tumor cellularity on formalin-fixed cell blocks for study inclusion. all 20 cases with benign mesothelial cells showed retained bap1 and mtap expression and retained or <50% loss of 5-hmc expression. mm in 7, 3 and 6 patients showed bap1 loss, mtap loss and/or >50% loss of 5-hmc expression (sensitivities of 58, 23 and 46%), respectively. bap1 icc was nc in one mm specimen. while sensitivity of mtap was low, mtap loss was seen in 2 mm with retained bap1 and either retained or <50% loss of 5-hmc expression. combined sensitivity of all 3 markers was 77%. cdkn2a deletions were detected by ngs in all 3 mm with mtap loss by icc. mtap icc was retained in 2 mm with cdkn2a deletions detected by ngs. the status of the intratumoral immune microenvironment is important to guarantee the effect of immune checkpoint (ic) blockade therapy, which has been broadly used in patients with non-small cell lung carcinoma (nsclc). glucocorticoid (gc) is a hormone well-known to act strongly on the immune system. therefore, we examined the correlation between intratumorally synthesized gc through 11β hydroxysteroid dehydrogenase (hsd) 1 and the immune microenvironment in nsclc. we evaluated 125 surgical specimens from patients with nsclc (95 adenocarcinoma and 30 squamous cell carcinoma), assessing mainly the immunoreactivity for 11βhsd1 and 11βhsd2 and the levels of tumor-infiltrating lymphocytes (tils) and cd3-or cd8-positive t cells. furthermore, we examined the correlations between 11βhsd1 immunoreactivity and the therapeutic efficacy of ic blockade therapy using nine biopsy specimens from patients with nsclc. subsequently, we explored the mechanisms of gc effects on the intratumoral immune microenvironment, focusing on cytokines. results: 11βhsd1 immunoreactivity was significantly inversely correlated with the numbers of intratumoral tils, cd3-positive t cells, and cd8-positive positive t cells. additionally, we found 11βhsd1 immunoreactivity tended to be inversely correlated with the efficacy of the ic blockade therapy. according to the in vitro study, gc reduced the expression of cytokines such as il-8 and il-6, resulting in an inhibition of monocytes migration. furthermore, production of cortisol, active gc, was confirmed in the cell lines expressing 11βhsd1. conclusions: this is the first study demonstrating the significant inhibitory effects of intratumorally synthesized gc through 11βhsd1 on tissue immune microenvironment in nsclc, and the possible correlation of intratumoral 11βhsd1 status with the efficacy of ic blockade therapy. our results provided new insights into the therapeutic strategies and the efficacy prediction of ic blockade therapy. these prognostic factors may be useful to the treating physician in crafting treatment plans. this study seeks to compare these parameters between metastatic and primary sites in mm. design: paired cases of pleural mm with metastatic and primary sites were identified from the pathology archives at the participating institutions with review of h&e stained sections. histologic subtype was noted in all cases. for epithelioid mm, ng (1, 2, or 3, as previously described in the literature by kadota et. al.) and necrosis were determined. results: 60 paired cases were identified with the primary site subtype comprised of 36 epithelioid, 24 biphasic, and 0 sarcomatoid cases. 57 of 60 (95%) metastases showed epithelioid morphology; 3 (5%) metastases were biphasic. the positive predictive value of epithelioid subtype at metastatic site was 63%, sensitivity 100%, and specificity 88%. ng and the presence or absence of necrosis at metastatic sites were not correlative with primary site histologic subtype (p=0.34 and p=0.78, respectively). thirty-three pairs of metastases and primaries with epithelioid morphology were graded; 3 metastases were too small to grade. pairs were more likely to show a higher ng at primary rather than metastatic sites (p<0.01) with 6 of 8 (75%) metastatic sites with ng 1 showing ng 2 at primary site, and 3 of 16 (19%) metastatic sites with ng 2 showing ng 3 at primary site. all metastases with ng 3 had ng 3 at primary site. there were no pairs with lower ng at primary site than metastatic site. the presence or absence of necrosis at metastatic site was not predictive of the presence or absence of necrosis at primary site (p=0.39). the application of pathologic parameters to metastatic mm may not accurately predict the parameters at primary site. while sensitive, epithelioid morphology at metastatic site is not specific. metastatic sites may underestimate nuclear grade at the primary site. the presence of necrosis in a metastatic site may not be predictive of necrosis at the primary site. biopsy of the primary site may be required to more accurately classify the tumor. background: immunotherapy has dramatically changed the treatment landscape of various malignancies including lung adenocarcinomas. the 2019 nccn guidelines initially recommended single agent immunotherapy as a first line treatment option for advanced lung adenocarcinoma with pd-l1 expression levels of 50% or greater. however, given the recent data suggest that pd-l1 monotherapy is less effective in patients with egfr or alk gene alterations, the nccn panel recently deleted the recommendation for subsequent immunotherapy in these patients. we retrospectively analyzed 124 cases of lung adenocarcinoma with pd-l1 expression (>1% expression) using pd-l1 ihc 22c3 pharmdx test and correlated pd-l1 expression with the presence of egfr mutations or alk gene rearrangement in the 120 cases where all 3 tests were performed. pd-l1 expression levels were subcategorized as low expression level (1%-49% of the tumor cells express pd-l1) and high expression level (50%-100% of the tumor cells express pd-l1). results: high expression levels of pd-l1 (50%-100%) were observed in 40% (50/124) of the cases. among the 5 (4%) tumors that harbored alk gene rearrangement, 4 showed high pd-l1 expression. among the 31 (26%) tumors with detected egfr mutations, only 6 showed high pd-l1 expression. these two alterations were mutually exclusive. results are summarized in the table 1. conclusions: in our study cohort 40% (50/124) of lung adenocarcinomas with pd-l1 expression showed high (50%-100%) expression levels. 20% of these high pd-l1 expressors were positive for either egfr or alk alterations: 8% (4/50) harbored alk gene rearrangement and 12% (6/50) showed egfr mutations. we corroborated the previously reported association of alk gene rearrangement with high expression levels, and egfr mutations with low expression levels of pd-l1 in lung adenocarcinomas that express pd-l1. our study showed a substantial number (30%) of lung adenocarcinomas that express (>1%) pdl-1 have egfr mutations or alk gene rearrangement, suggesting that it is important to consider the results of these tests simultaneously in order to be able to stratify patients according to current nccn therapeutic guidelines. background: pd-l1 is a predictive marker of anti-pd-1/pd-l1 therapies for non-small cell lung cancer (nsclc). heterogeneous pd-l1 expression may cause dilemmas in anti-pd-1/pd-l1 therapies when faced with discrepant biomarker results. our aim was to comprehensively analyze the heterogeneity of pd-l1 expression defined as intratumoral area, paired samples and clones of anti-pd-l1 antibody to optimize tumor sampling and improve its accuracy. we selected 1002 nsclc surgically resected specimens, 54 cell block and 73 biopsy specimens. we analyzed the associations of pd-l1 expression with histopathological characteristics, assessed the heterogeneity between paired cell block and biopsy samples (n=54), paired biopsy and resected samples (n=19), paired two blocks of the same resected sample (n=53), paired primary and metastatic lesions (n=29), and compared the consistency of clones of pd-l1 antibody (22c3 and sp263, n=66). background: recently, we identified two young adults with idiopathic acute respiratory distress syndrome (ards) histologically characterized by diffuse alveolar injury with marked alveolar denudation, a novel pattern termed daide. both patients were exposed to trimethoprim-sulfamethoxazole (tmp-smx). here, we report four additional patients with daide following tmp-smx exposure and detail the features of all six patients. the original 2 patients were identified through routine surgical pathology, and an additional 4 patients were identified in the consultation archives. daide was identified on surgical biopsy (n=4), autopsy (n=1), or both (n=1). six patients with dad on surgical biopsy were used for comparison. clinical information was obtained from the medical record. h&e and ihc for ae1.3/cam5.2, cd68, and ck5/6 were reviewed. six otherwise healthy patients (m:f=1:5, median age: 21 years; age range: 10-37 years) initially presented with uri symptoms that rapidly progressed to ards; extensive infectious and rheumatologic workups were negative. no patients had a history of vaping. all patients were intubated and placed on ecmo for a median of 93 days (range: 1 to 190 days). surgical biopsy was performed in 5 patients (6 days before to 10 days after intubation), and autopsy was performed in 2 patients (12 and 466 days after intubation). all 5 surgical biopsies showed marked alveolar denudation with histiocytes replacing alveolar lining and nested peribronchiolar metaplasia, consistent with daide. hyaline membranes were rare when present (n=3). the 2 autopsies showed patchy alveolar-filling fibrosis and reepithelialization of most alveoli, with focal areas of alveolar denudation and histiocytic lining, suggestive of partially resolving daide. in comparison, control surgical dad cases showed prominent hyaline membranes, only focal alveolar denudation, and no significant histiocytic lining. on follow-up, daide patients underwent bilateral lung transplant (n=2); overall, 3 patients are deceased (n=3). conclusions: daide appears to be a novel variant of dad with a rapid and severe clinical course involving previously healthy patients and is characterized by extensive alveolar denudation with a lining replaced by histiocytes and a paucity of hyaline membranes. although our patients share a history of tmp-smx exposure, the role of tmp-smx in daide is currently unclear. nonetheless, patients with daide appear to have a poor prognosis. lynette sholl 1 , adrian dubuc 1 , jason hornick 2 , david chapel 1 1 brigham and women's hospital, boston, ma, 2 brigham and women's hospital, harvard medical school, boston background: the entity diffuse idiopathic neuroendocrine hyperplasia (dipnech) is a clinical syndrome defined by the who as a generalized proliferation of neuroendocrine cells either confined to the bronchial mucosa or resulting in formation of carcinoid tumorlets or tumors. when thus defined, dipnech is a diagnostic entity seen in clinically symptomatic patients presenting with histologic alterations of neuroendocrine proliferations combined with radiological features of airtrapping and multiple bilateral nodules. the significance of incidentally discovered neuroendocrine cell hyperplasia in surgically resected specimens and its correlation to radiological and clinical features is not known. this study aims to characterize the clinicopathological and radiological patterns identified in patients with histologically identified diffuse neuroendocrine cell hyperplasia (nech). we searched the pathology database starting jan, 2009 till sept, 2019. for the for the combination of the following keywords "diffuse" and "neuroendocrine cell hyperplasia (nech)'' and "lung" and identified ten cases that met the search criteria. the radiological data was reviewed by one thoracic radiologist in a blinded fashion. clinical, radiological and pathological features of these patients are depicted in table 1 . there was female predominance in our study (9/10) and all patients were above the age of 45 years. 70% (7/10) of the nech cases were diagnosed incidentally during imaging workup or postoperative surveillance for other malignancies. the remaining 3 cases were biopsied for presentation of respiratory symptoms and or radiological finding of interstitial lung disease. radiologically, bilateral involvement of lung was seen in 9 out of 10 cases with air trapping in 2 cases. the radiological diagnosis of dipnech could be made only in three cases, one of which was incidentally discovered (case 5). nine cases showed presence of carcinoid tumor or tumorlet along with nech and 4 cases showed granulomas along with nech. in conclusion, in this study, vast majority of patients with nech were incidentally discovered during surveillance and follow up imaging for an unrelated malignancy or nodules. none of these incidentally discovered patients were clinically symptomatic. the criteria for evaluation of nech in incidentally discovered asymptomatic patients is not well defined and necessitates further investigation and appropriate follow-up for an evolving interstitial lung disease. elisabeth tabb background: recent studies have implicated local microbiota in activating gamma-delta t-lymphocytes (gdtl) and subsequently inducing neutrophilic infiltration to promote oncogenesis in murine models of kras-mutant lung adenocarcinoma. this study aimed to examine the translational relevance of these preclinical findings by evaluating gdtl and tumor-associated neutrophils (tan) in human lung adenocarcinomas, including those with and without kras mutations. we quantified the numbers of gdtl and tan using immunohistochemistry for t cell receptor-gamma chain and myeloperoxidase, respectively, from 3 high-power fields (hpf; each 0.24 mm 2 ) of viable tumor areas on tissue microarray (duplicate of 2-mm core) sections of 236 human lung adenocarcinomas resected in 2010-2012. high and low gdtl or tan were defined as above or below the respective median. data were correlated with demographics, histologic features (ajcc 8 th edition), immune parameters (tumoral pd-l1 expression, cd8+ tumor-infiltrating lymphocytes [til]), molecular alterations (using a multiplex-pcr based assay), and outcome via chi-square or logrank tests as appropriate (significance: p<0.05). results: gdtl ranged 0-177 (median 3.3) per hpf (in 228 tumors with evaluable data); whereas tan ranged 0-140 (median 1.3) per hpf (in 223 tumors with evaluable data). the numbers of gdtl and tan correlated with each other (p<0.0001). both high gdtl and high tan were associated with smoking history (p<0.01; p<0.05), solid/high-grade acinar histologic patterns (p<0.0001; p<0.001), the presence of tumor necrosis (p<0.05; p<0.001), and elevated cd8+ til (p<0.001; p<0.05). high tan -but not high gdtl -was also associated with greater total tumor size (p<0.01), invasive size (p<0.05), advanced stages 3-4 (p<0.05), and worse progression-free survival (p<0.05), with a trend toward worse overall survival (p=0.05). however, gdtl and tan were not associated with tumoral pd-l1 expression or molecular alterations including kras or egfr. in human lung adenocarcinomas, we identified correlations of gdtl and tan with smoking history, aggressive histology, and elevated cd8+ til, but no associations with mutation status. tan also appeared to be associated with worse patient outcome. increased gdtl and tan may reflect aggressive tumor biology; our data also suggests that their effects may be more general and not restricted to kras-mutant tumors. background: pd-l1 expression in non-small cell lung cancer (nsclc) is used as a biomarker to treat patients with pd1 blockade therapy. pd-l1 expression may be related to tumor stromal interactions guided by underlying genotypic/phenotypic characteristics of a tumor and tumor antigenicity. increasing evidence has shown that c-met pathway activation in coordination with interferon gamma can lead to pd-l1 upregulation. in order to determine whether the underlying genomic characteristics of a tumor, including c-met status is associated with pd-l1 status, we analyzed lung nsclc that had been genomically characterized and correlated with pd-l1 status. design: immunohistochemical staining for pd-l1 was performed with clone 22c3 on a dako autostainer and scored as no expression (<1%), low expression (1-49%) or high expression (50% or greater). flourescent in situ hybridization was performed for alk, ret and ros1 (break apart probes) and c-met amplification/polysomy. mutations were detected by next generation sequencing of a 26 gene panel (cmp26, based on illumina tst26) or multiplex hotspot mutation assay (snapshot, 10 gene panel). we evaluated 103 lung nsclc (85 adenocarcinomas/favor adenocarcinoma, 7 squamous cell carcinomas, 8 not otherwise specified, 2 adenosquamous and 1 combined large cell neuroendocrine carcinoma with adenocarcinoma). no pd-l1 expression was seen in 34% (n=35) while pd-l1 expression was seen in 66% (n=68) with low expression in 25% (n=26) and high expression in 41% (n=42). among the no expression cohort there was a slight increase in egfr mutated patients as compared to pd-l1 expression cohort (23% vs. 13%) while similar frequencies in kras mutations were seen in both cohorts (no expression 40%; pd-l1 expression 38%). c-met alterations in the form of amplification, high polysomy and mutations (exon 14 skip mutations and juxtamembrane mutations) were associated with pd-l1 positive status (no pd-l1 expression 9% (n=3) vs expression 25% (n=17)), p=0.05), especially when there is high pd-l1 expression (29% (n=12); p=0.03). only in the no pd-l1 expression category was concurrent egfr mutation and c-met amplification/polysomy observed (2 of 3). higher pd-l1 expression is associated with c-met amplification, polysomy and mutations. these results indicate that patients with abnormalities in c-met may benefit from combined inhibition of pd-1 pathway and c-met pathway. background: differentiating malignant pleural mesothelioma from reactive mesothelial processes can be quite challenging. ancillary tests such as bap1 immunohistochemisry (ihc) and p16 fluorescence in situ hybridization (fish) are very helpful tools to aid in this distinction. ihc for mtap has recently been proposed as an effective surrogate marker for p16 fish, and it is an attractive alternative test due to shorter turn-around time. there is little data regarding the specificity of mtap ihc for mesothelioma, or whether it may be useful to distinguish mesothelioma from other entities in the differential diagnosis. while there are many reliable markers to distinguish epithelioid mesothelioma from adenocarcinoma, this is not true of sarcomatoid mesothelioma, which can be very difficult to distinguish from sarcomatoid carcinoma. the goal of this study was to determine if mtap loss is present in pulmonary sarcomatoid carcinoma or only in sarcomatoid mesothelioma. design: well-characterized cases of sarcomatoid carcinoma (n=35) and sarcomatoid mesothelioma (n=62) were included; diagnoses were confirmed by two thoracic pathologists with incorporation of immunophenotype, clinical and radiographic features. each case was stained for mtap (clone 2g4) and bap1 (clone c-4). successful staining was confirmed by presence of internal positive control for both stains. results: loss of mtap expression by ihc was observed in 18 of 35 pulmonary sarcomatoid carcinomas (51%); 32 of these cases also had successful bap1 staining performed, which was retained in all cases. mtap expression was lost in 38 of 62 sarcomatoid mesotheliomas (61%); bap1 was successful in all 62 cases, and showed loss in 6 (10%). in the 6 cases of sarcomatoid mesothelioma with bap1 loss, 5 also had loss of mtap, while mtap expression was retained in 1 case. conclusions: loss of mtap expression by ihc is common in pulmonary sarcomatoid carcinoma, present in half of cases. this may reflect homozygous p16 deletion, which has been described in a few cases of sarcomatoid carcinoma studied by fish analysis. this rate is similar to what is observed in sarcomatoid mesothelioma (61%). therefore, mtap loss is not specific for mesothelioma, and this stain is not useful to distinguish between these two malignancies. mtap loss is more common than bap1 loss in the setting of sarcomatoid mesothelioma (61% vs 10%, respectively). basile background: metexon 14 skipping (metex14) mutations present in 4% of lung adenocarcinoma is now becoming an important alteration to test for targeted therapy, similarly to alk. the only commercially available way to test for metex14 mutations is through next generation sequencing. there is a need for a faster and more available method to be used for the detection and validation of metex14 mutations but traditional animal base monoclonal antibody (mabs) techniques are slow and difficult to scale. a newer, faster and animal-free approach using instead b-cells cloning to generate in vitrorecombinant antibodies (rabs) is increasingly popular. here we compare a novel rabs technique to the more traditional mabs generation approach in developing a mutation-specific monoclonal immunohistochemistry (ihc) to metex14 mutation. design: using the same amino acid sequence overlapping the fusion of metexon 13 and 15, we generated a total of 72 antibody clones: 68 rabs and 4 rabbit-based mabs. the clones were validated by enzyme-linked immunosorbent assay (elisa) and ihc using a combination of synthetic peptides, metex14 mutated cell line (h596) and archival lung adenocarcinoma tissue with metex14 mutation. of the 68 rabs screened for affinity by elisa, 37 were retained for ihc validation along with all 4 mabs. using the metex14 mutated cell line, strong (3/3 staining intensity) diffuse (100% tumor cells staining) membranous staining was achieved in 4 of the rabs. seven other rabs had weak-to-intermediate (1-2/3 staining intensity) non-diffuse (5-75% tumor cell staining) membranous staining and the remaining 26 rabs showed no membranous staining. the best mabs clone only showed focal (5%) weak-to-intermediate staining with substantial background staining and the other 3 mabs were completely negative. the rabs technique was an effective approach to generate metex14 mutation-specific ihc clones. it can be scaled up more readily as opposed to the traditional animal-based hybridoma technique, and as a result, it increases its rate of success and decreases cost. this technique might allow for easier transition of mutation-based biomarkers to ihc and improve turnaround time and access for predictive tests in oncology. the top 4 rabs are currently being tested on an extended cohort of lung carcinoma tissue. (0) and weak staining (1+) were classified as negative; focal moderate staining (2+) as equivocal; patchy and diffuse moderate staining (2+) and strong staining (3+) as positive. (figure 1 ) results: among 164 cases, insm1 was positive in 16 cases (9.8%) and equivocal in 14 cases (8.5%). of 97 adenocarcinomas, insm1 was positive in 6 cases (6.2%) and equivocal in 9 cases (9.3%). the positive adc cases included 1 case with focal 3+ nuclear staining, 2 cases with diffuse 2+ staining, and 3 cases with patchy 2+ staining. of 51 squamous carcinomas, insm1 was positive in 9 cases (17.6%) and equivocal in 6 cases (11.8%). the positive sqcc cases included 1 case with patchy 3+ staining, 2 cases with diffuse 2+ staining and 6 cases with patchy 2+ staining. focal 3+ nuclear staining was seen in 1 of 16 other nsclc cases. (table 1 ) other non-small cell carcinoma conclusions: our study demonstrates that insm1 is expressed in a subset of nsclcs and suggest that caution must be exercised in interpreting insm1 staining, especially with limited sample such as biopsy and cell block sections. although insm1 is useful for the diagnosis of neuroendocrine tumors, it should not be used as a stand-alone marker in differentiating primary lung tumors. background: egfr tyrosine kinase inhibitors (tkis) therapy is a validated approach in the treatment of egfr-mutated non-small cell lung carcinoma (nsclc), but resistance universally develops and it has become a major obstacle in prolonging the survival of patients. more novel molecular biomarkers are still urgently required to elucidate the underlying mechanisms of resistance. this study aimed to investigate the role of linc00520 in the acquired resistance of nsclc to egfr-tkis. design: gene expression profiles from geo dataset were analyzed to identify the genes associated with egfr-tkis resistance. egfrmutated nsclc cell line pc9 was cultured with gefitinib for more than 6 months to acquire gefitinib-resistance, which was designated as pc9r. the expression patterns of linc00520 were characterized using reverse transcription quantitative polymerase chain reaction (rt-qpcr), and lentiviral vectors were used to infect cells to regulate the expression. cytotoxicity of egfr-tkis on infected cells was determined by cell counting kit-8 (cck-8). survival follow-up time of 948 nsclc samples from tcga dataset were enrolled in this study. in addition, statistical analysis was mainly performed by r programming language and graphpad prism 7.0 (graphpad). results: linc00520 is highly expressed in gefitinib-resistant cell line pc9r relative to pc9 (p<0.05). inhibiting linc00520 with lentivirus vectors induces apoptosis in pc9r. linc00520 could promote cell proliferation and induce resistance. statistics from tcga dataset demonstrate there is no significant difference in linc00520 expression between luad tissues (483) and normal tissues (347), but the expression level in lusc tissues (486) conclusions: linc00520 is involved in acquired resistance of egfr-tkis in nsclc. it may serve as a predictor and a potential therapeutic target for egfr-tkis resistance. ilyas yambayev background: lung cancer is the most common cause of cancer death worldwide. screening by ldct is expected to increase the frequency of early-stage nsclc of which lung adenocarcinoma (luad) is the most common subtype. although the assignment of predominant histologic subtypes is now recommended, there remains no widely accepted prognostically relevant grading system. several grading systems have been proposed however there has been no direct comparison of these grading systems in an independent cohort. here we compare several previously published architecturally based grading systems in a large cohort of stage i luad. design: h&e slides were reviewed from stage i luad resection specimens form a multi-institutional cohort of 278 patients diagnosed between 2005-2015. the staging was reassigned using ajcc 8th edition after determining the invasive size and assessing for pleural invasion. comprehensive histologic subtyping in 5% increments was performed along with mitotic figure counts and assessment for angiolymphatic invasion. this data was applied to compare recurrence-free survival rates using 4 published grading systems. the demographic, smoking status and stage characteristics are summarized in table 1 . results: figure 1 shows the kaplan meier curves for 4 published grading systems. predominant architectural pattern assignment alone was prognostically significant in stratifying patients into low ( -1968 conclusions: predominant architectural pattern assignment alone is a valuable grading system. the addition of mitotic grade and angiolymphatic invasion allows for further refinement to identify higher proportions of low-risk and a small but significant subset of very high-risk luad which might aid in the precision clinical management of early-stage luad. harbored egfr mutations. 6 lung cancers with atm p.v2424g were identified, with generally higher vafs (55-92%). 2/6 cases with atm p.v2424g also harbored the egfr p.l858r variant, while 2 cases also harbored kras codon 12 mutations. interestingly, the two kras/atm-mutated cases were from a single patient, with different kras mutations. conclusions: disease-associated brca1/2 variants are rare in lung carcinomas, and many cases were associated with kras hotspot variants, suggesting that brca1/2 mutations may be somatic in origin, likely in the setting of significant smoking history. in contrast, while atm p.v2424g is equally rare, the genomic context and vafs of the atm variants suggest possible germline events. assessment of other genes in the hr pathway is currently underway. jingping yuan 1 , huihua he 2 , lin xiong 2 , li xu 2 background: pulmonary enteric adenocarcinoma (pea) is a rare histologic type of lung adenocarcinoma. pea is composed mainly of tall columnar cells arranged in an irregular acinar or cribriform pattern with extensive central necrosis, closely resembling the appearance of intestinal epithelial and colorectal carcinomas under the microscope. immunohistochemically, pea is usually positive for ck7. however, some cases lack ck7 expression and are positive for intestinal differentiation markers, such as cdx2, villin, and ck20. for these reasons, it is difficult to distinguish between pea and pulmonary metastases of colorectal carcinoma (mcrc), so new identification methods need to be explored. satb2 expression is tissue-specific, and the only epithelial cells expressing this protein in adult tissue are the glandular cells lining the lower gastrointestinal (gi) tract. the sensitivity of satb2 in colorectal adenocarcinoma reaches 80%-97%, and their low expression in primary pulmonary tumors. therefore, this study investigated differential diagnostic values of satb2 in pea and mcrc. design: according to the who primary pea diagnostic criteria, the cases of lung adenocarcinoma were collected from patients being treated at the renmin hospital of wuhan university from 2015.1-2019.9 were screened. the specimens were independently reviewed by two pathologists, and immunohistochemical staining of lung adenocarcinoma markers (ck7, ttf-1, and napsina) and intestinal cancer markers (ck20, cdx2, and villin) was performed to aid identification. finally, after excluding possible colorectal cancer metastasis by carefully analyzing the clinical histories and imaging examinations, we recruited 51 primary pea specimens and 17 mcrc specimens for study. the sensitivity and specificity of immunomarkers satb2, ck7, ttf-1, napsina, ck20, cdx2 and villin for distinguishing pea from mcrc are evaluated. the expression rates of satb2 in pea and mcrc were 0.00% (0/51) and 100.00% (17/17), respectively. the sensitivity of satb2-, ck7+, ttf-1+, napsina+, ck20-, cdx2-and villin-for distinguishing pea from mcrc were 100.00%, 98.04%, 49.02%, 45.10%, 66.67%, 58.82%, 47.06%, respectively. the specificity of satb2-, ck7+, ttf-1+, napsina+, ck20-, cdx2-and villin-for distinguishing pea from mcrc were 100.00%, 88.24%, 88.24%, 100.00%, 82.35%, 88.24%, 88.24%, respectively. conclusions: our study shows that the sensitivity and specificity of satb2, which can all reach 100%, is much higher than those of common lung adenocarcinoma immunomarkers (ttf-1, napsina) and intestinal cancer immunomarkers (ck20, cdx2 and villin). satb2 can be viewed as the best immunomarkers for distinguishing pea from mcrc. the diagnostic value of ck7 is slightly inferior to satb2, the results of ck7 can be used as a reference for differential diagnosis of satb2. lisi yuan results: of 2296 nsclcs tested between 2017-7/2019, met ex14 variants were present in 44 (1.9%). a recurring vus not expected to impact exon 14 splicing seen in 41 cases (c.2975c>t (p.thr992ile) was excluded from analysis. in positive cases, median age was 76 (59% men; 41% women), and 46.7% were fna specimens. 32 of 44 variants were met exon 14 skipping (previously reported and/or involve the canonical recognition site), while the other 12 mutations were significant missense (3) or vus (9). of 9 vus, 5 were adjacent to the canonical splice site and likely to impact splicing, and 4 were missense variants. average allele fraction was 30.2. four cases had concomitant mutations (3=kras, 1 =egfr). of 35 cases with known clinical staging, stage 1-2=20(57%), stage 3=3 (9%), and stage 4=12(34%). of 19 resected nsclss, histological types and growth pattern included 7 lepidic predominant, 6 acinar predominant, 2 micropapillary predominant, 1 solid predominant, 1 sarcomatoid, and 2 adenosquamous. pd-l1 expression in 27 cases is shown in table 1 . stage 4 pd-l1 <50% pd-l1 > 50% pd-l1 <50% pd-l1 > 50% 12 cases (67%) 6 cases (33%) 2 cases (33%) 4 cases (67%) figure 1 -1974 conclusions: most met variants identified in our cohort (73%) are met ex14 skipping. another 11% likely result in exon 14 skipping, while the other 16% are missense variants presumably unrelated to splicing. the prevalence of met ex14 variants is lower than previously reported (1.9% vs 3%), and a large percentage of tumors has lower clinical stage and less aggressive pathologic features, both possibly reflecting sampling differences attributed to universal testing of nsclc at our institution rather than testing of only advanced disease. background: the 2015 who classification of lung tumors provided the first specialized classification for small biopsies. this article aimed to apply the newest classification to reclassify a group of small lung biopsies and analyze their status of the main driver mutations. design: 5032 cases of small lung biopsies (bronchoscopic, needle, or core biopsies) were selected, which ranged from 2015 to 2018. we applied the newest classification to reclassify them and analyzed the relationship between the diagnostic subtypes of these biopsy specimens and the mutation rates of egfr and alk. the numbers of small lung biopsies each year during 2015-2018 were respectively 1068, 1299, 1511 and 1154. there were 3280 men and 1752 women, ranging in age from 11 to 93 years (median 63 years). the most common diagnosis was primary lung cancer (3130, figure 1 -1978 figure 2 -1978 conclusions: this study gave an panoramic view on pd-l1 expression and clinicopathological profiles based on the largest chinese nsclc cohort. the discrepancy of pd-l1 expression between surgically resected specimens and biopsy specimens and metastatic lesions may result from inter/intra-tumoral heterogeneity. background: accurate assessment of pd-l1 expression is critical for selection of patients of non-small cell lung cancer (nsclc) for immunotherapy with pd-l1/pd-1 inhibitors. however, only limited reports of pd-l1expression in population of nsclc in north america are available. this study reports pd-l1 expression level in a large patient population of nsclc in canada. design: pd-l1 testing of nsclc was performed for patients from the whole province of british columbia, canada in a centralized provincial pathology laboratory at bc cancer, vancouver centre. the test used dako pd-l1 ihc 22c3 pharmdx and dako autostainer link 48 immunostainer, as it was fda approved companion diagnostic test for pembrolizumab. pd-l1 protein expression is determined by using tumor proportion score (tps), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. results: from january 2017 to march 2018, 1,716 nsclc was tested, which included 1,301(75.8%) adenocarcinoma, 284 (16.6%) squamous cell carcinoma and 131 (7.6%) nsclc, nos. pd-l1 expression level in adenocarcinoma was similar to that in squamous cell carcinoma (p>0.05), but was significantly higher than in nsclc, nos (p<0.05, table 1 ). the overall percentage of high pd-l1expression (tps³50%) was 39%. the high pd-l1 expression (tps³50%) was found in 42% distant metastases, 45% mediastinal lymph nodes, and 35% lung primary tumors. the differences in the distribution of high pd-l1 expression among distant metastatic sites, mediastinal lymph nodes metastases and primary sites had statistical significance (c2=11.8, p<0.01). conclusions: using fda approved dako pd-l1 ihc 22c3 phamadx assay, we found that adenocarcinoma had a similar pd-l1 expression level to squamous cell carcinoma but significant higher expression than nsclc, nos. the percentage of high pd-l1 expression (tps³50%) was significantly higher in mediastinal lymph nodes and distant metastatic sites than in primary sites, which suggests that pd-l1 testing of metastatic nsclc could identify more patients eligible for immunotherapy. figure 1 -1980 figure 2 -1980 conclusions: the correlations among fs, fsc, and rt are low. fs stas+ cases remain stas+ only in 60% of fsc and 48% of rt. stas shows higher correlation with grade on rt than it does on fs. these results show a lack of reliability in the assessment of stas on fs, and do not support the proposal of reporting stas in fs to make intraoperative clinical decisions, as doing so may subject patients to unnecessarily aggressive surgery. ki67% when 6 classes were used (log-rank p=0.82 and p=0.21, respectively) or when 2 classes were used with either 20% ki67% (logrank p=0.95 and p=0.53) or 40% ki67% (log-rank p=0.78 and p=0.91) as cut-point conclusions: our findings do not support the use of 40% as the minimum ki67% in lcnec as suggested by who or the use of 20% as the minimum ki67% for lcnec as described for diagnosing entero-pancreatic neuroendocrine carcinoma expression of insulinoma-associated 1 (insm1) in non-small cell lung cancers: a diagnostic pitfall for neuroendocrine tumors none background: insulinoma-associated 1 (insm1) has recently been reported as a highly sensitive and specific marker of pulmonary neuroendocrine tumors. it has also been noticed that insm1 expression can be seen, although uncommonly, in non-neuroendocrine tumors. the aim of this study was to evaluate the expression of insm1 in non-small cell cancers (nsclcs) to avoid diagnostic pitfall uv genomic signature classifies lung melanomas of unknown primary as metastases from occult cutaneous melanomas grant or research support bristol myers squibb; advisory board member, immunocore; consultant, castle biosciences; marc ladanyi: none none alterations were identified for a total of 2 -5 shared alterations per pair (mean of 3 shared alterations). the probability of chance co among the primary lung cancer, the dominant type was adenocarcinoma (1421, 28.2%), followed by nscc, favor adenocarcinoma (501, 10.0%), squamous cell carcinoma (368, 7.3%), and nscc, favor squamous cell carcinoma (360, 7.2%). the tests of the main driver mutations using arms-pcr technology demonstrated that egfr was positive in 56.1%(499/889, in adenocarcinoma and nscc, favor adenocarcinoma) 6%, 35/148) and p.s768_d770dup (20.9%, 31/148) were most frequent. 94.4% (320/339) of erbb insertions were found in adenocarcinoma, among which surgical samples were more common than small biopsies (10.1% vs 7.3%, p=0.016). among adenocarcinoma, female have higher frequencies of erbb insertions than male (11.3% vs 6.8%, p<0.001). the median age of egfr insertion carrier, erbb2 insertion carrier and non-erbb insertion carrier was 54-, 49-and 62-year old respectively (p<0.001). compared with invasive adenocarcinoma (ia) (5.6%), adenocarcinoma in situ (ais) (28.4%) and minimally invasive adenocarcinoma (mia) (20.0%) were more likely to harbor an erbb insertion (p<0.001). the pleural invasion frequency of egfr insertion carrier, erbb2 insertion carrier and non-erbb conclusions: the insertion mutations in kinase domain of egfr and erbb2 were more common in younger, female and adenocarcinoma patients. ais and mia were more frequent to harbor an erbb insertion than ia, which suggest erbb insertion may be related to the evolution of adenocarcinoma. erbb2 insertion carrier tend to have a lower pleural invasion rate while a higher lymph node metastasis rate correlation between pd-l1 expression and clinicopathological and molecular characteristics of non-small cell lung cancer: a large scale multi-centric real-world study of chinese cohort the first affiliated hospital of none background: programmed cell death ligand-1 (pd-l1) is a predictive marker of anti-pd-1/pd-l1 immune therapies for non-small cell lung cancer (nsclc).the definite relationship between pd-l1 expression and clinicopathological, molecular profiles of nsclc design: a total of 6126 nsclc specimens were enrolled from 6 centers in china. we analyzed pd-l1 (22c3) expression by immunohistochemistry on dako autostainer link 48 platform. the status of egfr was defined by rt-pcr or ngs in 2382 samples and alk was tested by ihc, fish or ngs in 1716 samples pd-l1 high expression was more frequent in egfr-wild type than in mutant type (12.9% vs. 4.7%, p < 0.001). furthermore, pd-l1 high expression was more prevalent in rare egfr mutant types than in common mutations (42.1% vs. 20.8%, p=0.031). besides, pd-l1 high expression was more frequently identified in alk fusion cases (14.6% vs. 6.5%, p= 0.001). a total of 1665 small biopsy cases included 1454 primary specimens and 211 metastatic specimens. the prevalence of pd-l1 high expression in surgical samples was much lower than in primary biopsy samples. among of them, pd-l1 high expression was also prevalent in egfr-wild type than in mutant type (32 we found that high pd-l1 expression was more prevalent in metastatic specimens than in primary biopsy specimens (30.8% vs. 21.8%). in metastatic adc specimens, the rate of high pd-l1 expression was greater than in primary adc navneet narula 4 , mari mino-kenudson 3 , andre moreira 5 1 nyu school of medicine none background: spread through air spaces (stas) has been reported to be associated with a worse prognosis in adenocarcinoma of lung. recently it has been proposed that stas be reported on frozen sections (fs) as an indication for more aggressive surgery (lobectomy vs sublobar resection). we undertook this study to evaluate the reliability of stas assessment on fs compared to fs controls (fsc) design: cases of adenocarcinoma that had fs of the tumor were identified retrospectively from two institutions. for each case, the following was recorded: presence(+)/absence(-) of stas on fs, fsc, and rt; and % of tumor patterns: lepidic(l) cross-tabulations and spearman's correlations (rs) were performed in spss (see table) of which 28/47(60%) had stas+ on fsc (rs=0.39) and 22/46(48%) had stas+ on rt (rs=0.34) (1 tumor was only present on fs/fsc). of the 40 stas+ cases on rt, 18/40(45%) did not have stas (stas-) on fs (rs=0.34). 118/165 of cases were stas-on fs; of these of the 15 g2 cases with stas+ on fs, 7 had 10% to <20% high grade pattern (m/s) we performed a retrospective review of patients with imas who had genomic analysis performed on tumors in different lobes. molecular assays included dna-based targeted next-generation sequencing (ngs) for 410-468 cancer genes combined with rna-based ngs fusion assay (archer) and non-ngs panels for a subset of cases. tumor clonal relationships were assessed by comparing somatic alterations between the separate tumor sites.results: twenty-one patients with genomically-profiled imas involving contralateral (n=19) or ipsilateral different lobes (n=2) were identified. in most patients (n=14), tumors had discrete nodular presentation. second ima presented metachronously in 11 patients with a mean latency of 4.2 years. notably, in 3 patients, contralateral spread manifested ≥8 years (up to 11 years) after initial tumor resection. genomic analysis was performed on 2 separate imas in 19 patients and 3 separate imas in 2 patients, resulting in a total of 44 genotyped tumors. comparative genomic analysis revealed that tumors in all patients shared matching driver alterations including kras (n=17), nrg1 (n=2), erbb2 (n=1) and braf (n=1). in addition, in tumor pairs profiled by ngs and archer, other shared we have encountered a group of patients with melanomas involving the lung in the absence of a clinically known primary melanoma elsewhere. a subset of patients presented with solitary large tumors. while primary pulmonary melanomas (ppm) is a category included in the thoracic who classification, given the absence of normal melanocytes in the lung its existence has been questioned. herein we investigate genomic profiles of melanomas of unknown primary origin involving the lung. in particular, we sought to determine whether uv genomic signature -a characteristic feature of most cutaneous melanomas -is present in such tumors.design: cases of melanomas involving the lung with no known primary elsewhere were identified retrospectively. the clinicopathologic characteristics of each case were annotated. all cases included in the study underwent targeted dna next-generation sequencing (ngs) interrogating up to 468 cancer genes. genomic signatures were analyzed based on a method described by alexandrov lb et al. (nature 2013; 500:415-421) .results: ten ngs-profiled melanomas involving the lung were identified. five patients had solitary lung lesions with the median size of 5.2 cm (range 2.6 to 10.1 cm). of those, 3 tumors were endo/peri-bronchial, thus meeting the suggested criteria for ppm. hilar nodes were involved in 2 patients, and 8 tumors had epithelioid morphology resembling non-small cell carcinoma. no evidence of primary melanomas was found for any patients on clinical follow-up (median 28 months; range 5 to 42 months). genomic testing revealed the following driver mutations commonly found in melanomas: braf (n=6), nras (n=1) and kit (n=1). genomic signature analysis was feasible for 8 cases harboring >20 mutations required for reliable analysis, including 4 patients with solitary masses. this revealed the presence of a dominant uv signature in all cases. in contrast, none of the primary lung carcinomas tested by the same method (n = 255) had a uv signature. the consistent presence of a uv signature provides strong support for an occult or regressed cutaneous origin of melanomas involving the lung, and argues against the concept of ppm. clinical presentation as solitary large (reaching >10 cm) masses occasionally with hilar adenopathy and epithelioid morphology may closely mimic primary lung carcinomas both clinicoradiologically and pathologically, representing a major potential diagnostic pitfall. background: pulmonary invasive mucinous adenocarcinoma (ima) commonly presents as a multifocal disease. it is widely recognized that diffuse 'pneumonic-type' ima represents aerogenous spread of a single tumor. however, imas may also present as discrete nodules in different lobes, raising the possibility of separate primary tumors. here, we explored the clonal relationship of imas involving different lobes using comparative molecular profiling.conclusions: molecular profiling supports that multifocal imas involving different lobes represent intrapulmonary spread of a single tumor rather than separate primary tumors, including tumors presenting contralaterally after a remarkably long latency (>8 years). overall, these findings reinforce the unique biology and clinical behavior of imas, and draw a sharp distinction with multifocal non-mucinous lung adenocarcinomas, which recent molecular studies confirm to represent predominantly separate primary tumors. we addressed these issues in human non-small cell lung cancer (nsclc). design: pd-l1 and b7-h3 expression in tumor cells were evaluated using immunohistochemistry. composition of tumor-infiltrating immune cells, including lymphoid cells, macrophages and dendritic cells, was analyzed using flow cytometry for fresh tissues from a prospective cohort of 71 patients with nsclc and was compared according to pd-l1 and b7-h3 expression status. ju-yoon yoon 1 , jason rosenbaum 2 we examined the local cohort of patients with the referral diagnosis of "lung cancer", sequenced by the in-house 152 gene massively parallel sequencing (mps, also known as next-generation sequencing or ngs) assay. all alterations were filtered and reviewed for disease-associated variants. for atm, our assessment was limited to p.v2424g, a variant with the highest penetrance among the atm variants, associated with increased breast cancer risks at levels comparable to disease-associated brca1/2 variants.results: 1,473 cases were successfully sequenced by the in-house solid mps assay, among which 23 patients (1.6%) were found to harbor disease-associated brca1 (14/23) or brca2 (9/23) alterations, with variant allele fractions (vafs) ranging 4-69%. these cancers were mostly adenocarcinomas (17/23, with 5 carcinoma nos and 1 scc). 10/23 harbored kras hotspot mutations, and no cases the criteria for small lung biopsies proposed by the 2015 classification of lung tumors should be applied to pathologists' daily work. it can improve the diagnostic efficiency and quality of small lung biopsies and assist oncologists in accurately understanding the pathologic diagnosis. in this way, accurate treatment and improved prognosis are more available to the patients. zhihong zhang background: to study the differential diagnosis of multi-focal lung cancer and lung cancer with pulmonary metastasis by detecting the different lesions of the same patient. to explore the differences in prognosis between mplc and im, and to explore the factors affecting the prognosis of multi-focal lung cancer and the tumor heterogeneity of multi-focal lung cancer in combination with histopathology and molecular biology.design: fifty patients with multi-focal lung cancer were screened, and the relevant clinical information was noted; the patients were diagnosed by accp standard. mutations of the lesions were detected by arms-pcr, and the detected genes included egfr, alk, ros1, met, kras, ret, her-2, braf, nras and pik3ca. the results of genetic testing were compared with those of accp standard diagnosis. we analyzed a total of 101 tumors from 50 patients. classification based on gene testing contradicted the clinicopathologic diagnosis in 10 (20%) of the comparisons, identifying independent primaries in 6 cases diagnosed as metastasis and metastases in 4 cases diagnosed as independent primaries. another 7(14%) tumor pairings were assigned an "equivocal" result based on gene testing. the results of gene testing of the remaining 33(66%) tumor pairings were consistent with the clinicopathologic diagnosis. the mutant heat map indicated that im patients have a higher rate of mutation consistency than mplc patients. the difference of prognosis between patients with mutations and those with wild-type genes patients was statistically significant (p=0.002). the difference of prognosis between patients with lymph node metastasis and those with no metastasis of lymph nodes was statistically significant (p=0.006). the difference of prognosis between patients with mplc and those with im was statistically significant (p=0.038). the difference of prognosis between patients who had different condition was statistically significant (p=0.038). multi-gene detection of multi-focal lung cancer has a certain auxiliary effect on the differential diagnosis of multiple primary lung cancer and lung cancer with pulmonary metastasis, which can complement the clinical standards, but also has some limitations. key: cord-277173-zdft23q8 authors: cauchemez, simon; ferguson, neil m. title: methods to infer transmission risk factors in complex outbreak data date: 2012-03-07 journal: j r soc interface doi: 10.1098/rsif.2011.0379 sha: doc_id: 277173 cord_uid: zdft23q8 data collected during outbreaks are essential to better understand infectious disease transmission and design effective control strategies. but analysis of such data is challenging owing to the dependency between observations that is typically observed in an outbreak and to missing data. in this paper, we discuss strategies to tackle some of the ongoing challenges in the analysis of outbreak data. we present a relatively generic statistical model for the estimation of transmission risk factors, and discuss algorithms to estimate its parameters for different levels of missing data. we look at the problem of computational times for relatively large datasets and show how they can be reduced by appropriate use of discretization, sufficient statistics and some simple assumptions on the natural history of the disease. we also discuss approaches to integrate parametric model fitting and tree reconstruction methods in coherent statistical analyses. the methods are tested on both real and simulated datasets of large outbreaks in structured populations. data collected during field outbreak investigations are essential to better understand the clinical and epidemiological features of an infectious disease. they can also provide useful insights for outbreak management and control. for example, evaluating the risk factors governing transmission is important to design efficient control measures, and identify those individuals that are most at risk of infection or are the main contributors of infection and should therefore be targeted first. however, characterizing transmission from outbreak data can be challenging. first, the transmission process is usually imperfectly observed. for example, we may observe the date of symptoms onset of a case, but we rarely know where, when and by whom a case was infected. inference, therefore, requires integrating over 'missing data', which may quickly become cumbersome. over the last 15 years, data augmentation methods have been used to tackle this problem: data are augmented with missing data (e.g. dates of infection) that are needed to write down the likelihood; in a bayesian setting, the joint posterior distribution of parameters and augmented data is explored usually via markov chain monte carlo (mcmc) sampling [1] . this methodology is now well established in the field and has been successfully applied to analyse a range of complex datasets. however, for relatively large outbreaks with detailed data, this approach may require very long computational times. interested readers can, for example, read references [2 -11] . the second challenge is that the type of dependency between observations that is typically observed in an outbreak (i.e. the risk of infection of an individual depends on the infection status of other individuals) is specific to communicable diseases and needs to be accounted for with dedicated methods. this usually requires that the statistical model used to analyse the data is explicitly based on a mechanistic model of disease spread [12] . transmission parameters of interest, for example, the reproduction number (the number of individuals infected by a case), are usually mathematically defined in those models. fitting such parametric mechanistic models to outbreak data can give useful insights on transmission [7, [13] [14] [15] , but is subject to the same limitations as parametric fitting in other fields. for example, although there are some exceptions [10] , the approach usually requires to predefine time intervals on which transmission rates are constant. this can sometimes be difficult to achieve in a non-ad hoc way. an alternative approach that has become increasingly popular is to reconstruct the transmission tree and derive important summary statistics from it, for example, the temporal trends in the reproduction number [16 -18] . this may give greater flexibility (for example, there is no need to specify time intervals with constant transmission rates), potentially at the cost of larger variance of the estimates [15, 18] . however, since these methods generally only consider disease *author for correspondence (s.cauchemez@imperial.ac.uk). cases rather than the uninfected, but potentially susceptible bulk of the population, they can say little about the risk factors for infection or provide estimates of transmissibility in different contexts (e.g. households, schools or as a function of distance between a susceptible and an infected individual). overall, the two methodologies (fitting of a parametric mechanistic model and tree-reconstruction methods) are largely complementary. fitting a mechanistic model seems to be the only way to account for the depletion of susceptibles, the information on uninfected individuals leading to a quantification of relative risks; and it may ensure a better control of the variance of the estimates. tree-reconstruction methods can provide further insights on what effectively happened during the outbreak with summary statistics on who was infected by whom, when and where and temporal change in the reproduction number. they can also provide a framework to detect abnormal features in the data that are not initially accounted for in a mechanistic model. it is therefore important that the two approaches can be integrated in a coherent way. in this paper, we discuss strategies to tackle some of the ongoing challenges in the analysis of outbreak data. we present a relatively generic statistical model for the estimation of transmission risk factors, and discuss algorithms to estimate its parameters for different levels of missing data. we look at the problem of computational times for relatively large datasets and show how they can be reduced by appropriate use of discretization, sufficient statistics and some simple assumptions on the natural history of the disease. we also discuss approaches to integrate parametric model fitting and tree reconstruction methods in coherent statistical analyses. the methods are tested on both real and simulated datasets of large outbreaks in structured populations. assume that we observe the spread of a disease in a population of size n from day 0 to day t. for each individual i ¼ 1, . . . , n, let y t i ¼ 1; if individual i is infected between 0 and t; 0 otherwise. each individual i is characterized by a vector of q covariates z i (t) ¼ fz i 1 (t), . . . , z i q (t)g such as age, gender, location, household id, etc . . . that may vary with day t. we want to quantify the transmission risk factors. we first consider the situation where day t i of infection of each case i is observed (by convention, . this assumption is relaxed in §5. for a directly transmitted disease, the first step to estimate transmission risk factors is usually to propose a model for transmission hazard l i!j ðtjqþ from case i to subject j on day t, i.e. define l i!j ðtjqþ as a function of the individual covariates z i (t) and z j (t) and a set of parameters q. for example, the transmission hazard l i!j ðtjqþ may depend on: -the time lag t-t i between day t and the day of infection t i of case i since infectiousness of a case may vary during the course of infection. the functional form between l i!j ðtjqþ and t-t i will depend on the assumed natural history of the disease; -the individual characteristics of subjects i and j. for example, some subjects may be more infectious while others are more susceptible; and -the type of interactions that exist between subjects i and j. the contact rate might, for example, depend on whether the subjects live in the same household or go to the same school, etc. it could also depend on the spatial distance between them. examples of specifications for the transmission hazard l i!j ðtjqþ are given in § §6 and 7. the force of infection exerted on individual j on day t is then the sum: l i!j ðtjqþ: the contribution to the likelihood of case j is: where the first term is the probability of infection on day t, and the second term is the probability to escape infection up to day t (the link between the continuous time and discrete time transmission model is discussed in appendix a). the contribution of non-case j is: the log-likelihood is therefore: the number of pairs of farms to be considered per calculation of the likelihood is over 10 8 [19] . even with the recent increase in computational power, brute force exploration of the system, though feasible, is very time consuming. fast and efficient algorithms are necessary to provide real-time support to decision making. here, we explore the extent to which the discretization of the transmission risk factors can reduce the computational burden associated with the evaluation of equation (2.3). therefore, we now restrict our analysis to the situation where each transmission risk factor takes a finite set of values. we will then explore how the approach can be used to investigate continuous risk factors. assume that the transmission hazard between two individuals depends on k risk factors x ¼ fx 1 , . . . ,x k g and that the k-th risk factor x k (k ¼ 1, . . . , k ) takes a finite number (¼c k ) of values fv k 1 ; . . . ; v k c k g. the set c of possible values for risk factor vector x ¼ fx 1 , . . . , x k g has size q k k¼1 c k . for example, the list of risk factors might include the (discretized) distance between the individuals (either spatial or social, e.g. members of the same household), the time lag since infection or individual characteristics, such as age. we model the transmission hazard between a case and a susceptible individual in the population by: where the specific effect of x k on the transmission hazard is measured by function b k ðx k ; u k þ ! 0, and u k ¼ fu k 1 ; . . . ; u k l k g is a parameter vector of size l k . this expression makes the simplifying assumption that the effect of all risk factors on the hazard can be expressed as the product of the impacts of each factor. parameters of the model are remembering that z i denotes data available for individual i, we assume here that risk factors for transmission from case i to individual j on day t are a function of triplet fz i , z j , tg: x ði;j;tþ ¼ fx 1 ði;j;tþ ; . . . ; x k ði;j;tþ g, where for k ¼ 1, . . . , k, x k ði;j;tþ ¼ g k ðfz i ; z j ; tgþ. the transmission hazard l i!j ðt j jqþ from case i to individual j on day t is therefore: l i! j ðtjqþ ¼ bðx ði;j;tþ ; qþ: the total hazard of infection for individual j on day t is then: where here d a,b is the kronecker delta or identity function (¼1 if a ¼ b and 0 otherwise). m x counts the number of day-transmission events of type x, which might have occurred before time t but did not. equation (3.2) has important implications in terms of computational speed since it shows that the computational burden can be substantially reduced without loss of information. the first term of equation (3.2) only involves disease cases; the second term characterizes the probability of escaping infection up to time t. so, data needs for inference reduce to: -'case data'; -table fm x g x[c of sufficient statistics that characterize the interaction of cases with any individual of the population. it can be pre-computed and stored once, given they do not functionally depend on the parameters. tree reconstruction [7, [16] [17] [18] is a useful complement to the likelihood-based estimates of the parameters derived from equation (3.2) . here, we present and discuss different strategies to perform tree reconstruction and parameter estimation in a coherent statistical framework. given parameter q and given that case j was infected on day t j , the probability that case j was infected by case i (t i , t j ) is simply (see appendix a): a natural way to integrate tree reconstruction and parameter estimation in a coherent setting is to proceed sequentially. for example, in a bayesian setting, a sample fq m g m¼1;...;m can be drawn from the posterior distribution of q via mcmc sampling relying on equation (3.2) . then, for each parameter value in the sample m ¼ 1, . . . ,m, a source of infection r j can be drawn from its distribution f p i!j ðt j jq m þg i for each case j. this gives a sample of m transmission trees drawn from their predictive distribution. for example, we used this strategy when analysing detailed data from an influenza outbreak in a school [7] . here, we explore an alternative strategy where the two tasks are performed simultaneously. the method formalizes those introduced in work undertaken in 2001 on the uk fmd epidemic of that year [13] . the idea is that the source of infection r j for each case j is considered as 'augmented' data. the augmented loglikelihood is: in many situations, where the force of infection exerted on individuals is relatively small, the likelihood simplifies to: the analysis of the augmented likelihood can be performed in a frequentist or in a bayesian setting. in the frequentist setting, it is straightforward to implement an expectation conditional maximization (ecm) algorithm [20, 21] to both derive maximum-likelihood estimates of the parameters of the model and reconstruct the transmission tree. the pseudo-code for this algorithm is given in box 1. in the common situation, where one is interested in relative risks (i.e. comparison with a reference group) and where the force of infection exerted on individuals is relatively small, no maximization routine is needed since the maximum value is simply a ratio of two sums (appendix a) [13] . confidence intervals can be obtained from the fisher information matrix derived at the maximum value for the incomplete log-likelihood (equation 3.2). the approach, therefore, requires that the second derivative of the log-likelihood with respect to q exists. alternatively, inference can be performed in a bayesian setting via mcmc [1] , with a pseudo-code presented in box 2. the key difference is in the way missing data are handled: in the bayesian approach, one realization of the missing data is drawn from its expected distribution; by contrast in the em algorithm, the whole expected distribution of the missing data is used in the expression of the augmented likelihood. often, the day of symptom onset s j of case j is observed but not the day of infection t j . as a consequence, box 1. the em algorithm when the days of infection are observed. assume that at the beginning of iteration n, parameter vector is q n21 : -expectation step: (i) for each case j, compute probabilities f p i!j ðt j þg i given q n21 (equation 4.1; see appendix a), and (ii) for x in c, compute the expected number of transmission events of type x : other parameters being fixed (see appendix a): bayesian algorithm when the days of infection are observed. at iteration n: -update missing data: (i) for each case j, compute probabilities f p i!j ðt j þg i given q n21 (equation 4.1; see appendix a), (ii) for each case j, draw the source r j of case j from distribution f p i!j ðt j þg i , and (iii) for x in c, compute the number of transmission events of type x: -update parameters: (i) for k ¼ 1, . . . , k, and (ii) for j ¼ 1, . . . , l k : mcmc update u k j relying on the augmented likelihood l c (see appendix a): methods to infer transmission risk factors s. cauchemez and n. m. ferguson 459 likelihood as shown in equation (3.2) is no longer available. if the incubation period (time lag between the day of infection and the day of symptoms onset) has a known density f (s j jt j ), a common strategy to tackle the problem is to augment the data with the day of infection of each case. a particular computational burden is then that updating the day of infection of a single case may require re-calculation of the whole likelihood as the update may affect the risk of infection of all other individuals. in order to avoid this computational cost, we introduce the additional assumption: (h1) given the day of symptoms onset s j , infectiousness over time is independent of the day of infection t j . this is, for example, the case if infectiousness starts with symptom onset. this assumption seems acceptable for a relatively wide range of diseases since infectivity is often triggered or influenced by symptoms. under h1, there is no need to re-compute the whole likelihood each time a day of infection is updated. it would be possible to extend the em approach to the situation when the days of infection are unobserved. inference would work as in the previous section except that one would have to take the expectation on both the contact tracing information and the day of infection. however, for this second application, it is no longer possible to easily derive the variance of the estimates. this is because, although maximum-likelihood point estimates could be derived from the likelihood for the 'complete' dataset, estimation of the variance of the estimates has to rely on the likelihood of the observed dataset (equation 3.2). this expression cannot be computed here since days of infection are not observed. the bayesian approach does not suffer from this limitation and makes it possible to easily obtain bayesian credible intervals (box 3). we explore how the discretization of risk factors and the algorithms introduced in §4 can reduce the computational burden of inference. for simplicity, farms are partitioned into three groups on the basis of the number of cattle n c and the number of sheep 3n s : cattle (cattle farm: n c ! n s ), sheep (sheep farm: n s . n c ) and small (small farm: n c þ n s , 100) [13] . we assume that the latent period of fmd is 3 days and that infectious farms remain so until the time of slaughter. the transmission hazard b between case farm i and susceptible farm j that is introduced in equation (3.1) is modelled as a function of the following characteristics: -the type (cattle, sheep or small) of case farm i. we estimate the relative infectivity of sheep farms ðg sh i þ and small farms ðg sm i þ relative to cattle box 3. bayesian algorithm when only days of symptom onset are observed. at iteration n: -gibbs sampling for missing data: (i) for each case j, compute probabilities f p j ðtþg t for the day of infection of the case given q n21 (see appendix a), (ii) for each case j, draw the day t j of infection of the case from distribution f p j ðtþg t , (iii) for each case j, compute probabilities f p i!j ðt j þg i given q n21 (equation 4.1; see appendix a), (iv) for each case j, draw the source r j of case j from distribution f p i!j ðt j þg i , and (v) for x in c, compute the number of transmission events of type x: -update parameters: (i) for k ¼ 1, . . . , k, and (ii) for j ¼ 1, . . . , l k : mcmc update u k j relying on the augmented likelihood. the augmented likelihood is slightly different from the case when days of infection are known (equation 3.2). in particular, since days of infection of cases change during the inference procedure, it is no longer possible to pre-compile and store the contribution of cases to matrix fm x g (i.e. number of day-transmission events of type x that could have occurred but did not); but one can still pre-compile and store the contribution fmm x g of non-cases, which is usually the key computational burden. the augmented likelihood is: farms. so the multiplicative term on the transmission hazard is g sh i ; if case farm is a sheep farm, g sm i if it is a small farm and 1 if it is a cattle farm; -the type (cattle, sheep or small) of susceptible farm j. we estimate the relative susceptibility of sheep farms ðg sh s þ and small farms ðg sm s þ relative to cattle farms; and -the distance d ij between farm i and farm j. two models are considered: m1: discrete model. we assume that the transmission kernel is a step function with k þ 1 change points fd k g k¼0, . . . ,k and where the multiplicative term on the transmission hazard is g k d if d k21 , d ij d k . in practice, we take 18 change points f0, 0.5, 1, 1. ð6:1þ where qða; bþ ¼ 1= ð 2000 d¼0 ð1 þ u=aþ àb du is a normalizing constant. here, we introduce a discretized version of this kernel. consider k þ 1 change points fd k g k¼0, . . . ,k . we define d k ; the mean distance between farms i and j satisfying d k21 , d ij d k : the transmission kernel is the step function: in for the ecm algorithm, we consider that convergence is achieved at iteration n if the relative change in parameter values between iteration n and iteration n þ 1 is smaller than 10 27 for all parameters. in the bayesian implementation of the model, we specify the following priors for our parameters. parameters g sh i ; g sm i ; g sh s ; g sm s ; g 1 d ; . . . ; g k d all have a gamma prior g(a, a) with a ¼ 10 22 . we also do a sensitivity analysis for a ¼ 10 21 , 1. for model m2, we specify a uniform prior u [0,100] for kernel parameters a and b and a gamma prior g(a, a) for parameter c. the mcmc is run for 8000 iterations with a burn in of 500 iterations. transmission parameters are estimated for time interval 23rd february (when the national ban on animal movements was introduced) to 5th october 2001, conditional on the state of the epidemic on 23rd february. computation times are given for single threaded code running on an intel xeon x5570 system. we first use the ecm algorithm (box 1) to estimate model m1 (table 1) . convergence is achieved in only 91 iterations (figure 1). total computational time is 1 min 14 s with most of the time (58 s) spent reading the data and computing the table fm x g x[c of sufficient statistics (equation 3.2). in particular, there is no need to use maximization routines since there is an analytical solution to the conditional maximization step (see appendix a). computational times are also very short (4 min 28 s) for the bayesian algorithm (box 2). estimates of transmission kernel m2 are obtained in 6 min 41 s (table 2) . those computational times contrast with those needed through brute calculation of the likelihood (equation 2.3). replacing model m2 by the exact continuous parametric kernel (equation 6.1) and using equation (2.3) does not affect estimates (table 2); but computational times move to a month for the same number of iterations of the mcmc, on the same machine and with no serious attempt to optimize the code. while algorithmic optimization and parallel programming allows this to be reduced to a few days [19] , the algorithms presented here still give comparable estimates two orders of magnitude more rapidly than brute force approaches. we simulate an epidemic in a city that is structured in households and hospitals and where community transmission can happen. table 3 summarizes the structure of the city. we consider a city of size 400 000 with an average household size of 2.2 persons and with household demographics consistent with the 1999 french census [22] . the city has three hospitals with 2240 staff members and 800 beds each, a bed occupancy of 70 per cent outside the epidemic period for a duration of hospitalization of 10 days. we simulate the spread of a disease in this population and would like to assess how the techniques described above can be used to evaluate and monitor transmission in the different settings (community, hospital and household), infectivity and susceptibility of different types of individuals (here: children versus adults) along with the efficacy of the interventions that are put in place in the different settings. we are interested in a scenario like the severe acute respiratory syndrome (sars) rather than, for example, an influenza scenario; that is a disease for which it is possible to detect and identify a substantial proportion of cases. we assume that the incubation period of the disease has a geometric distribution (with probability 0.3, truncated after 10 days); that individuals start to be infectious on the day of symptoms onset with an infectivity profile following that time which has an exponential shape with mean 3 days (truncated after 20 days). we assume that 20 per cent of cases are hospitalized with equal probability of hospitalization occurring 1 or 2 days after symptoms onset. we assume that children are 1.5-fold more susceptible and more infectious than adults. following cauchemez et al. [8] , we assume that the person-to-person household transmission rate is inversely proportional to the size of the household. the epidemic starts with five cases infected on day 0. control measures targeting community, household and hospital transmission each with an efficacy of reducing transmission of 50 per cent are implemented on day 60 of the outbreak. we consider different scenarios for the proportion of cases that are detected in the population. initially, we assume that all cases are detected. in alternative scenarios, we investigate the situations where 50 or 25 per cent of cases are randomly detected in the community/hospital, but where follow-up of households with detected cases is good (90%) and, last, the situation where detection of cases among household members is of the same quality as detection of cases in the community and in hospitals. the transmission hazard b between case i and individual j that is introduced in equation (3.1) depends on the following characteristics: -setting, i.e. whether individuals i and j are (i) members of the same household, (ii) have visited the same hospital or (iii) other (i.e. community transmission)-the multiplicative term on the transmission hazard is b hous /n (n: size of the household), b hosp /n hosp (n hosp : number of staff members plus average hospital occupancy outside an epidemic) and b com /n com (n com : size of the city). we specify a gamma prior g(10 25 , 10 23 ) for b hous , b hosp and b com ; -infectivity profile from symptom onset of case imodelled with a normalized discretized exponential distribution with a mean to be estimated. we specify a uniform prior u [1,10] for the mean value; -whether or not case i is a child-we estimate the infectivity of children relative to adults. we specify a lognormal prior distribution with log-mean 0 and log-variance 1 for the relative infectivity of children g i . this ensures that g i has the same prior as 1/g i ; -whether or not individual j is a child-we estimate the susceptibility of children relative to adults, g s , assuming a lognormal prior distribution with log-mean 0 and log-variance 1; and -efficacy of interventions implemented in the different settings (i.e. household, hospital and community). after implementation of the intervention (day 60), the transmission rate in the household, hospital and community is multiplied by parameter g hous , g hosp and g com , respectively; where g hous , g hosp and g com have the same prior as the relative infectivity and susceptibility of children. figure 2 summarizes the data that would need to be collected during the outbreak with the age and dates of symptom onset of cases (figure 2a), a follow-up of households with cases and tracking of hospitalizations and more generally of hospital occupancy (figure 2b) and a follow-up of epidemics in hospitals (figure 2c). inference also requires having information on the age distribution of the population. in the simulated outbreak, there were a total of 1842 cases with 631 (34%) child cases. in the scenario, where all cases are identified, figure 3 shows how estimates change in real-time. on day 20, only 31 cases have been detected and credible intervals of parameters are therefore wide. the credible interval includes the true value for all parameters except the relative infectivity of children. on day 30, with 80 cases detected, posterior means are always relatively close to the true simulation value although credible intervals remain wide for some parameters like the relative infectivity and susceptibility of children and the mean generation time. on day 40 (182 cases detected), we would rightly conclude that children are more infectious and susceptible than adults although here again the credible intervals remain relatively wide. properly characterizing the infectivity profile requires substantially more data (849 cases detected by day 60). only 10 days after control measures are implemented, fairly accurate estimation of the efficacy of interventions in different settings becomes possible. when only 50 per cent of cases in the community and in hospitals are detected, performance of the approach remains satisfying although as expected credible intervals are wider and it takes longer for accuracy to be acceptable (figure 4). when 25 per cent of cases in the community and in hospitals are detected, precision of estimates starts to break down (electronic supplementary material, figure s1 ). although estimates of transmission rates in the community and the hospital are not strongly affected by under-reporting in those settings, this is not true of estimates of transmission rates in small closed settings such as households (electronic supplementary material, figure s2 ). the method also allows disaggregated monitoring of the reproduction number and the number of cases infected in different settings (figure 5). in this paper, we have presented strategies to tackle some of the challenges associated with the estimation of transmission characteristics of infectious diseases and the risk factors affecting transmission patterns. the dependency that is typically observed in outbreak data (i.e. the risk of infection for an individual depends on the infection status of other individuals) can potentially lead to long computational times. we showed that if risk factors are discretized, the inferential problem can be simplified to the analysis of (i) a dataset on cases only and (ii) a pre-compiled summary table on interactions between individuals of the population and cases. in the fmd application, discretization reduced the computational time from few weeks to few minutes with no change in the estimates of the transmission kernel. it is likely that with substantial effort and parallel programming, we could have reduced the computation time of brute force approach by one or two orders of magnitude. even so, it seems unlikely that computational times could have gone much below few days. this has to be compared with the few minutes needed to run our algorithms on the fmd dataset. for small datasets, discretization may provide no computational gain if it takes longer to explore the set c of transmission risk factors than to sum over the pairs fcase i, individual jg. discretization may be difficult to implement on particularly complex transmission models [19, 23] in which case brute force calculation of the likelihood may be needed. we presented two strategies to perform parameter estimation and tree reconstruction in a coherent way. the first one is a sequential approach that we used to analyse data from an influenza school outbreak [7] . here, we implemented an alternative strategy where transmission parameters and the transmission tree are estimated simultaneously. there are pros and cons for each of those strategies. a nice feature of simultaneous inference is that the information on the transmission for situations when the dates of infection are unknown, we presented models which assumed that infectivity depends on the time elapsed since symptom onset, and is independent of the time of infection. this assumption reduces the computational burden since it implies that the infection hazard an individual is exposed to solely depends on measured quantities plus the parameters of the model, rather than on the unobserved days of infection of other cases. in practice, assumption h1 seems acceptable for a relatively wide range of diseases since infectivity is often triggered or influenced by symptoms. however, where the assumption is invalid, more computationally intensive methods accounting for the unobserved times of infection become necessary [23] . in the simulation study, we considered an epidemic for which it would be possible to detect and identify a substantial proportion of cases; this would therefore be more applicable to a sars-like scenario rather than pandemic influenza. the simulation study showed that even in situations where under-reporting is substantial (e.g. 50%), it would still be possible to obtain informative estimates of key characteristics. we found that estimates of the transmission rate in the community and in the hospital were relatively robust to under-reporting in those settings. this can be explained by the fact that in a large population, the exponential growth rate of the epidemic is not affected by under-reporting. but estimates of transmission rates in small social units such as households were-as might be expected-strongly affected by under-ascertainment of cases. estimates of relative infectivity were particularly sensitive to underreporting, with large variance and sometimes important bias. estimating relative infectivity is in general quite challenging because it requires that one can compare the offspring of one group of individuals (e.g. adults) with that of another group (e.g. children) and this becomes very difficult as under-reporting increases. it is likely that estimates would be less robust to underreporting, if reporting rates changed over time, as probably happens in real epidemics. here, we have presented relatively simple approaches to reduce computational burden in the estimation of transmission parameters and to integrate parameter estimation and tree reconstruction in a coherent way. however, the analysis of outbreak data is subject to many other challenges. for example, it may be difficult to infer which parametric distribution should be used for the infectivity profile and the incubation period; data augmentation strategies may fail in a context when data are not missing at random or when there are false-positives or false-negatives [23] . a particular challenge in outbreak data is that they are rarely informative about the incubation period of the disease. in the simulations study, for example, we made the assumption that the distribution of the incubation period of the disease was known. if it was not the case, one would require extra data to estimate it. for example, in the past, data from outbreaks in an aeroplane [24] or in a bus [25] were used to estimate the incubation period of influenza. a key practical challenge to implement the methods presented here in real time is the rapid collection and digitization of sufficiently detailed epidemiological data. however, recent experience demonstrates that it is possible to collect very detailed epidemiological data even during large outbreaks [10, [13] [14] [15] 25, 26] . cleaning and processing those data so that they are ready for analysis close to real-time remain a huge challenge, but the recent examples of the 2001 fmd outbreak in the uk [13, 14] , the 2003 sars epidemic [15, 26] and the 2009 h1n1 pandemic [25] show that this is increasingly feasible. however, reporting delays should always be expected and it will be important to account for those delays in future developments of the statistical method presented here. last, a key limit on the more widespread use of the type of methods presented in this paper is the relatively high technical hurdle to implement them, given there is currently no user-friendly statistical software package that allows easy implementation of this type of analysis. developing such tools is therefore a priority. we thank the mrc methodology programme, the nigms midas initiative, the eu fp7 emperie consortium and research council uk for research funding. we explain here the link between the continuous time and discrete time transmission models. at any (continuous) time point u during day t (i.e. u [ [t;t þ 1)), the instantaneous hazard of infection exerted on individual j is l ã j ðuþ ¼ l j ðtþ for t u , t þ 1; where l j (t) is defined in the main text. so, we make the assumption that the instantaneous hazard of infection is a step function with daily steps. conditional on the fact that individual j has not been infected up to day t, the probability that individual j is infected on day t is equal to in the continuous time model, conditional on the (continuous) time of infection u j of case j with u j [ [t j ; t j þ 1), the probability that case i is the case source is: p ã i!j ðu j jqþ ¼ l i!j ðu j jqþ p k:t k ,t j l k!j ðu j jqþ ¼ l i!j ðt j jqþ p k:t k ,t j l k!j ðt j jqþ : and we note that this probability is constant for u j [ [t j ; t j þ 1). conditional on the day t j of infection, the probability that case i is the case source given in equation (4.1) is: u j ¼t j p ã i!j ðu j jqþpðu j jt j þdu j and p i!j ðt j jqþ ¼ l i!j ðt j jqþ p k:t k ,t j l k!j ðt j jqþ ð t j þ1 u j ¼t j pðu j jt j þdu j ¼ l i!j ðt j jqþ p k:t k ,t j l k!j ðt j jqþ : let consider the common situation where -we are interested in relative risks (i.e. comparison with a reference group). for example, the population is partitioned in c k groups fn k 1 ; . . . ; n k c k g on the basis of risk factor k so that the multiplicative term associated with risk factor k in equation ( for the conditional maximization of the likelihood with respect to u k m in the ecm algorithm (box 1), the log-likelihood can be re-written: markov chain monte carlo in practice stochastic epidemic models and their statistical analysis statistical studies of infectious disease incidence a tutorial introduction to bayesian inference for stochastic epidemic models using markov chain monte carlo methods analyses of infectious disease data from household outbreaks by markov chain monte carlo methods bayesian inference for partially observed stochastic epidemics role of social networks in shaping disease transmission during a community outbreak of 2009 h1n1 pandemic influenza a bayesian mcmc approach to study transmission of influenza: application to household longitudinal data investigating heterogeneity in pneumococcal transmission: a bayesian-mcmc approach applied to a follow-up of schools temporal variability and social heterogeneity in disease transmission: the case of sars in hong kong statistical inference and model selection for the 1861 hagelloch measles epidemic infectious diseases of humans: dynamics and control transmission intensity and impact of control policies on the foot and mouth epidemic in great britain dynamics of the 2001 uk foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions estimating in real time the efficacy of measures to control emerging communicable diseases different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures transmission parameters of the 2001 foot and mouth epidemic in great britain maximum likelihood from incomplete data via the em algorithm (with discussion) maximum-likelihoodestimation via the ecm algorithm-a general framework estimating the impact of school closure on influenza transmission from sentinel data epidemiological inference for partially observed epidemics: the example of the 2001 foot and mouth epidemic in great britain an outbreak of influenza aboard a commercial airliner the early transmission dynamics of h1n1pdm influenza in the united kingdom. version 19 transmission dynamics and control of severe acute respiratory syndrome key: cord-167454-ivhqeu01 authors: battiston, pietro; gamba, simona title: covid-19: $r_0$ is lower where outbreak is larger date: 2020-04-16 journal: nan doi: nan sha: doc_id: 167454 cord_uid: ivhqeu01 we use daily data from lombardy, the italian region most affected by the covid-19 outbreak, to calibrate a sir model individually on each municipality. these are all covered by the same health system and, in the post-lockdown phase we focus on, all subject to the same social distancing regulations. we find that municipalities with a higher number of cases at the beginning of the period analyzed have a lower rate of diffusion, which cannot be imputed to herd immunity. in particular, there is a robust and strongly significant negative correlation between the estimated basic reproduction number ($r_0$) and the initial outbreak size, in contrast with the role of $r_0$ as a emph{predictor} of outbreak size. we explore different possible explanations for this phenomenon and conclude that a higher number of cases causes changes of behavior, such as a more strict adoption of social distancing measures among the population, that reduce the spread. this result calls for a transparent, real-time distribution of detailed epidemiological data, as such data affects the behavior of populations in areas affected by the outbreak. the basic reproduction number, or r 0 , represents the average number of secondary cases produced by a single infected case in an otherwise susceptible population, and it is typically used as a reference value to assess the transmissibility of an infectious disease in a given population. given a number of individuals susceptible to infection, a disease with higher r 0 will infect a larger number of individuals. there is hence an obvious positive relationship between the r 0 and the resulting size of an outbreak (tildesley and keeling, 2009 ). however, the value of r 0 during an outbreak does not only depend on exante features of a virus or a population, but potentially also on the response of both population and authorities to the outbreak. this is particularly true in the context of the covid-19 pandemic, to which most countries in the world have reacted with some form of social distancing measures, or lockdown. in absence of a vaccine or effective drugs, these measures are the best weapon to reduce the number of deaths, as well as the number of intensive care unit beds required (kucharski et al., 2020; flaxman et al., 2020; ferguson et al., 2020; greenstone and nigam, 2020) . in the present study, we analyze data on the diffusion of covid-19 in lombardy, the region of italy most heavily affected by the pandemic provide an accurate description of the early phase of the outbreak in such region). specifically, we employ daily data on the number of individuals positive to covid-19 at the municipality level, focusing on a period in which the entire country was subject to a lockdown. all municipalities under analysis share the same public health system and, in the period considered, were subject to the same social distancing regulation. however, at the start of the period, they were characterized by a strong heterogeneity in the number of cases, both in absolute terms and in terms of cases per capita. we study a period beginning on march 25, 2020, that is, more than two weeks after the lockdown regulation was put in place, and ending with april 14, when such regulations still held: this means that movements across municipalities are severely restricted, requiring any travelers to present a valid (typically work or health related) justification for their journey. we fit a susceptible-infected-recovered (sir) model on data from each municipality and find that the estimated r 0 is negatively correlated with the prevalence in the municipality at the beginning of our period. this result holds both when considering the absolute and per capita number of cases and is robust to different specifications and sample disaggregations. we present and compare different complementary explanations for this finding. early and widespread testing increases the reported number of cases and might allow the authorities to slow the spread of the pandemic by isolating known cases. at the same time, where the number of cases is higher, the population might comply more strictly with the lockdown measures, thus reducing the rate of spread: we show in section 4.1 why the latter mechanism is most likely to drive our results. we employ count data of per-municipality recorded cases, updated daily and distributed by regional authorities. we do not rely on data on recovered and deceased individuals, as such data are not available with the required geographical disaggregation. data are available starting from march 25, 2020 and cover a period of twenty-one days during which lockdown measures were always in place. we verify that only minimal deviations appear between regional data and the aggregation of municipal data. out of 1507 municipalities in lombardy, 960 had at least one recorded covid-19 case as of this date. figure 1a displays the number of cases (size of the dots) and the cases per capita (color of the dots) as of march 25 for each of these 960 municipalities. similarly, figure 1b displays the number of new cases (size of the dots) and the number of new cases per capita (color of the dots) recorded in each municipality between march 25 and april 14. it should be noted that official data concerning the covid-19 outbreak in italy has been found to be strongly incomplete, both in terms of positive individuals and of casualties: several researchers have estimated an outbreak size much higher than that suggested by official numbers (flaxman et al., 2020) , while others have corroborated this with an analysis of anomalies in death rates. 1 moreover, local testing strategies are known to have deviated from who guidelines and have changed over time, also depending on available resources: towards the end of our period of interest, more subjects with mild symptoms were tested. for this reason, some researchers have put forwards adaptations of the sir model that account for a threshold in the note: dot size represent absolute numbers, colors represent cases per one thousand inhabitants. capacity of the health system. 2 such problems are not specific to italy, as official data from a number of countries have been questioned. more in general, the difficulty in obtaining reliable data on the number of infected, deceased and recovered individuals calls for refinements of traditional epidemiologic models (atkeson et al., 2020; riccardo et al., 2020) . given our research question, these caveats are of limited importance. indeed, the focus of the present work is to document differences in response across municipalities, rather than to precisely estimate the epidemiological parameters or expected duration of the covid-19 outbreak in lombardy. data on population size is obtained from the italian national istitute of statistics (istat). we fit a sir model on each municipality in the period of twenty-one days beginning in march 25. given the short time span considered, we employ a simplified sir model which does not account for natural rate of mortality. hence, the model is entirely defined by setting few parameters: β, which determines the rate at which susceptible (s) individuals become infected (i); γ, which determine the rate at which infected individuals become recovered (r); the initial number of infected and recovered individuals, and the population size n (= s + i + r). we take population size from official statistics. we hence consider a discretized version of the continuous sir model -each period corresponding to a day -and automatically explore the parameter space for β, γ, and the initial value for i and r, looking for the combination that provides the best fit. 3 specifically, the goodness of fit is maximized by minimizing the sum of square residuals between the cases count and the sum of the i and r pools sizes. 4 the initial values for the free parameters are set to those calibrated on the entire lombardy region. note: fit between data and the corresponding sir model for lombardy region (left) and the most affected municipalities at the beginning of our period of interest in absolute and per capita terms, respectively (center, right). given that the sir model assumes a non-null initial population of infected individuals, we only consider the 960 municipalities satisfying this condition. we further drop 47 municipalities which had new cases recorded on only one or two dates, hence reducing to 913 municipalities. 5 although this sample selection might in principle affect our results, we show in section 4.2 that this is not the case. figure 2a displays the fit between data at the regional level and the corresponding simulated sir model. figures 2b and 2c are the equivalent for milan and castiglione d'adda: these are the two municipalities which, at the beginning of our period of interest, had been most heavily hit in absolute and per capita terms, respectively. note that a weekly fluctuation can be observed for all municipalities: this is in line with documented evidence that less tests are processed during the weeekend, and the effect reverberates on the number of positive detected cases with a delay of two to three days. we expect these fluctuations to affect the entire region homogeneously. once we find the best sir parameters for each municipality, we regress the estimated r 0 (the ratio of the estimated β and γ) on the outbreak size within the municipality as of march 25. we focus on the per capita number of cases, as we expect any effect to be related to the prevalence of the outbreak -a same number of cases will be perceived in a very different way in milan or in a small municipality. figure 3 shows the distribution of the estimated values of r 0 : the mean estimated value is 0.83 (0.85 when weighted on population), while the median is 0.70a strong heterogeneity (which can be partly attributed to statistical noise -several municipalities count only a few cases each) can be observed across municipalities: in what follows, unless differently specified, we trim data by dropping 0.5% of outliers on each side of the distribution of r 0 , hence reducing to 903 municipalities. in only few of these (179) the value of r 0 appears to be larger than the critical threshold of 1: that is, in the vast majority of municipalities, the outbreak is expected to spontaneously extinguish without requiring herd immunity. table 1 presents the results of the regression analysis. we see a negative and strongly significant relationship between the initial number of cases per one thousand inhabitants and the estimated r 0 (column (1)); this relationship is robust to controlling for population size (column (2)), and to both the absolute number of cases and the inverse of population size (column (3)), i.e., a full interaction model where the the per capita count represents the interaction term (kronmal, 1993) . the coefficient for the per capita number of cases can be interpreted as the reduction in r 0 resulting from an increase of one case per one thousand individuals in the prevalence of the outbreak. the value of -0.023 observed in column (2), which we consider as our baseline specification, indicates a sizeable effect: for reference, given that the prevalence in milan as of march 25 was of around 1.7% , the above mentioned result suggests that had it been 2.7% , the average r 0 would have been around 0.88 instead than the observed 0.90. the same negative and strongly significant effect is observed if we focus on the absolute number of cases as explanatory variable, controlling for the population size (column (4)). it should be noted that any intrinsic characteristic of municipalities -such as demography, location, structure of the economy -which might explain a larger outbreak size should also favor a larger r 0 (mills, 2020) . thus, controlling for such characteristics is expected to further reduce the coefficient for cases% . there are a few reasons that might explain why a larger outbreak should result in a subsequent lower r 0 . the first might be related to herd immunity, by which areas where the outbreak is initially more present have less scope for further spread because a large share of individuals have already caught, and possibly developed immunity to, the virus. this is in principle not a problem of our approach, as the sir model accounts for this effect and should estimate an r 0 net of it -in other terms, r 0 describes the evolution of the outbreak in an hypothetical situation in which the pool of susceptible individuals is never reduced. however, the problem might still arise if the count data employed severely underestimate the actual spread of the virus: the number of positive cases could actually be much larger than the detected one, leading to an estimated r 0 lower than the real one because of the undetected effect of herd immunity in reducing the rate of contagion. the underestimation of infected population might also suggest an alternative explanation of the result related to test capacity: to the extent that a lower detected prevalence reflects a lower ability of authorities to identify infected individuals, it should then correlate with a lower ability to isolate, hospitalize and cure them, and hence to a faster outbreak growth. a third, social, explanation is instead that wherever the local population is aware of a larger prevalence of the disease, it reacts by changing its behavior towards a stricter application of social distancing rules, thus leading to a lower r 0 . in what follows, we provide evidence in favor of this hypothesis. we start by analyzing the first possible explanation: several sources have argued that the actual size of the infected population might lie between four and ten times the official reported numbers. in the most affected muncipalities in our sample during the period analyzed, 57 infections per one thousand inhabitants have been recorded, and according to the most pessimistic estimates this would mean that up to 57% of the population was infected. while most municipalities have a number of recorded cases per one thousand inhabitants which is orders of magnitude lower, to avoid the possibility that an even partial herd immunity effect might be driving the results, we reestimate our main model on subsamples of municipalities according to their initial number of cases per capita. specifically, we split the sample according to quartiles of cases per capita on march 25. the results, presented in columns (1) to (4) of table 2 , show that our findings are not driven by herd immunity, as the coefficient for cases% is negative in each quartile. the absolute value of such coefficient is actually much larger for municipalities with a low prevalence than for those with a higher prevalence, and is strongly significant in the first two quartiles, hence including municipalities with 2 cases per one thousand inhabitants or less. we then consider the second possible explanation: that a lower detected prevalence signals a lower detection ability, and that this naturally correlates with lower ability to track and quarantine infected subjects, hence raising the subsequent rate of diffusion. in order to disentangle this test capacity explanation from the third, social, one, we sketch two simple models of how these would be expected to affect r 0 . let us represent with u t the unknown real number of infected subjects per one thousand inhabitants, at time t in a given municipality, and with i t the corresponding known number. we are interested in the extent to which unidentified infected subjects (which are u t −i t cases per one thousand inhabitants) will raise the r 0 for the municipality in the subsequent period. more specifically, we can assume that identified and unidentified patients form two different pools of infected subjects and that the latter has a much higher β -probability of infecting susceptible individuals -that leads to a corresponding higher r 0 . since β enters linearly in r 0 -and assuming for simplicity that γ is constant -the relationship between u t − i t and r 0 would be expected to be linear. moreover, it is well known that not only identified patients are subject to a stronger form of isolation, but also close contacts of such patient (some of which are not infected) are recommended to selfquarantine: this does not happen in municipalities with a larger number of undetected cases, which implies that the effect of each unidentified patient should be more than linear in increasing the r 0 . this would imply a linear or concave relationship between cases% and r 0 . vice-versa, any social explanation is based on the assumption that inhabitants react to the news of the cases in their municipality. given any concave function describing this reaction, a same increase in per capita cases will be perceived as more important if the initial number of cases is lower. that is, we can expect inhabitants of two towns with respectively 1 and 11 known cases per one thousand inhabitants to differ in their compliance with social distancing prescriptions more than inhabitants of two towns with respectively 20 and 30 known cases per one thousands inhabitants: a same difference of one percentage point in prevalence will have a weaker effect on people behavior were prevalence is higher. this alternative explanation predicts a convex relationship (given the negative sign) between cases% and r 0 . to disentangle between the test capacity and the social explanation, we enrich our basic model by introducing a quadratic term in cases% . this is done in column (5) of table 2 . we see that the quadratic term has a positive sign and is strongly significant, while the sign of the linear term is still negative and has increased in absolute terms. hence, while this does not allow us to exlude that the other explanations might play a role, we can conclude that the social explanation is the main driver of the negative relationship between cases% and r 0 . (jones et al., 2020) describes two possible opposite reactions to the covid-19 oubreak: a precautionary attitude that leads to a stricter adherence to guidelines, and a "fatalism effect" according to which an individual who is more likely to be infected in the future "reduces her incentives to be careful today". our results provide strong evidence in favor of the first mechanism. in addition to the quantile analysis previously described, we verify that our main result also holds consistently across the 12 provinces (lower level administrative regions) of which lombardy is composed. results are displayed in figure 4a . we see that, for each province, the effect of cases% on r 0 is negative: although the small sample size results in only few provinces reaching statistical significance, it is clear that no specific area of lombardy is alone responsible for our findings. in order to verify that our results do not strictly depend upon the period considered, we replicate our analysis over different 10-days moving windows within our period of analysis. for each subperiod, we fit the r 0 for each municipality and regress it on the number of cases per thousand individuals at the beginning of that subperiod. in accordance with the selection procedure described in section 3, we reduce this analysis to the 713 municipalities that feature at least one case on march 25 and, in each window, have new cases recorded in at least two dates. the results are shown in figure 4b . for comparability, we also display the value of the coefficient estimated for the entire time period on the same restricted sample of 713 municipalities. we find that the effect of interest is robust, that is, the coefficient for the cases% is consistently negative and strongly significant for each subperiod. its absolute value is significantly decreasing over time; that is, the effect of the number of cases on the r 0 in the following days appears to be stronger in the earlier days of the outbreak. while there might be multiple explanations for this, we only remark that the rate of growth of the epidemic has been consistently decreasing: whether individual behavior reacts not just to outbreak size, but also to its change over time, is an issue for further research. finally, we verify that all results reported in table 1 , including statistical significance, are virtually unchanged if we do not trim the data as previously described. while an accurate predition of the date of extinction of the outbreak deserves more sophisticated epidemiologic models riccardo et al., 2020) that are out of the scope of the present paper, we can analyze to some extent the relationship between the predicted date of extinction and the number of initial cases. (1) and (2), 0.1 cases in columns (3) and (4). * p<0.1; * * p<0.05; * * * p<0.01 in general, the relationship between infected population at time t and expected date of extinction of the outbreak within a sir model depends on the size of r 0 : if the latter is smaller than 1 -i.e., the outbreak is spontaneously slowing -then a smaller outbreak will extinguish sooner; viceversa, if r 0 > 1, a larger outbreak will sooner reach a level of herd immunity, and hence die out. since, according to our data, most municipalities in lombardy display an r 0 < 1, we focus on this case. while for a same level of r 0 we expect the predicted time to extinction to increase with the initial outbreak size, the fact that the r 0 is negatively related to initial outbreak size -and that a lower r 0 leads to a quicker extinction -leaves theoretically undetermined the relationship between initial outbreak size and duration of the outbreak. in order to shed light on this indeterminacy, we proceed to simulating the sir model for each municipality until the predicted size of the infected population decreases below either (i) 0.1 cases for one thousands inhabitants or (ii) 0.1 cases 6 and we consider the number of periods elapsed as the outbreak duration. we then regress the outbreak duration, defined in these two different ways, over the initial (i) number of cases per capita (columns (1) and (2) of table 3 ) and (ii) absolute number of cases (columns (3) and (4) of table 3 ), respectively. results from table 3 show that the relationship between outbreak size and extinction date is non-trivial. first, the relatively few municipalities with r 0 > 1 do influence significantly the results -as already discussed, the expected effect of outbreak size for a same r 0 is reversed in such cases. second, if we restrict to r 0 < 1, the relationship is positive and significant when reasoning in per capita terms, but not in absolute terms. it should also be mentioned that the results depend on the thresholds adopted in the definition of outbreak extinction. in general, given that r 0 < 1 determines the exponential decay, a lower threshold will mean that the date of extinction is further away for municipalities with a relatively low number of cases and relatively high r 0 . summing up, the results of predicting the extinction date are to be interpreted as cautionary: municipalities with smaller outbreaks might get rid of them sooner than others with more infected individuals (column (2) of table 3 ), but this result does not generalize to the absolute outbreak size (columns (3) and (4) -the former even featuring a negative sign). plans for a gradual exit from lockdown should take into account that the relationship between outbreak size and expected outbreak duration is difficult to pinpoint -as well as the possibility that a larger outbreak might bring the population closer to herd immunity, making it more resistent to a new outbreak. we show that in lombardy, during a lockdown, the basic reproduction number for covid-19 reacts negatively to the initial size of an outbreak at the municipality level, an effect which cannot be explained by the population having reached herd immunity. limited test capacity -and hence a limited ability by health authorities to isolate and treat affected individuals -appear 6 sir models by design tend to 0 infected subjects only for t → ∞ and different authors pick different thresholds as denoting outbreak extinction. notice that the most appropriate value crucially depends also on the extent to which the outbreak is underestimated by available data. to have at most a marginal role in explaining our result. instead, we show that the population's behavior is key to slowing down the contagion and in particular that information about local outbreaks impacts on diffusion rates. this effect is consistent across all provinces and it is robust to the sample period considered. the fact that the effect is particularly strong in municipalities characterized by a smaller outbreak suggests that individuals react more strongly to the first few cases. this aspect is confirmed by the convex relationship we find between the initial size of the outbreak and the r 0 : the marginal effect on behavior of each new case seems to decrease in the number of cases. our results provide evidence in favor of a precautionary rather than fatalistic individual attitude towards the outbreak. they call for considering the population as an integral part of the decision making process, and for a timely and transparent provision of epidemiologic data. how deadly is covid-19? understanding the difficulties with estimation of its fatality rate impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand estimating the number of infections and the impact of nonpharmaceutical interventions on covid-19 in 11 european countries. imperial college covid-19 response team 30 does social distancing matter? university of chicago, becker friedman institute for economics working paper potential short-term outcome of an uncontrolled covid-19 epidemic in lombardy, italy optimal mitigation policies in a pandemic: social distancing and working from home spurious correlation and the fallacy of the ratio standard revisited early dynamics of transmission and control of covid-19: a mathematical modelling study. the lancet infectious diseases demographic science covid-19 is r0 a good predictor of final epidemic size: foot-and-mouth disease in the uk compute values of the parameter π i−l = π i × (1 − δ π,i ) and π i,r = π i × (1 + δ π,i ) (the right and left candidate values for parameter π), 2. compare count data and the simulation obtained with each of the three candidates π i,l , π i and π i,r by computing the sum of squared residuals, 3. select the candidate value which results in the smallest error as new parameter value π i+1 , 4. if the value did not change (that is, π i+1 = π i ) optimization procedure for simplicity, the procedure for fitting the sir model is implemented over the parameters r 0 and γ rather than β and γ, where r 0 = β γ . for each parameter (including the initial values i =î and r =r), the procedure is initialized by deriving reasonable values based on tuning the model to regional aggregated data.then the procedure works as follows (i denotes an iteration, δ π,1 is set to 0.2 for each parameter π): key: cord-259984-csdf1a69 authors: raffiq, azman; seng, liew boon; san, lim swee; zakaria, zaitun; yee, ang song; fitzrol, diana noma; hassan, wan mohd nazaruddin wan; idris, zamzuri; ghani, abdul rahman izaini; rosman, azmin kass; abdullah, jafri malin title: covid-19 pandemic and its impact on neurosurgery practice in malaysia: academic insights, clinical experience and protocols from march till august 2020 date: 2020-10-27 journal: malays j med sci doi: 10.21315/mjms2020.27.5.14 sha: doc_id: 259984 cord_uid: csdf1a69 the newly discovered coronavirus disease 2019 (covid-19) is an infectious disease introduced to humans for the first time. following the pandemic of covid-19, there is a major shift of practices among surgical departments in response to an unprecedented surge in reducing the transmission of disease. with pooling and outsourcing of more health care workers to emergency rooms, public health care services and medical services, further in-hospital resources are prioritised to those in need. it is imperative to balance the requirements of caring for covid-19 patients with imminent risk of delay to others who need care. as malaysia now approaches the recovery phase following the pandemic, the crisis impacted significantly on neurosurgical services throughout the country. various emergency measures taken at the height of the crisis may remain as the new normal in the provision of neurosurgical services and practices in malaysia. the crisis has certainly put a strain on the effective delivery of services and as we approach the recovery era, what may have been a strain may prove to be a silver lining in neurosurgical services in malaysia. the following details are various measures put in place as the new operational protocols for neurosurgical services in malaysia. the novel coronavirus, otherwise known as coronavirus disease 2019 (covid-19) pandemic in malaysia, is part of the worldwide ongoing pandemic which was first reported in wuhan china in december 2019. malaysia's first case was confirmed on 25 january 2020, detected among travellers from china entering malaysia via singapore. malaysia's response overseen by the national crisis preparedness and response centre (cprc) under the health ministry began as early as 6 january 2020 following world health organization (who) initial report of the disease in china. various hospital was designated specifically as covid-19 pandemic hospitals, gazettement of quarantine centres, reorganisation of health services, formulation of new operational protocols, stockpiling of essential equipment and redeployment of essential medical workforce and manpower to these centres; as well active detection, monitoring and treating covid-19 patients. these early response measures were escalated to a total lockdown of movement on 18 march 2020, intended to mitigate the spread of covid-19, following the declaration of a worldwide covid-19 pandemic by who on 11 march 2020. the movement control order (mco) in malaysia lasting for a period of 6 months, coupled by the intense proactive measures by the health ministry has successfully kept the number of cases relatively low comparatively, with a total recorded case of 8,556 with 121 deaths. malaysia's proactive and stringent measures have and continue to receive international praise and recognition in successfully mitigating the spread and containing covid-19 transmission. as malaysia now approaches the recovery phase following the pandemic, the crisis impacted significantly on neurosurgical services throughout the country. various emergency measures taken at the height of the crisis may remain as the new normal in the provision of neurosurgical services and practices in malaysia. the crisis has certainly put a strain on the effective delivery of services, and as we approach the recovery era, what may have been a strain may prove to be a silver lining in neurosurgical services in malaysia. the following details the various measures put in place as the new operational protocols for neurosurgical services in malaysia. role and importance of dedicated operating theatre, instruments and icu care all tertiary hospital should have a dedicated operating theatre (ot) for suspected covid-19 patients (1) . the ot requirements should be fully equipped with negative pressure ventilation (recommended). the maximal number of staff is six: i) one specialist; ii) one medical officer; iii) one anaesthetist; iv) one anaesthetist mo; v) one scrub nurse; and vi) one circulating nurse. for elective surgery, kindly refer to its section further below (2) . limiting ot staff to essential members will help preserve the surgical workforce. the types of neurosurgery cases done are either cranial surgery or spine surgery. endonasal surgery should be avoided during covid-19 pandemic (3) . table 1 shows tiers for neurosurgery cases (4) . a neurosurgical emergency includes cerebral haemorrhages (subarachnoid and intraparenchymal), acute hydrocephalus, tumours at risk of intracranial hypertension, spinal cord compressions with, or at risk of neurological deficit, and traumatic cranial and spinal trauma emergencies (5) . (table 2 ) (6) for green level, all elective cases proceed as scheduled. for yellow level, the ot schedule is capped for 3 weeks to 75% of capacity, yielding a 25% reduction in all elective and procedural cases. all outpatient procedures black level, significant state or federal resources are needed to fight the outbreak. all urgent scheduled surgical cases will be cancelled. this 'volume limiting approach' encourages maximal adaptability, in which the supply of hospital capability meets the demand for scheduling needs. should be designated to an off-site hospital where covid-19 patients are not expected to be admitted. there is a hard cap on the number of cases requiring post-operative admission; the 12-patient limit for all surgical cases (including non-neurosurgical cases). for red level, there is a 50% reduction in all elective case scheduling. for the team-based paired coverage will go into effect during red levels of covid-19. in this model, each hospital (columns) will have three groups of providers. there will be two teams that switch coverage on a 3-day cycle. each team will cover for 3 days, and then have 3 days off while the second team covers. the transition between teams will occur virtually, avoiding unnecessary team-to-team contact. a backup group substitutes for any team member who shows signs of illness. if a team becomes contaminated, the other team will take over and the alternates will fill the gap. contact between teams and alternate is prohibited. each team will only rotate at one hospital (no cross-covering) and will only have contact with members within their team. teams at the same site will not have any overlapping clinical time with each other. this system ensures adequate coverage, minimises hand-off issues, and, most importantly, minimises transmission risk across teams. due to the likelihood of infection among inpatient providers, there will be a designated 'alternate pool' of providers that will substitute for those who show covid-19 symptoms. activation of paired coverage system (pcm) (6) is triggered by a red level of surge. all residents are aware of their role in the pcm ahead of time. site-specific needs are addressed within the team. for example, teams at the main hospital are larger than teams at other satellite hospitals. the pcm is adaptable such that the number of team members can vary, along with the experience level of the team. the core function of the pcm (limiting healthcare worker transmission of the virus) remains. it is also important that neurosurgeons provide their anaesthesia colleagues, nursing staff and the ot with objective data about which cases should be expected to proceed during a covid-19 outbreak. neurosurgeons need to predict what cases should be classified as an emergency. a checklist that can be applied to neurosurgical cases during the covid-19 pandemic is as per figure 1 . the checklists help organise surgical staff during times of crisis, such as guiding action during 'red alerts' from neuromonitoring during spinal surgery. the checklist strategy organises surgical staff around the common goal of booking cases during the outbreak. distribution of the checklist to all surgical staff will facilitate effective communication and the ease with which appropriate neurosurgical cases can be scheduled. surgical workforce (7) the surgical workforce must be able to maintain emergency surgery capabilities including major trauma. it is important to protect and preserve the surgical workforce as well. these will include rotas where some members of the team do not come into work and act as a healthy reserve (refer to a 3-group setting [surgical team]). when not vital to the effort, keep surgical and anaesthetic staff out of hospital and self-isolating at home to preserve human resources. this will also allow personnel to rest before they return to clinical work. non-surgical solutions to be used to avoid surgery where possible. personal protective equipment (ppe) must be used correctly in line with national guidance. rest, recuperation and psychological support should be factored into all planning. (3, 8, 9) pre-operatively, the patient must be tested for covid-19 and proceed if negative. full powered air purifying respirators (paprs) for surgeons and all team members in the ot for any of these cases that do actually need to move forward, either for cases in which we cannot wait for test results or for cases that test positive but still need to proceed. for urgent cases (that should be done within 1 week), two covid-19 tests separated by 24 h with the patient quarantined in the interval between tests before the surgery, with the surgery proceeding only if the results are negative for both tests. if covid positive, papr for all ot staff may be necessary until further data is available. for emergent/unavoidable case for a known or undetermined covid-19 patient, the surgeon and all ot personnel in the surgical suite should use papr, which filter the air being breathed in addition to face shields and other standard ppe. it is also vital for a cessation of positive pressure ventilation in ot during the procedure until 20 min after the patient leaves (10). the patient should be isolated and medical personnel should wear full ppe when nursing post-operative patients until covid-19 status is known. (1, 3) regional anaesthesia (ra) is preferred than general anaesthesia (ga) to reduce aerosols. in ga, a negative pressure setting (risk of aerosol transmission) is required. the intubation and extubation are done with full ppe including papr or its equivalent. only essential staff should be present in the ot. postop management is done in the isolation room. thromboprophylaxis should be considered throughout the hospital stay. non-invasive ventilation should not be used. (1) proper droplet precautions or proper decontamination processes should be followed. there should be an escalation of standard of practice during airway management for all patients to reduce exposure to secretions. hand hygiene should be taken care of with frequent hand washing using alcohol-based hand wash gels, which should be available near every anaesthesia station. the number of staff members present for intubations/ extubations should be limited to reduce the risk of unnecessary exposure. it is also recommended to strongly consider prophylactic antiemetics to reduce the risk of vomiting and possible viral spread. (3, 9, 12) rapid sequence induction (rsi) and use a video laryngoscope (vl) with the goal of a high first pass success rate (fps) is recommended. iv sequence induction without bag mask ventilation is preferred to minimise exposure risks and aerosolisation. preoxygenation for a minimum of 5 min with 100% oxygen and perform a rsi to avoid manual ventilation of patient's lungs and potential aerosolisation of virus from airways (3) . rsi (ensure a skilled assistant is available to perform cricoid pressure) or a modified rsi should be performed as clinically indicated. if manual ventilation is required, apply small tidal volumes (3). placement of a high quality heat and moisture exchanging filter (hmef) rated to remove at least 99.97% of airborne particles 0.3 microns or greater in between the facemask and breathing circuit or in between facemask and reservoir bag is required. awake fibreoptic intubations are essentially contraindicated unless specifically indicated. ppe should be provided when performing an aerosol generating procedure. preoxygenation using a bag-valve-mask (bvm) that can be purposely modified for covid-19 patients with a viral filter, without squeezing the bag. the most experienced anaesthesia professional available should perform intubation if possible. a trainee should avoid intubations of sick patients during this time. the laryngoscope should be resheathed immediately post-intubation (double glove technique). used airway equipment should be sealed in a double zip-locked plastic bag and must then be removed for decontamination and disinfection. there should be no airway carts in the room. thus, appropriately sized equipment should be pre-packed for that patient. the use of deeper sedation extubation to prevent coughing is also preferable as long as the airway is safe. (3, 12, 13) all anaesthesia professionals should utilise ppe appropriate for aerosol-generating procedures for all patients when working near the airway. 'rescue like' crash intubations where ppe cannot be fully adhered to should be avoided. correct donning and doffing of ppe should be adhered to. properly fitted n95 masks or paprs should be used for all patient. at a minimum, n95 masks should be used for all patients. for those who are not n95 fit-tested, have facial hair or fail n95 fit-testing, paprs should be used if possible. issuance of n95 masks or availability of paprs for all clinical anaesthesia personnel should be a priority. extended use and/or limited reuse of n95 masks should follow the centres for disease control and prevention (cdc) and institutional guidelines. a papr provides superior protection and may be warranted for airway procedures in patients with known or suspected covid-19. for aerosol-generating procedures, this includes eye protection (goggles or a disposable face shield that covers the front and sides of the face), a gown and gloves, in addition to airway protection with n95 masks or paprs. effective hand hygiene before putting on and after removing ppe must be ensured. procedures for proper donning and doffing, disposal of contaminated ppe, and cleaning of contaminated reusable ppe and anaesthesia equipment should be established following cdc and institutional recommendations. the double gloving technique is used during intubation. the outer gloves are used to sheath the laryngoscope blade and change the inner gloves as soon as possible afterwards. after removing protective equipment, remember to avoid touching your hair or face before washing hands. during extubation, maintain strict hands hygiene, wear a mask with a face shield and carefully dispose of contaminated equipment. the use of two providers for ppe donning and doffing procedures should be encouraged, to allow one person to observe and coach the other through the steps of the routine. appropriate ppe and the procedures for donning and doffing ppe are available at the cdc webpage. approaches to conserve supplies (12, 13) the administration should minimise the number of individuals involved. where feasible, use alternatives to n95 masks (e.g. other classes of filtering facepiece masks, facepiece air purifying respirators and paprs). n95 masks should be allowed for extended use and/or limited reuse. the use of n95 masks should be prioritised for that personnel at highest risk of covid-19 exposure and/or those anaesthesia professionals in high risk categories (e.g. those with prior health conditions, older age). staff should receive training in the appropriate donning and doffing of ppe taught through simulation and videos without using precious resources. in resource-limited situations, extended use of n95 masks (continuous wearing while seeing multiple patients) is preferred to limit the reuse of n95 masks (doffing and redonning between patients). n95 mask life may be lengthened and surface contamination reduced by wearing a plastic face shield or a surgical mask over the n95 (cdc respirator guidance). use of chlorine or alcohol solution to sanitise n95 masks is not recommended as it damages mask integrity. heating n95 masks to 70 â°c (160 â°f) in a dry oven for 30 min seems a promising solution to disrupt viral particles and maintain mask integrity for reuse. further guidance from cdc and partners in health (pih) on reuse of n95 masks or best practices when no respirators are available (such as wearing two surgical masks) are available. in routine clinical care of covid-19 suspected or confirmed infections, surgical masks are acceptable ppe, except in the case of aerosol generating procedures (intubation, high flow nasal cannula, non-invasive ventilation, bronchoscopy, administration of nebulised medications, etc). facilities which do not have disposable surgical attire, theatre garb in the form of cloth scrub hats or bonnets should be washed between each use if possible and no less than daily. theatre gowns and drapes should be washed and sterilised between each patient as is currently expected. if theatre gowns are repurposed for isolation units, they should be washed after each prolonged care routine. if surgical ppe is not impermeable, consider wearing rubber aprons under linen gowns and always perform handwashing after doffing surgical ppe and before touching clean items or self. ot management and preparation during covid-19 (14, 15) surveillance on possible further transmission to patients and other personnel should be done. covid-19 in a patient receiving surgery is sporadically reported with a special focus of management technique. surgical infection usually focuses on patient but it is important to give attention to the practitioner who works in the operation room. an ot with a negative room pressure environment located at a corner of the operating complex, and with separate access, is designated for all confirmed (or suspected) covid-19 cases. the ot consists of five interconnected rooms, of which only the anteroom and anaesthesia induction rooms have negative atmospheric pressures. the ot proper, preparation and scrub rooms all have positive pressures. understanding the airflow within the ot is crucial to minimising the risk of infection. the same ot and the same anaesthesia machine will only be used for covid-19 cases for the duration of the epidemic. an additional heat and moisture exchanger (hme) filter are placed on the expiratory limb of the circuit. both hme filters and the soda lime are changed after each case. the anaesthetic drug trolley is kept in the induction room. no unnecessary items should be brought into the ot, including personal items such as mobile phones and pens. before the start of each operation, the anaesthesiologist puts all the drugs and equipment required for the procedure onto a tray to avoid handling of the drug trolley during the case. nevertheless, if there is a need for additional drugs, hand hygiene and glove changing are performed before entering the induction room and handling the drug trolley. a fully stocked airway trolley is also placed in the induction room, as far as possible, disposable airway equipment is used. if single-use plastic anaesthesia or surgical equipment (endotracheal tubes, ventilator circuit tubing, plastic suction tubing, electrocautery handpieces) must be reused, ensure that disinfection aiming for 'high-level disinfection' or 'sterility' is employed. this includes immersion in appropriate concentration glutaraldehyde, phenol, or hydrogen peroxide solution for the recommended duration. the airway should be secured using the method with the highest chance of first-time success to avoid repeated instrumentation of the airway, including using a video-laryngoscope. equipment in limited supply such as bispectral index monitors or infusion pumps may be requested but need to be thoroughly wiped down after use. hospital security is responsible for clearing the route from the ward or intensive care unit (icu) to the ot, including the elevators. traffic should be minimised, especially opening and closing of theatre doors. patients with known or suspected covid-19 infection should wear surgical masks when being transported through hospital spaces or in rooms without negative pressure isolation. the transfer from the ward to the ot will be done by the ward nurses in full ppe including a well-fitting n95 mask, goggles or face shield, splash-resistant gown and boot covers. for patients coming from the icu, a dedicated transport ventilator is used to avoid aerosolisation, the gas flow is turned off and the endotracheal tube clamped with forceps during the switching of ventilators. the icu personnel should wear full ppe with a papr for the transfer. in the induction room, a papr is worn during induction and reversal of anaesthesia for all personnel within 2 m of the patient. for operative airway procedures such as tracheostomy, all staff keep their papr on throughout the procedure. during the procedure, a runner is stationed outside the ot if additional drugs or equipment are needed. these are placed onto a trolley that will be left in the anteroom for the ot team to retrieve. this same process in reverse is used to send out specimens such as arterial blood gas samples and frozen section specimens. the runner wears ppe when entering the anteroom. personnel exiting the ot discard their used gowns and gloves in the anteroom and perform hand hygiene before leaving the anteroom. any papr will be removed outside the anteroom. patients who do not require icu care post-operatively are fully recovered in the ot itself. when the patient is ready for discharge, the route to the isolation ward or icu is again cleared by security (using an advance runner to clear the way). a minimum of 1 h is planned between cases to allow ot staff to send the patient back to the ward, conduct thorough decontamination of all surfaces, screens, keyboard, cables, monitors and anaesthesia machine. surfaces in the ot should be thoroughly cleaned between cases including pulse oximeter probes, thermometers, blood pressure cuffs and other reusable materials (70% alcohol solution or 0.5% chlorine solution). as part of minimising contamination in ot, in addition to surface cleaning, using clear plastic sheets (to be changed in between patients) to cover the anaesthesia machine, the monitors as well as the patient's face, especially during aerosol producing airway manoeuvres like intubation and extubation, is recommended. all unused items on the drug tray and airway trolley should be assumed to be contaminated and discarded. all staff must shower before resuming their regular duties. as an added precaution, after confirmed covid-19 cases, a hydrogen peroxide vaporiser will be used to decontaminate the ot. clear instructional posters for ppe donning/doffing should be prominently displayed. a taped off area just outside of the ot door should be clearly marked for donning and doffing activities. (16) few steps must be emphasised to reduce the risk of contracting covid-19 among hcps. some precaution that needs to be taken note are: staff who are more at risks such as older adults (e.g. those over 60 years), those with underlying medical conditions (e.g. heart disease, chronic respiratory diseases, cancer), those at risk due to an immunocompromised from a medical condition or treatment (e.g. chemotherapy) and pregnant staff should take care of the lower risk stream of non-covid-19. (16) ot is potentially a high exposure zones given manipulation of the airway and aerosolisation of respiratory particles, with anaesthesia providers at particularly high risk. it comes with the additional risk inherent in the presence of multiple staff members. the perioperative personnel are at an advantage given their familiarity with maintaining sterility. however, the ancillary staff such as ot cleaners, instrument reprocessing staff and laundry personnel may be at risk. hence, they should take appropriate precautions and wear ppe (goggles or face shield, surgical mask, heavy duty gloves, long sleeved gown, boots) to avoid exposure to contaminated materials. there are no special decontamination methods other than machine laundering with detergent are required for laundering linens; all surface areas should be disinfected with 0.5% chlorine or 70% alcohol solutions. immediate surgical plan during pandemic (13) a clear plan should be made to conduct essential operations. elective operations should be indefinitely postponed to preserve vital resources including hospital beds and ppe. exceptions are for cancer or highly symptomatic patients and as such the current guidance is not to postpone (table 1) . each hospital must make a plan based on the current availability of resources. to facilitate decision making and avoid conflicts between patients and providers, a triage algorithm for identification of nonemergent conditions can be used. surgical emergencies still require prioritisation. funding must be adequate to support the hospital and staff with critical surgical services that will continue to be required despite the pandemic response. using a checklist to ensure appropriate precautions are taken for operations with suspected or known covid-19 infection is also important. simulation has been also helpful in establishing new routines in the ot. aerosolgenerating procedures that can be provided using other mechanisms should be avoided if at all possible (e.g. metered-dose inhaler instead of nebuliser treatment). further planning for the repurposing of ots to support critical care whilst not precluding the ability to provide lifesaving operative care is needed. surgical services are already underfunded and poorly prioritised in many health systems, so the commandeering of ots for use as icu, which has been proposed in many high-resource settings, must be done with extreme caution. emergency surgery will still be necessary for obstetrics and to save life (neurotrauma) and limb, and these capacities should not be compromised by taking up all available ot space and ventilators with covid+ patients. as the average reported time spent on mechanical ventilation has been up to 13 days, critical resources and space will be occupied for many weeks and will be difficult to reclaim once repurposed. repurposing of staff for managing covid-19 cases should be taken into consideration as well. guidance and training should be provided to make the best use of the technical and clinical skills of all perioperative personnel while protecting them from exposure. hospitals, professional societies and ministries of health could also provide physician and nursing staff with basic icu and ventilator management refresher education to improve their capacity to care for covid-19 patients. up to date guidelines on covid-19 management should be provided as knowledge and evidence around best management evolves. it is also vital to maintain and support staff wellness. it is important to recognise that doctors, nurses, cleaners and other hospital support staff have significant fears and concerns (the fears of transmitting to family or becoming infected oneself, the increase in work hours and the need for childcare coverage) that must be acknowledged and managed. providers may also be understandably nervous about providing care outside of their normal scope of practice or working beyond their area of competence. leadership can help manage these by providing information in a transparent way, expressing gratitude for the commitment to patients and colleagues, and reassurances that the system will help protect them and support them and their family. national cprc mentioned that severity of the situation and the availability of resources may change on a daily basis. thus, communication is critical, and an effective communication plan both within and between facilities and health system planners, as well as between providers across the health system and even between countries, is essential and should be established immediately. the preparation of healthcare facilities at large for the safe triage, testing and management of patients with confirmed or suspected covid-19, and managing surge conditions are needed. ethical considerations in resource management are also very important. in many places, the number of ventilators available for persons requiring ventilatory support will be inadequate. in some settings, it is common to reallocate resources from terminal patients or patients with brain death or very low likelihood of recovery (e.g. severe traumatic brain injury) to those with a higher likelihood of recovery. in settings where resources are severely limited and must be rationed, consider creating a committee or utilising standardised risk assessments to determine allocation decisions in advance. this avoids placing the burden of decision making on the frontline health care workers, as these decisions should be not be made ad hoc by the bedside clinician but through careful deliberations by the institution. cultural and medicolegal context should be taken into account to determine the most appropriate allocation and potential protocols for rationing medical resources and care in advance. critical testing, ppe, icu beds, therapeutics, and vaccines should go first to front line health care workers and others who keep critical infrastructure functioning; these workers should be given priority not because they are more worthy but due to their instrumental value in the pandemic response and difficulty of replacing (instrumental value). should the surgical instrument be reused? (17). instruments and devices that have been used in procedures for patients with known or suspected covid-19 should be handled the same as other instruments. reprocessing should follow manufacturer's instructions for use (ifu) and be consistent with recommendations in the local infectious disease unit. covid-19 is an enveloped virus and is susceptible to the environmental protection agency (epa)registered disinfectants that are used in the health care setting (18) . there are no additional recommendations by cdc for disinfection and sterilisation of these items used for covid-19 patients. instruments should be cleaned, decontaminated, dried and stored in a manner that reduces the risk of exposing patients and personnel to potentially pathogenic microorganisms (19). high-level disinfect or sterilise them according to the manufacturer's written ifu. they should be packed and stored in individual packs that avoid contamination. (20) patients should perform second pcr + rapid test prior to icu admission. if rapid test positive, then to discuss with covid-19 hospital. if accepted, then to transfer to covid-19 hospital. if not accepted, then send to negative pressure isolation room icu using designated walkway (only suspected covid-19) while awaiting second pcr results. if require urgent resuscitation, to transfer to negative pressure isolation room icu using specific walkway (only suspected covid-19). if rapid test is negative, then to send to negative pressure isolation room icu using designated walkway (only suspected covid-19) while awaiting second pcr. if two negative pcr results, then it is considered a non-covid-19 patient and to be admitted normal icu cubicle. if pcr is not done on admission or only rapid test is done on admission, then a critically ill patient should have 2 pcr done 48 h apart to rule out disease. there is an exception for planned elective admission to icu after elective surgery. for this group of patients, a negative first pcr is adequate, and the patient is to be admitted to a non-isolation cubicle in icu. (21, 22) full ppe must be readily available in icu. prepacked full ppe sets including hazmat suit should be put into a bag, to ease donning in the case of emergency. two sets will be readily available in the emergency trolley with additional sets kept at specific storage area in the icu together with papr. two nurses to attend with full ppe first. one person to be exclusively in charge to help donning of ppe of medical staff to ensure that all are properly fitted, with hazmat suit and papr if required. papr and hazmat suit will be needed for procedures like intubation and bronchoscopy. donning and doffing of ppe instructions should be pasted on the wall. nurses and doctors to undergo training on where and how to don and doff in icu isolation room, as well as in other non-isolation cubicles. designated area to doff ppe in non-isolation icu cubicles/wards to be assigned. walkie talkie to be available in isolation room for outside communication (to get medications, additional materials). nurses outside will prepare required materials/medication and put on a trolley, leave it in the airlock to be collected by nurses in the isolation room. (13, 18, 20) glidescope and ultrasound must be fully covered with plastic when in a room. ultrasound transducer to be covered with transducer cover if in use. cleaning should be per biomedic advice. person cleaning equipment should be made aware of the infectious component and be protected with full ppe. outside nurse to be fully protected when removing equipment outside isolation room for cleaning. the same ventilator should be used for negative pressure isolation room icu and should not with ventilator in the other rooms. (23) the viral filter should be on both inspiratory and expiratory ends. once intubated, to clamp the ett, remove the viral filter for connection with ventilator tubing. etco 2 monitor i.e. capnograph and closed suction catheter to be connected to patient immediately upon intubation. disconnection of ventilator should be reduced. (23) nurses should be divided into covid-19 team and non-covid-19 team. the covid-19 team to take care of covid-19 suspect or covid-19 positive patients. the non-covid-19 team to take care of the non-covid-19 patients. the covid-19 team nurses should not be rotated to non-covid-19 patients. (e.g. patient a is managed by nurse x, y and z in three shifts. this team should take care of patient a until second pcr results are available) container for contaminated ppe for covid-19 suspect or positive should be clearly labelled for cleaners to take extra precautions. decontamination of the pathways and negative pressure isolation room icu used by these patients will be per hospital protocol. the whole icu need to be close and decontaminated in the presence of a covid-19 positive case in nonisolation icu cubicle with no other patients around. for the presence of a covid-19 positive case in non-isolation icu cubicle with existing non-covid-19 patients in icu, the whole icu must be closed and decontaminated plus to consider testing these patients (existing non-covid-19 patients) and transferring to other hospitals. potential covid-19 case in non-isolation room, to limit one visitor/patient. the covid-19 positive in non-isolation room, then no visitors allowed in icu. one staff member in front of icu is stationed to monitor visitor flow in and out of icu (get visitor's name and phone number in the case of contact tracing). in the event of a covid-19 positive case in non-isolation room in icu, contact tracing will include, (1) existing non-covid-19 patients in icu during that period, (2) all visitors who enter icu during that period, (3) all icu staff entering icu during that period, and (4) all doctors entering icu during that period. (27) triage may not be implemented by a facility without clear sanction from appropriate public health authorities. systems for sharing information about the number and severity of cases, equipment availability, and staffing shortages could be activated throughout hospital groups and regional networks. patients are assessed on medical/ clinical factors alone, regardless of their work role. the ontario health plan for an influenza pandemic (ohpip) protocol and the sequential organ failure assessment (sofa) score should be used. candidates for extubation during a pandemic would include patients with the highest probability of mortality. initial assessment using the sofa scoring system (points based on objective measures of function in six domains: lungs, liver, brain, kidneys, blood clotting, and blood pressure) with best score of 0 and worst score of 24. time trials assessment at intervals of 48 h and 120 h. those showing improvement would continue ventilator use until the next assessment, whereas those who no longer met the criteria would lose access to mechanical ventilation. chronic care facilities will have to provide more intensive care on-site as part of the general process of expanding care beyond standard locations. the proposed justification for such a strategy would be that more patients could ultimately survive if these ventilators were used by the previously healthy victims of a pandemic. setting aside the small number of ventilators in chronic care facilities for use by chronically ill people, who likely will have severely limited access to ventilators in acute care facilities. clinicians providing direct care will relay data to a supervising clinician serving as a triage officer, who will calculate the sofa score and make triage decisions but will not provide direct care. terminal weaning in response to patient preferences can include sedation so that the patient need not experience air hunger. patients who are extubated against their wishes should be offered appropriate palliative care based on their clinical conditions and preferences. because transparency is a crucial element of adherence to ethical standards, clinicians must document decisions regarding sedation with extubation. the guidelines do not support the use of manual ventilation devices for patients who do not meet criteria for ventilator access. daily retrospective review of all triage decisions should be made, to ensure consistency and justice. physicians will need to discuss altered standards of care in a disaster, especially for scarce resources such as ventilators. patients and families must be informed immediately that ventilator support represents a trial of therapy that may not improve the patient's condition sufficiently and that the ventilator will be removed if the patient does not meet specific criteria. elective surgeries and other elective procedures that could result in the use of mechanical ventilators should be cancelled (27) . ventilators, supplies, and personnel from ambulatory surgery centres and other facilities not being utilised for covid-19 patients or not experiencing covid-19 outbreaks should be transferred. anaesthesia ventilation machines capable of providing controlled ventilation or assisted ventilation may be used outside of the traditional use for anaesthetic indication (20, 27) . the asa and fda provide specific guidance on how to convert anaesthesia machines for use on covid-19 patients in respiratory failure (27) . transport ventilators may be used for prolonged ventilation in certain patients. continuous ventilators labelled for home use may be used in a medical facility setting depending on the features of the ventilator and provided there is appropriate monitoring (as available) of the patient's condition. non-invasive ventilation (niv) patient interfaces capable of prescribed breath may be used for patients requiring such ventilator support, including niv patient interfaces labelled for sleep apnoea. channelling exhalation through a filter is recommended to prevent aerosolisation. continuous positive airway pressure (cpap), auto-cpap and bilevel positive airway pressure (bipap or bpap) machines typically used for treatment of sleep apnoea (either in the home or facility setting) may be used to support patients with respiratory insufficiency. bipap may be used for invasive ventilation. if all other alternatives are exhausted, care providers could consider ventilation of two patients on a single ventilator for short-term use, although there are significant limitations to this strategy. alternatively, manual bag-valve-mask ventilation done by ancillary providers can be considered as a bridging option to mechanical ventilation. the summary of head and neck examination and procedure recommendations can be viewed in table 4 (28) . (28) in general, most tracheostomy procedures should be avoided or delayed (even beyond 14 days) because of the high infectious risks of the procedure and subsequent care until such time as the acute phase of infection has passed, when the likelihood of recovery is high, and when ventilator weaning has become the primary goal of care. avoiding early tracheostomy in patients with covid-19 is suggested because of the higher viral load that may be present at this time. early tracheostomy was not found to be associated with improved mortality or reduced length of intensive care unit stays in a randomised clinical trial of patients on mechanical ventilation. select the patients carefully. if the tracheostomy is assessed as difficult because of anatomy, history, comorbidities or other factors, consider postponing the procedure. considerations may be given to percutaneous dilatational tracheostomy. allow it to be done safely with minimal or no bronchoscopy, endotracheal suctioning and disruption of the ventilator circuit. adequate sedation provided including paralysis to eliminate the risk of coughing during the procedure. ventilation should be paused (apnoea) at end-expiration when the trachea is entered and any time the ventilation circuit is disconnected. choose a non-fenestrated, cuffed, tracheostomy tube on the smaller side to make the tracheostomy hole smaller overall (shiley size 6 for both men and women is adequate). keep the cuff inflated to limit the spread of virus through the upper airway. tracheostomy suctioning is performed using a closed suction system with a viral filter. heat moisture exchanger device is used instead of tracheostomy collar during weaning to prevent virus spread or reinfection of patients. changing the tracheostomy tube should be delayed until the viral load is as low as possible. case series of open tracheostomies performed during the severe acute respiratory syndrome (sars) outbreak can be viewed in table 5 (29) (30) (31) (32) . making in times of covid-19 crisis: a proposal to safe and sustainable practice in times of crisis neurotrauma (33) neurotrauma forms the bulk of emergency neurosurgical cases presenting or referred to neurosurgical centres, varying from cases of concussion to severe head injuries requiring urgent surgical intervention. in the current climate of a pandemic crisis, neurotrauma poses various management and logistics issues to the neurosurgical team in the following aspects as listed. time between injury to intervention determines outcome. urgency of intervention outlines management of patient. ongoing dynamic pathological process in trauma may alter clinical picture between referral to presentation. pre-emptive management plan is essential to ensure optimum outcome from intervention where feasible. pre-operative screening is vital, particularly those requiring surgical management (34) . all cases undergoing surgery poses high risk to operative team due to potential aerosol generating procedure that may occur from intubation/extubation, positioning of patient to prone/park bench position, tracheostomy procedures and prolonged proximity of surgeon to the head region of patient (33, 36) neurotrauma cases should be managed in a neurosurgical centre. most neurosurgical departments in malaysia are situated in major hospitals, often managing high volume of high risk or covid-19 cases. principle logistic consideration is to whether trauma cases referred from a non-covid-19 designated hospital should be managed in covid-19 designated hospital as this may increase risk of exposure to patients. alternative options should be taken into consideration where feasible, such as team deployment to manage patients in referring hospitals, training and privileging general surgeons in management of cases and transfer of patients back to referral hospitals for continuity of intensive care management postoperatively. resource availability (37) neurotrauma patients require postoperative ventilation with icu care, often for prolonged duration which may limit availability in case of urgent needs. ventilators, ppe and icu are precious commodities in this current pandemic, and fluctuate with time depending on epidemiological dynamics. available resources are commonly and rightly so; prioritised to patients afflicted by the ongoing pandemic, health care workers (hcws) involved in their care. rational balance and anticipation of resource need is essential to optimise usage and sustainable availability in times of crisis. early prognostication (37, 38) outcome from neurotrauma depends on various well-defined parameters. brain damage incurred in primary injury remains irreversible in majority of patients; compounded by secondary factors. early prognostication is essential in times of crisis for resource allocation. it is paramount to ensure that optimum patient care and outcome remains priority of intention to treat. determining long term outcome using available prognostic models for decision making in proceeding with active treatment or withdrawing treatment is essential; albeit exceptionally difficult to ensure continuous availability of limited resource. prognostication based decision is best made with team consensus using all available scientific evidence present. (6) all brain trauma management is in accordance with the brain trauma foundation (btf) guidelines recommendations. all neurosurgical emergencies must be referred to the respective neurosurgical team for consultation and management plans (table 6 ) (39). brain trauma requiring urgent surgical intervention (decompression) with or without intensive care monitoring is best managed in hospitals with dedicated neurosurgical facilities or available neurosurgical services (level ii). brain trauma not requiring urgent decompressive surgical intervention, but which may require or benefit from intracranial pressure (icp) monitoring and intensive care management (level iib) is best managed in centres with available resources to provide objective assessment and management plan, reduce the duration of icu stay and intensive management and early weaning from intensive therapy. brain trauma not requiring urgent decompressive surgical intervention, but which may benefit from icp monitoring and intensive care management in situations where resource availability is limited may be managed with cerebral perfusion pressure (cpp) based target therapy (level iii) and serial ct scan at 6 h-12 h intervals in an intensive care setting where feasible. brain trauma requiring surgical intervention but with limited resources available at dedicated neurosurgical facilities; the following may be considered (anecdotal evidence based on local/regional practice): brain trauma not requiring any surgical intervention but requiring close observation is best managed in dedicated neurosurgical centres or available neurosurgical services if the risk of potential deterioration is deemed to be high (e.g. burst temporal/frontal lobes) and duration and distance of transfer may result in a delay of treatment. brain trauma requiring multidisciplinary management is best transferred and managed in dedicated i) deployment of neurosurgical team to primary referral hospital where feasible to facilitate timely intervention ii) surgical intervention in neurosurgical facilities with subsequent transfer back to primary referring hospitals for continuity of intensive care management (40) screening for traumatic brain injury cases is strongly recommended for safety of hcws. screening recommendations are in accordance with moh guidelines (figure 2 ). risk of covid-19/pui/severe acute respiratory infections (sari) should be ruled out as per moh guidelines and hospital protocols prior to transfer of cases for further management (figure 3 ). secondary screening should be done by attending neurosurgical team on arrival. relatives must accompany for confirmation of history of potential exposure as per moh protocol and directives. confounding factors must be taken into consideration during screening, including: i) potential aspiration in patients with low glasgow coma scale (gcs); ii) metabolic response resulting in abnormal white blood cell count (wcc) and elevated temperature; iii) co-existing chest injuries; and iv) post-intubation changes on chest radiograph (cxr) common. note: all ventilated cases pose a high risk of aerosol exposure to hcws. it is vital to be meticulous and vigilant for potential risk: critical resources for optimum neurosurgical services remain limited and may continue to fluctuate in time of crisis. these include ventilators, ppe, icu availability and operative instruments. the rationale for early prognostication is recommended for optimum resource usage and allocation to ensure beneficial outcome and sustainable supply. prognostication is based on available scientific evidence to guide in management options and rationale of resource allocation. early triage is required for timely and appropriate treatment and enables surgeons to prioritise management according to available resources and the potential outcome. this will help in limiting the proportion of patients in a vegetative state and limiting burden to family and available resources at the time of crisis. in the end, it will help to prepare family with a realistic outlook on the potential outcome. prognostic factors include: i) age > 60; ii) gcs post-resuscitation: motor score m2 -poor outcome; iii) pupils-bilateral fixed/ dilated pupils; iv) systolic blood pressure < 90 mmhg -sustainable/multiple episodes; and v) marshall ct grade. age, gcs motor score and pupillary changes are the three main prognostic factors determining the outcome. the caveats to prognosticating outcome include: aneurysmal sah is a devastating clinical entity. the natural history of aneurysmal sah remains unfavourable with a cumulative rerupture rate at 50% at 2 weeks after initial presentation and overall mortality of 60%. prognostication of aneurysmal sah is well defined according to the world federation of neurosurgery (wfns) grade system predictive of figure 4 . proposed workflow of patients referred for sah outcome following aneurysmal rupture and hunt & hess grade which predicts mortality rate in patients. management of aneurysmal sah during times of crisis must be guided by expected prognosis as defined using these grading systems. this is essential in times of pandemic crisis as difficult decisions need to be made to preserve life and function in face of limited and precious resources essential to the management of patients (41). the proposed recommendations can be viewed in figure 4 . aneurysmal sah (44) adequate resuscitation measures should be instituted on admission where required. these include the airway, breathing and circulation (abc) and mechanical ventilatory support as required with correction of fluid and electrolyte abnormalities if present at primary referral centres. the physician receiving the referral should get an accurate assessment of wfns, and hunt & hess grades on presentation. if hydrocephalus is present on ictus, urgent ventricular drainage should be performed at centres with neurosurgical facilities or trained and privileged surgeons at the designated non neurosurgical centres. a noncontrast ct brain and ct angiogram (cta) are the first line investigative parameter at the admitting hospital before transfer to a centre with neurosurgical facilities or services. when perimesencephalic sah is the likely diagnosis after confirmation by a neuroradiologist, then the patient should be managed expectantly. there will be no further imaging required. however, if aneurysmal rupture cannot be ruled out, then the recommendation is based on the wfns grading. for wfns 1-3, a digital subtraction angiography (dsa) is recommended at the primary centre if available or transfer to centres with available facilities and neurosurgical services. for wfns 4 and 5, the patient should stay at the primary centre if feasible with a repeat cta in 1 week. a continuous neurological assessment should be documented and if whenever an improvement is noted, then the patient should be considered for transfer. poor grade aneurysm cases may benefit from continued neuroprotection. however, multiple factors should be taken into accounts such as age, comorbidities and available resources to sustain prolonged care in such patients. the decision to consider conservative management if no further improvement in wfns score is the prerogative of attending consultants. the quality of cta is important in determining the next treatment of care. for patients with wfns grade 1-3, if cta deemed adequate by attending consultants for safe and effective definitive management by surgical clipping, then the recommendation is to proceed for surgical treatment as urgently possible at the centre with available neurosurgical services. if cta deemed inadequate for definitive management, then the patient should be transferred for dsa at centres with available radiological and neurosurgical services. the choice of treatment between surgical treatment and endovascular treatments should remain similar to current standards. however, interventional neurovascular services in malaysia are limited to a few major hospitals. transferring patients across states in times of limited resources may result in unnecessary delays and worsening outcomes from potential deterioration during the interim period. thus, various factors should be taken into consideration when deciding the best treatment options in times of crisis. they include: if dsa is required, the option of definitive management of endovascular coiling at the same setting should be considered. there are several reasons which include: i) minimising the risk of aerosol disbursement that is highest during intubation/extubation; ii) early definitive management can be achieved at the same setting with reduced risk of re-rupture; iii) treatment of vasospasm if present for applicable cases done at the same setting; and iv) reducing the risk of exposure to personnel from a second ga procedure. patients with wfns grade 4 may benefit from neuroprotective measures. definitive management should be considered in selected cases such as: i) young age; ii) no morbidities/comorbidities; and iii) choice of treatment depends on available services and following discussion between attending consultant and family. for patients with wfns grade 5, conservative management should be considered if no further improvement achieved following a period of neuroprotection. the decision is made through a collective discussion between attending physician and family members. a thorough history should be elicited to determine any evidence of sentinel haemorrhage that may appear trivial to patients. an appropriate diagnostic modality is required to look for radiological evidence of recent i.e 'teat sign'. within an applicable timeframe, a lumbar puncture should be considered. an aneurysm with neurological symptoms and signs should be planned and treated in a timely manner as a semi-emergency case; an example is a patient with posterior communicating artery aneurysm presenting with ptosis, rather than a delayed surgery in this current pandemic of uncertain duration to avoid potential irreversible neurological compromise or worsening deterioration. in cases of multiple aneurysms, the treatment should be undertaken for ruptured aneurysm as well as aneurysm with increased risk of rupture, if deemed feasible at the same sitting. arteriovenous malformations (avms) are a heterogeneous group of neurovascular abnormalities with an incidence of 1.1/100000 population. anatomically avms are defined as a complex of abnormal arteries and veins that communicate directly without an intervening capillary bed. avm presents with haemorrhage in as many as 50% of cases. the natural history of avms is more favourable as compared to aneurysmal sah, with an annual rupture rate of 4%, and recurrent haemorrhage rate of 6%-17% in the first year following a rupture (45) . cases of avm presenting with haemorrhage may require urgent surgical management. the proposed recommendations are divided into three categories: i) avm rupture with mass effect; ii) avm rupture without mass effect; and iii) avms not presenting with haemorrhage (46) (47) (48) . any ruptured avm with mass effect should be transferred to a centre with neurosurgical services or facilities. a cta should be performed on admission to confirm the diagnosis of ruptured avm and to determine the location of avm in relation to clot. this will aid a safe surgical access planning. following an urgent surgical evacuation of a clot, the patient should be managed in an intensive care setting for neuroprotection. a post-operative dsa/cta is warranted to look for potential high risk factors for haemorrhage, such as nidal aneurysm/varix and to consider an endovascular treatment if feasible and required. definitive treatment should be deferred safely to a later date if feasible. the patient should be transferred to a centre with neurosurgical facilities, particularly when avm presents with intraventricular haemorrhage (ivh) or cta shows evidence of nidal aneurysm or varix. if no ivh present and cta shows no high risk factors of haemorrhage, the patient may be managed at a non neurosurgical centre with early follow up scheduled in the clinic for review. the management is mainly medical treatment to optimise seizure control or headache. all definitive treatment should be deferred to a later date. (49) these are rare conditions that comprise of fistulas connecting branches of dural arteries to dural veins or venous sinuses. dural arteriovenous fistulas (davfs) are typically stable lesions with a reported annual haemorrhage risk ranging between 6%-8% (10), and mortality rate ranges at 6%-20%. endovascular modalities remain the diagnostic and therapeutic modality of choice. the recommendations are proposed for the management of (1) ruptured cranial davf and ruptured spinal davf. patients who presented with ruptured cranial davf should be transferred to a neurosurgical centre with endovascular facilities for urgent surgical management of clot with mass effect, if present, and definitive endovascular management. patients who presented with ruptured spinal davf requires transfer to a neurosurgical centre with endovascular facilities for urgent treatment, especially when there is rapid neurological deterioration. some patients presented with spontaneous hypertensive haemorrhage that may or may not require surgical intervention. patients that are not a candidate for surgical interventions include: i) small, deep haemorrhage; ii) large haemorrhage without hydrocephalus, ivh or neurological deterioration; and iii) those with supratentorial haemorrhage with a gcs score below 8 unless this is because of hydrocephalus (50) . a poor prognosis is expected in the following conditions: ii) candidate with gcs score is 5 or less; and ii) the haematoma is very large and death is expected (50) . in this condition, a careful consideration should be taken and a family meeting should be done sooner. surgical interventions are recommended in the following categories: the diagnosis of middle cerebral artery (mca) infarction depends on the clinical presentation, neurological findings, followed by radiological imaging. patients with suspected transient ischaemic attack (tia) should be assessed by a specialist physician before a decision on brain imaging is made, except when haemorrhage requires exclusion in patients taking an anticoagulant or with a bleeding disorder when noncontrast ct should be performed urgently (50) . further imaging, such as carotid imaging is essential for any patient presenting with symptoms suggesting of an anterior circulation cerebral ischaemia who might be suitable for intervention for carotid stenosis. patients with tia or acute non-disabling stroke with stable neurological symptoms who have symptomatic severe carotid stenosis of 50%-99% (nascet method) should receive an urgent carotid endarterectomy (within 7 days). the treatment tends to happen in a vascular surgical centre routinely participating in national audit (50) . the indications for mechanical thrombectomy (mt) (50) are when there are proximal large artery occlusion as an adjunct to intravenous thrombolysis (ivt), and for those patients with contraindications to ivt but not to mt. another indication is when major vessel occlusion is in the posterior circulation, up to 24 h from known onset. the indications for decompressive hemicraniectomy are as follows (50) (53), ventriculostomy is recommended in the treatment of obstructive hydrocephalus. concomitant or subsequent decompressive craniectomy may or may not be necessary on the basis of factors such as: i) the size of the infarction; ii) neurological condition; iii) degree of brainstem compression; and iv) effectiveness of medical management. there are times when an emergency carotid endarterectomy (cea)/ carotid angioplasty and stenting will be useful as clinical indicators or brain imaging suggests (53) . an example is when a small infarct core with large territory at risk (e.g. penumbra), compromised by inadequate flow from critical carotid stenosis or occlusion. however, in patients with unstable neurological status (e.g. stroke-in-evolution), the efficacy of emergency or urgent cea /carotid angioplasty and stenting is not well established. stroke complicated covid-19 infection in 5.9% of patients at a median 10 days after symptom onset. stroke mechanisms may vary and could include hypercoagulability from critical illness and cardioembolism from virusrelated cardiac injury. the clinical presentation in stroke patients typically manifested as cns involvements. the most common neurological manifestations in covid-19 patients were dizziness (16.8%), headache (13.1%) and encephalopathy (2.8%). the most common peripheral signs and symptoms were anosmia (5.1%), dysgeusia (5.6%) and muscle injury (10.1%, detected by elevated creatine kinase). patients with stroke were older, had more cardiovascular comorbidities, and more severe pneumonia. ideally, every stroke patient would be treated as potentially infected, hence the requirement of ppe. many teams have begun using telemedicine both within their own ed and regionally. this solution avoids the use of needed ppe, allows a reasonable stroke evaluation, avoids unnecessary interfacility transfers, and reduces exposure risk for the stroke team. in the setting of the pandemic, full compliance to clinical practice guidelines has become a goal, not an expectation. each team must use their judgement, guided by local realities, and continue to try to treat as many acute stroke patients as possible. patients with large intracerebral haemorrhages, sah or large ischemic strokes at risk for herniation must be monitored in an intensive care setting with appropriately trained personnel, where possible. appropriate resource should be allocated for critically ill stroke patients. appropriate intensive care of these seriously ill patients with haemorrhagic stroke, some of whom are also young and with an excellent long term outcome, should be maintained. however, in each locality, specialists from all intensive care specialities e.g. pulmonary, cardiology, neurology, neurosurgery must discuss the relative merits of prolonged icu care for any particular patient. (55) the establishment of stroke networks and care systems can deliver a high quality emergency stroke care at all times, particularly at times of crisis. although there is a strong case for such centres to be the system of care, it is particularly important to have services that can continue to function. the hospital should inform the emergency medical system and the public that these centres will be protected and will remain fully operational even during crises. the hospital or stroke team should regularly update and educates the health professionals and the public, especially those who are at high risk of stroke to recognise a stroke and call emergency medical services immediately. those patients should be taken to one of the designated stroke centres to avoid significant delay in transferring patient from one hospital to the other. categorising elective neurosurgical cases at a time of covid-19 pandemic is adapted and as per guidelines (with minimal modification) -perioperative mortality review (pomr): prioritisation of cases for emergency and elective surgery (2nd revision) (56) ( table 7 ). the tier status of each case is according to the urgency and the decision will be based on: i) natural history of the disease; ii) patient's neurological status; and iii) availability of manpower and equipment's for surgery ( figure 6 ). to summarised, all elective neurosurgery should be postponed (1, 21) . in patients with suspected covid-19, the surgery should be deferred for at least 14 days, with an appropriate test taken to confirm the status (1, 21) . the elective urgent inpatient diagnostic and surgical procedures should be shifted to outpatient settings, when feasible (2). dangers during neurosurgical procedure (2, 18, 19, 24, 28, 57) according to limited data from cdc, covid-19 has been detected in blood specimens and it is unknown whether the virus is viable or infectious in extrapulmonary (outside the lungs) specimens. there have been some reports that covid-19 is present in stool and maybe transmissible through the faecal-oral route. bronchoscopy, tracheostomy and thoracic cases may have a higher risk for airborne transmission of covid-19 because the nature of the procedures involves the respiratory tract, which could lead to aerosolisation of the virus. procedures that may aerosolise blood and body fluids during surgery include: i) electrocautery of blood or tissue; ii) laparoscopy; iii) endoscopy; iv) use of intraoperative debridement devices with irrigation (e.g. hydrosurgery, pulse lavage or low frequency ultrasonic debridement); and v) use of high speed powered equipment (e.g. saws and drills). surgical smoke represents another important issue to tackle during surgery. it is recommended for the evacuation of all surgical smoke as it contains hazardous chemicals, ultrafine particles, viruses, bacteria and cancer cells. the earliest detected case of covid-19 was in china on 17 november 2019. as such, there is currently no research on the transmission of the virus through surgical smoke. however, there is no indication or proof that covid-19 is not transmissible through surgical smoke. research studies have demonstrated the presence of viruses (e.g. human papillomavirus) in surgical smoke with documented transmission to health care providers. according to limited data from the cdc, sars-cov-2 rna has been detected in blood specimens and it is unknown whether the virus is viable or infectious in extrapulmonary (outside the lungs) specimens. in similar coronaviruses, viable and infectious sars-cov was isolated from blood specimens, although infectious mers-cov was only isolated from the respiratory tract. of importance to neurosurgeons, the use of high speed drills and also electrocautery during surgery will cause aerosolised blood and body fluid. thus, increasing exposure of neurosurgeons to the virus. however, the risk of transmission of covid-19 through aerosolised blood and body fluids is unknown. thus, extra precautionary measures must be taken during procedures for protection. proper ppe must be worn during any neurosurgical procedure to prevent transmission. (58, 59) covid-19 was declared a pandemic by who on 11 march 2020 because of its rapid worldwide spread. covid-19 has achieved effective and sustained human-to-human transmission via contact, droplet and likely airborne routes. as with previous outbreaks such as severe acute respiratory syndrome (sars), influenza a (h1n1) 2009 infection and the middle east respiratory syndrome, this would require heightened precautions and tailoring our anaesthetic practice to reduce exposure to patients' respiratory secretions and the risk of perioperative viral transmission to healthcare workers and other patients. in particular, this should involve minimising the many aerosolgenerating procedures we perform during ga, such as bag mask ventilation, open airway suctioning and endotracheal intubation. during the sars outbreak, intubation was one of the independent risk factors for super-spreading nosocomial outbreaks affecting many healthcare workers in hong kong and guangzhou, china. nevertheless, to avoid any airway manipulation, the use of ra techniques (e.g. peripheral nerve blocks and/or central neuraxial blocks) may be consideration should be given to the available resources, facilities, equipment, consumable and real time logistic capability and feasibility. designated covid-19 hospitals may not be able to support all elective cases, in particular those that require post-operative intensive care or significant use of blood and blood products surgeons, in consultation with anaesthetist, nursing colleagues as well as patients (or legally accepted next of kin), should weigh the risks of proceeding (exposure, lack of resources) against those of deferment, (progression of disease, worse patients outcomes) including the expectation of delay of 2-3 months or more or until the covid-19 is less prevalent figure 6 . availability of manpower and equipment for surgery preferred. thus, ra manipulation should be considered whenever surgery is planned for a suspected or confirmed covid-19 patient or any patient who poses an infection risk. ra has benefits of preservation of respiratory function, avoidance of aerosolisation and hence viral transmission. there is no proper guideline and recommendation as of today regarding the use of ra in covid-19 patients. however, general precautions and ppe should be applied for all the healthcare workers even though the patient is undergoing ra. this is because, in case of failed ra, ga must be used for the surgery. anaesthesia providers for these patients should be well-versed in both ga and ra techniques. for neurosurgery patients, ra such as scalp block must be considered in simple procedures such as borehole. for emergency neurosurgery cases, most of it would be involving patients with poor gcs thus making ra not as feasible as the patient would be already intubated. covid-19 is an infectious disease introduced to humans for the first time. individuals can be infected by breathing in the virus within 1 metre of a person who has covid-19, or by touching a contaminated surface and then touching their own mouth, nose, or possibly their eyes. on 30 january 2020, who declared the outbreak as a public health emergency of international concern (pheic), and by 11 march 2020, the outbreak has rapidly accelerated to become pandemic. until 3 april 2020, there were 976, 249 confirmed cases, with 50,489 deaths, affecting 207 countries, including malaysia (60). following the pandemic of covid-19, there is a major shift of practices among surgical departments in response to an unprecedented surge in reducing the transmission of disease. with pooling and outsourcing of more hcws to emergency room, public health care services and medical services, further in hospital resources are prioritised to those in need. along with slowing and breaking the transmission of covid-19 by social distancing, the neurosurgical outpatient clinic, elective and non-emergency surgery are delayed. this will reduce the face-toface contact with potential covid-19 cases, and shields patients and hcws from the virus. every neurosurgical team has to reevaluate the timing of operation in those patients with neurosurgical disease that are in need of treatment. the real risk of proceeding and the real risk of delay should be carefully assessed. when considering a delay in treatment to a time where covid-19 is less prevalent, the decision making process must always take into account each patient's courses of disease, social circumstances and needs. it is imperative to balance the requirements of caring for covid-19 patients with imminent risk of delay to others who need care (61). currently, patient's screening process is crucial. the moh recommendations of screening involve questionnaires to identify suspected patients (1, 62) . patients who meet certain criteria should be evaluated as a patient under investigation (pui). these general questions to all patients include: i) do you have any fever or acute respiratory infection (sudden onset of respiratory infection with at least one of: shortness of breath, cough or sore throat)? ii) do you have any history of travelling to or residing in affected countries in the past 14 days? iii) did you have any contact with a confirmed covid-19 case within the past 14 days? there are two laboratory tests that can be used to detect covid-19 (1, 61): rt-pcr. the sample commonly taken is upper airway specimens (pharyngeal swabs, nasal swabs, nasopharyngeal secretions). among patients with confirmed positive in respiratory tract, ~ 50%-60% of patients have detected viral load in faeces, ~30%-40% in the blood, while the lowest positive rate is in urine samples. ii) rapid test kit (rtk) serology for serum antibody igm and/or igg. this can be used as diagnostic criteria for suspected patients with negative pcr detection. during follow-up monitoring, igm is detectable 10 days after symptom onset and igg is detectable 12 days after symptom onset. the viral load gradually decreases with the increase of serum antibody levels. urgent/emergent cases are previously defined as patients requiring access to surgical treatment within 24 h of the decision to operate (63) . however, with the current pandemic, access to surgical treatment may be delayed and some patients could face increasing morbidity/ mortality by the time surgery happens. other countries have now come up with guidelines for the triage, or ranking in order of priority, of surgical patients. the american college of surgeons (acs) describes the acuity scales based on tier classification, with most cancers and highly symptomatic patients considered tier 3a (do not postpone) (64) . other than trauma and life threatening condition, other treatments are recommended to postpone, if possible. then what will happen to all elective cases that will be pushed back for a further few months? will that add to the mounting burden of long waiting lists that is already stretched? to delineate the current situation, we propose a few steps that will in future, limit and protect both patients and surgeons from the risk of transmission (figure 7) . special consideration is given to pre-operative patients needing endoscopic transnasal surgery, even for asymptomatic patients (8, 57) . the transmission of covid-19 is predominantly via respiratory droplets (e.g. coughing and sneezing) and contact with contaminated surfaces (18, 60, 65) . however, earlier studies have shown the presence of the virus in conjunctival secretions and even stool (65) . hence all body fluids except for sweat should be considered as potentially infectious (66) . contamination of the surrounding environment may also occur following aerosol-generating procedures (agp) which include the use of high-speed devices or when splashing or spillage of bodily fluid is expected (66) . appropriate steps especially in these environments need to be taken to disrupt the transmission of covid-19 and reduce risk of infection. these environments are considered as high-risk environments and include the icu, high-dependency unit (hdu) and ots. the emergency rhesus areas where suspected or confirmed cases of covid-19 are managed are also considered as high-risk areas (66) . all highrisk areas require level iii ppe (table 8 ) (66) . ppe is only a part of the safe system of working (67) . clinical staff must be trained and competent in the use of ppe in their respective hospitals. table 8 shows the recommended ppe for clinical setting (15, 61, 68) . ppe should be located close to the point of use and should be stored in a clean and dry area to prevent contamination. ideally, ppe is for single use and changed between patients unless in a situation of ppe shortage or when re-usable ppe is used. the used ppe must be disposed of in designated waste streams. the practice of donning (putting on) and doffing (taking off) of ppe must be done in designated areas safe for the respective procedures (18, 24) . different hospitals may have different arrangements of clinical areas (ot, wards, clinics, etc.). hence, the physicians must make sure to be aware of the designated areas for donning and doffing prior to putting on ppe. ideally, each clinical staff donning must be supervised by another competent clinical staff especially to assist in donning and to make a final visual inspection of ppe. recommended ppe components: i) protective medical gown should be long in length, long sleeves, rear-fastening and fluid-resistant. also, include protective, fluid-resistant boots with disposable fluid-resistant covers when appropriate (high-risk procedures). for the gloves, the recommendation is to use disposable, non-sterile latex gloves for non-sterile procedures, whereas for sterile procedures is to use disposable sterile latex gloves. double gloving is essential in those procedures. (15) ii) for the face/eye protector, ideally a disposable visor which covers the whole face including chin will give better protection than glasses and contact lenses. iii) for the respirator/mask, papr is recommended and preferred for high-risk procedures including agp. a minimum of n95 mask with face protector (i.e. visor) for agp (28, (69) (70) (71) . for sars-cov, there is limited evidence (from observational studies) showing a protective effect of up to 80% of n95 masks (equivalent to ffp2 masks) used by healthcare workers. hence, although the cdc recommends n95 masks or higher level respirators for agp, n95 respirators are not recommended for agp in the uk (19, 69) . the masks used should be fitted to the face without airleaks. in general clinical setting (other than aerosol-producing procedures), a fluidresistant mask is required. while fabric masks are widely available, this should not be used in any clinical setting. figure 8 shows how to perform a particulate respirator seal check (72) . i) essential to sanitise hand with alcohol gel before, in between each step and after donning of ppe ii) ppe must be put on in an order that ensures adequate placement of ppe equipment and prevents self-contamination and self-inoculation while using ppe and when taking off ppe (69, 71) . figure 9 shows levels of ppe. figure 10 shows the recommended ppe to be used at the primary triage and non-sari area, while figure 11 shows the recommended ppe in the ot, the ideal pressure system is a negative pressure room and if available, the recommended location is at the corner of the operating complex (57, 61). the theatre should have a separate entrance. for intubation, the surgeons and personnel that are not involved in intubation should wait outside of the ot until anaesthetic induction and intubation are completed (12, 15, 61) . during surgery (except for endoscopic endonasal procedures), procedures involving high speed devices are considered as aerosol-producing procedures and are high risk. the number of personnel in an ot should be minimised (58) and full ppe should be applied. a n95 mask or masks that offer a higher level of protection should be used (28, 65, 66) . endoscopic endonasal procedures are not safe and should be avoided (28, 33) . if the surgery cannot be postponed, consider a craniotomy or microscope-based transsphenoidal procedure. during a shortage of ppe, the hospital or physicians are encouraged to minimise the use of ppe (16, 69) . there are options available, such as considering telemedicine, where appropriate, to avoid direct contact with patients hence removing the necessity of ppe. the use of sterile gloves should be reserved only for procedures requiring sterility (61, 65, 66) . all elective or non-urgent procedures, which usually require components of ppe should be delayed (65) . certain areas should be monitored with restricted access, such as areas where suspected or confirmed covid-19 patients are being treated. some activities that require to be done at proximity to the patient (e.g. at bedside) must be planned early and bundle them together to minimise the number of times entering the room. visitors should not be allowed unless necessary, with restricted numbers and amount of time spent in the area. visitors must have clear instructions and guidance when donning and doffing with strict hand hygiene (20, 61) . the use of appropriate ppe should be prioritised (19, 61) and rationalised according to the risk of exposure and transmission dynamics of pathogens (air droplets, contact). overuse of ppe will further impact on supply shortages. (table 8) the physician usually have to tailor the ppe usage based on the setting and activity being performed. below are some recommendations: extend the use of surgical gowns, masks and face protectors between patients with the same disease, who were confined in the same area without changing in between patients and whilst performing low risk procedures (14, 19, 61) ii) consider re-usable face protectors (i.e. visors or googles) ( the ultraviolet (uv) spectrum is best known for uva, uvb and uvc (germicidal radiation). the spectral ranges for uva, uvb and uvc are 315 nm-400 nm, 280 nm-315 nm and 100 nm-280 nm, respectively. uvc is the one with the strongest antimicrobial/antiviral properties (74, 75) . with the rising healthcare awareness, some industry has demonstrated the effectiveness of radiation disinfection, especially uv light disinfection system on surface contamination, such as floors and equipment after the manual chemical disinfection process is completed (76) . uv light disinfection is an implementation of 'no-touch' technology, is chemical free, does not require changes in the room's ventilation and will not leave a residue after treatment. in healthcare facilities such as icu and ot, this may be an adjunct to disinfection process (77) . a laboratory study has shown that coronavirus could effectively be inactivated by uvc light (74, 75, 78) . nevertheless, the uv light device is not a substitute for handwashing, mask-wearing and distancing. moreover, the international commission on non-ionizing radiation protection (icnirp) does not recommend the usage of lamps in the home. this is due to lack of adequate instructions of installation, duration of disinfection and increasing cases of skin and eye burns (79) . who has come out with a fact that uv lamps should not be used to disinfect hands or other skin areas. furthermore, reiteration was made that 'cleaning your hands with alcohol-based hand rub or washing your hands with soap and water are the most effective ways to remove the virus' (80) . the malaysian medical council has formed an advisory group to define and monitor virtual consultation (during the covid-19 pandemic). this advisory is guided by the medical act 1971 (amended 2012) which regulates the registration and practice of medicine in malaysia and the malaysian medical council's code of professional conduct. a virtual consultation is a form of telemedicine. telemedicine (teleconsultation, video conferencing, teleworkers, televideo) is a medical service provided remotely via information and communication technology. when the consultation is conducted without physical contact and does not necessarily involve long distances, then it is known as remote consultation. the role of the council is to regulate physicians, not technology. the council reminds physicians that the use of technology does not alter the ethical, professional and legal requirements in the provision of care. the malaysian medical council's jurisdiction is within this country only and physicians must ensure appropriate liability protection is in place to provide indemnity for malpractice. when the health care delivery is affected by any national epidemic or global pandemic, or any other movement restrictions imposed on the public by the government, the use of communication technology can improve the access to care. in this unprecedented time where the situation is seen to be very urgent, rapidly changing and where there is a fine balance between public safety and individual health, it is equally important for medical practitioners to have the virtues of accountability and truth telling. the code of professional conduct clearly says a physical examination is ethically mandatory. a non-physical contact virtual consultation makes a physical examination incomplete other than the visual and auditory observation. however, if a physician under current circumstances conducting such telemedicine virtual consultation feels this is so in good faith, then appropriate treatment can be initiated based on such, without the need for a physical examination in person. in providing medical care using telecommunications technologies, physicians are advised that they must possess adequate training and competency to manage patients through telemedicine. the ethical and legal requirements such obtaining valid informed consent from the patient should be taken, at the same time ensuring that the physician's identity, place of practice and registration status are made known to the patient, and the identity of the patient is confirmed at each consultation. the identities of all other participants involved in the telemedicine are disclosed and approved by the patient, and documented in the patient record. both the physician-site and the patient-site are using appropriate technology that complies with legal requirements regarding privacy and security and accreditation standards where required. considerations must be given to safety and maintaining a high standard of patient care. the physicians must consider whether the telemedicine medium affords adequate assessment of the presenting problem, and if it does not, an arrangement for a timely in-person assessment should be taken. the physician should be prepared to advise remote patients about how and where to arrange for necessary care when follow-up is indicated. with the limitation of telemedicine, the physicians should exercise caution when providing prescriptions or other treatment recommendations to patients whom they have not personally examined. when carrying out a diagnostic evaluation or prescribing medication, a physician conducting a remote interaction should: i) verify the patient's identity; ii) confirm that the remote interaction is appropriate to the patient's situation and medical needs; iii) write any prescriptions in keeping with best practice guidelines and formulary restrictions (and in keeping with ethics guidance on prudent stewardship); and iv) document the clinical evaluation and prescription, as well as any instructions given to the patient. a medical record of the consultation, in accordance with professional and legal requirements, are kept and available to other health care professionals for the provision of ongoing patient care. this is especially important when there is a followup and referral to other specialities. hence, the physician must ensure adherence to the same obligations for patient follow up in telemedicine as is expected with in-person consultation. many centres have implemented the telemedicine in neurosurgical consultation, specifically in patients with confirmed covid-19, or recovered patients (pcr negative and beyond 14 days incubation period) that may need comprehensive clinical assessment to be performed. throughout all levels, neurosurgeons are encouraged to convert meetings (with staff, colleagues and patients) to teleconsultation and/or video conferencing (6) . teleconsultation minimises face-to-face clinic visits for all doctors and patients, including neurosurgeons and their patients (3). staff and patients over the age of 65 are encouraged to avoid coming to the hospital and clinic. the conversion of many clinic visits as medically appropriate to this new modality allow patients to stay safe at home and allows clinic nurses and staff to help care for covid-19 patients. another speciality that is using telemedicine is neurology, mainly for the assessment of patients with a suspected stroke. the telemedicine should enable the physician to discuss the case with the assessing clinician, talk to the patient and/or family/carers directly and review radiological investigations (50) . hence, it is important that a high-quality video link is maintained to enable the remote physician to observe the clinical examination. the physician providing care (at both ends of the system) should be appropriately trained in the hyperacute assessment of people with suspected acute stroke, in the delivery of thrombolysis and the use of this approach and technology. the impact on the quality of care, efficacy of telemedicine and decision-making using telemedicine should be regularly audited (81) in keeping with physician's fiduciary obligations to patients across that continuum (82) . timely updates from trusted sources about the relative risk of contracting the novel disease versus a more common one are critical (83, 84) . strategic social media use (e.g. hashtags) may be an effective way for agencies to communicate accurate information to the public during times of crisis. residents may be advised to connect with and follow local health agencies and service providers for the most geographically relevant information. researchers may use publicly available 'big data' (e.g. localised tweets) to gauge the risk of communication efforts of local agencies. (84) (85) (86) in the emergency department, there is a surge control for 'forward triage', meaning utilisation of sorting the patients before they arrive in the emergency department. this allows patients to be efficiently screened, is both patient-centred and conducive to self-quarantine, and it protects patients, clinicians and the community from exposure. the physicians and patients are still able to communicate 24/7, either by using smartphones or webcam-enabled computers. the respiratory symptoms (which may be early signs of covid-19) are among the conditions most commonly evaluated with this approach. telemedicine consultations for oncologic patients may not be suitable and individual clinicians must be able to make an appropriate judgment. however, patients will greatly benefit from such virtual clinic consultations over a cancellation. (87, 88) the presence of virus within csf fluid and during autopsy can be tested via electron microscopy, immunohistochemistry and realtime reverse transcriptional. there are 36.4% of patients had neurologic manifestations due to neurotropic potential in the covid-19 virus found in one study. during an early or later phase of the infection, the dissemination of covid-19 in the systemic circulation or across the cribriform plate of the ethmoid bone takes place. the ability to cross the blood brain barrier into the cerebral circulation is due to the properties of the covid-19 virus spike protein with angiotensin-converting enzyme 2 (ace2) receptors expressed in the capillary endothelium. the receptor has been detected over glial cells and neurons. covid-19 virus exploits the ace2 receptors to gain entry inside the cells (89) causing neuronal death in mice by invading the brain via the nose close to the olfactory epithelium. in an uncomplicated early stage, findings like an altered sense of smell or hyposmia can be found. once the virus caused respiratory manifestation, there will be neurological involvement with loss of involuntary control over breathing (90) . tuberculosis (91) the physician should maintain continuity of essential services for people affected with tuberculosis (tb) during the covid-19 pandemic. it is anticipated that ill patients with both tb and covid-19 may have poorer treatment outcomes, especially if tb treatment is interrupted. therefore, accurate diagnostic tests are essential for both tb and covid-19. simultaneous testing of the same patient for both tb and covid-19 would generally be indicated for three main reasons (subject to the specific setting in the country): i) clinical features that are common to both diseases; or ii) simultaneous exposure to both diseases; or iii) presence of a risk factor for poor outcomes to either disease. in tuberculous meningitis with communicating hydrocephalus, the recommendation is to treat with furosemide with or without acetazolamide. some institutions favour daily lumbar punctures with icp monitoring through manometry (92) . in tuberculous meningitis with noncommunicating hydrocephalus, this will involve invasive neurosurgical procedures such as an external ventricular drain (evd), ventriculoperitoneal shunting or endoscopic third ventriculostomy (etv) (92) . acute necrotising encephalopathy is a condition that can be triggered by viral infections like influenza and herpes. a case report of a woman who tested positive for covid-19 developed acute necrotising encephalopathy (93) . the patient presented altered mental status and a noncontrast head ct images demonstrated symmetric hypoattenuation within the bilateral medial thalami with a normal cta and ct venogram. brain mri demonstrated a haemorrhagic rim enhancing lesions within the bilateral thalami, medial temporal lobes, and subinsular regions. the patient was started on intravenous immunoglobulin. this is the first reported case of meningitis associated with sars-cov-2 who presented with convulsion followed by unconsciousness (94) . the patient had transient generalised seizures that lasted about a minute. the specific sars-cov-2 rna was not detected in the nasopharyngeal swab but was detected in a csf. brain mri showed hyperintensity along the wall of right lateral ventricle and hyperintense signal changes in the right mesial temporal lobe and hippocampus, suggesting the possibility of sars-cov-2 meningitis. paediatric neurosurgery accounts for up to 15% of neurosurgical admissions. in this current crisis, covid-19 infection among children remains limited in numbers. however, paediatric patients are at risk of exposure from potential family members and caretakers that may harbour the virus and remain asymptomatic. the main consideration is therefore to be vigilant and screen caretakers and family of patients for risk of exposure based on current and updated guidelines provided by the moh (1). the priority as for all cases in the pandemic crisis remains the protection of healthcare personnel and other non-infected patients as well as the working environment at healthcare facilities. the guidelines provided by moh (1) applies equally for paediatric neurosurgery cases; principally in deciding for the need of surgical care and intensive management in times of reduced availability of resources. this guiding principle forms the core basis of decision making for paediatric neurosurgery in malaysia as the availability of specialised paediatric intensive care units and specialised instruments are typically present in major hospitals with dedicated neurosurgical units and facilities. with the ongoing pandemic crisis, these subspecialised resources are crucial for the treatment of patients afflicted by covid-19. (95) all non-essential outpatient cases should be postponed. some new cases with symptoms and signs of raised icp or neurological compromise should be reviewed urgently by a neurosurgeon. whereas, new cases with no symptoms or signs of raised icp or neurological compromise should be reviewed via teleconsultation. any pending surgeries should be reviewed with team and mdt to prioritise cases based on the urgency of surgical intervention, taking into account symptoms and signs, radiological evidence of mass effect or vital structure compression, expected radiological progression over time and expected histology along with the availability of vital specialised resources of intensive care, equipment requirements and ot availability. certain logistic factors, such as patient travel and family economic factor that requires one or both parents to be away from work should also be taken into account in the decision making process to ensure compliance of patient and caretakers to the management plan. paediatric neurotrauma (38) all cases must be screened for risk of covid-19 as per hospital protocols. the management of paediatric neurotrauma is based on the brain trauma foundation (btf) guidelines. all cases undergoing emergency surgery must be performed under full ppe as per hospital/moh protocols (1, 62) . the initial referral should be reviewed by the neurosurgical team or consulted via teleconferencing with the neurosurgical team on admission. the modified paediatric gcs score system (table 9 ) must be applied and made available to referring or primary management team where applicable (96) . cases of neurotrauma with surgical lesions must be transferred to a hospital with a neurosurgical facility and or services for management. frequent repeats of ct scan should be avoided where feasible and should be factored into the decision making process for applicable cases. there are certain cases of neurotrauma that has positive ct findings but with a nonsurgical lesion (97) . if the treatment requires an icp monitoring, then the patient should be transferred to a centre with neurosurgical services or facilities. if the treatment does not require an icp monitoring but there is a potential risk of progression and deterioration, then the patient should also be transferred over. however, for patients who do not require intensive care with low risk of deterioration or progression, it is advisable to transfer to centres with available neurosurgical services. if it is not feasible, then the patient should be managed by attending paediatrician/surgeon at primary referral centres with regular consultations to the neurosurgical team. cases with no positive ct brain findings (97) may be managed at primary referral centres if deemed suitable with a consultation to the neurosurgery team. in a non-traumatic paediatric neurosurgery case, the approach is the same; all cases must be screened for risk of covid-19 as per hospital protocols. suitable use of ppe must be adhered to for all cases undergoing surgery. the surgeon should: i) consider the option of fastest and simples access route to the lesion; ii) consider biopsy if deemed appropriate over surgical guidelines covid-19 management in malaysia covid-19: recommendations for management of elective surgical procedures american association of neurological surgeons. covid-19 and neurosurgery cms adult elective surgery and procedures recommendations neurosurgery during the covid-19 pandemic: update from lombardy, northern italy letter: the coronavirus disease 2019 global pandemic: a neurosurgical treatment algorithm guidance for surgeons working during the covid-19 pandemic letter: precautions for endoscopic transnasal skull base surgery during the covid-19 pandemic just the facts: airway management during the coronavirus disease 2019 (covid-19) pandemic adoption of a safe or cautious approach towards surgery covid-19 preparedness within the surgical, obstetric, and anesthetic ecosystem in sub-saharan africa 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developments focusing on prognostic factors for outcome covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features a first case of meningitis/encephalitis associated with sarscoronavirus-2 impact of the coronavirus (covid-19) pandemic on surgical practice -part 2 (surgical prioritisation) performance of the pediatric glasgow coma scale score in the evaluation of children with blunt head trauma the society of british neurological surgeons. clinical guide for the management of paediatric neurosurgery patients during the coronavirus pandemic early lessons in the management of covid-19 for the pediatric neurosurgical community from the leadership of the american society of pediatric neurosurgeons anaesthesia and sars infographic for principles of airway management in covid-19 world federation of societies of anesthesiologists this publication is not a proposed national guideline for the covid-19 management of covid-19 neurosurgical patients but a review of management and protocols from local and international peer reviewed and non-peer reviewed publications, web-based information and data until the month of august 2020. we hope that this publication will be used to improve care of neurological or neurosurgical patients and their caregivers/frontlines globally especially in developing countries. none. none. appendix 2. principles of airway management in coronavirus covid-19 (100) www.mjms.usm.my 189 key: cord-273601-icituitn authors: liu, m.; thomadsen, r.; yao, s. title: forecasting the spread of covid-19 under different reopening strategies date: 2020-05-29 journal: nan doi: 10.1101/2020.05.26.20113993 sha: doc_id: 273601 cord_uid: icituitn we combine covid-19 case data with demographic and mobility data to estimate a modified susceptible-infected-recovered (sir) model for the spread of this disease in the united states. we find that the incidence of infectious covid-19 individuals has a concave effect on contagion, as would be expected if people have inter-related social networks. we also demonstrate that social distancing and population density have large effects on the rate of contagion. the social distancing in late march and april substantially reduced the number of covid-19 cases. however, the concave contagion pattern means that when social distancing measures are lifted, the growth rate is considerable but will not be exponential as predicted by standard sir models. furthermore, counties with the lowest population density could likely avoid high levels of contagion even with no social distancing. we forecast rates of new cases for covid-19 under different social distancing norms and find that if social distancing is eliminated there will be a massive increase in the cases of covid-19, about double what would occur if the us only restored to 50% of the way to normalcy. as covid-19 spreads across the world and the united states, governments and individuals have worked to slow the growth of the disease by reducing the extent to which people leave their homes. in the united states, these actions have largely been acted on by households who have voluntarily stayed home unless they needed to travel, but they have also been bolstered by orders of local and state governments. these orders have occurred over different time periods, and have taken many different forms, but they have had a similar flavor of limiting gathering sizes, closing schools, and shutting down non-essential businesses or shifting their operations to a contact-free experience. that said, many areas of the country never had any form of stay-at-home orders. ultimately, the purpose of the stay-at-home orders is to reduce the amount of contact between people in order to slow the growth of covid-19, which is thought to be spread primarily through droplets that require being within a relatively small distance of an infected person. in this paper, we first measure the extent to which social distancing reduces the speed at which covid-19 spreads. we then run simulations of how covid-19 will spread over time under different policy regimes. we find that covid-19 spreads less than proportionately with the number of contagious individuals. we also observe that social distancing during late march and april significantly reduced the spread of the disease. higher population density also leads to an increased spread of covid-19. our model gives good out-of-sample forecasts of the disease for the two and half weeks after the end of our mobility data, assuming that the country continues the nearly 50% return to normalcy observed at the end of april (as compared to the observed peak social distancing levels). we forecast that completely opening up the country to 100% of the pre-shutdown levels of social interaction will lead to 4 million additional covid-19 cases (officially diagnosed) by the end of september 2020, corresponding to a doubling of the cases than we would expect if the country continued with the path of 50% return to normalcy we observe at the end of april. however, there is a great heterogeneity among counties, and according to our simulations 44% of the counties could open up while still experiencing a low infection rate of less than 0.1% over a 3-month period. these counties all have low population densities. the model we estimate is a simplified version of a susceptible-infected-recovered (sir) model. we assume that where y i,t is the number of individuals who are infected in county i on day t, r i,t is the rate at which infectious individuals in the county transmit the disease, s i,t is the percentage of the county population that is susceptible to covid-19 (i.e., the share of people who have not yet had , and y i,t is the number of cumulative individuals who have been infected by day t. the y i,t−2 −y i,t−8 term reflects our assumption that infected individuals are contagious from the second day after they catch the virus through the seventh day, leading to a serial interval of 4.5 days 1 . this treatment of the infectious population is an approximation to the standard sir models, where the infectious population is typically modeled as a stock that has an outflow at a constant rate. this assumption makes the estimation much easier with the large number of fixed effects we include in our model, and as a practical matter this assumption only has a minimal impact on our estimates of the contagion of covid-19. in the supplemental appendix we show that we get extremely similar results if we take the time of contagiousness to be 14 days the main difference between this model and a standard sir model is that a standard sir model constrains ω = 1. we show in the supplemental appendix that the estimated model with this constraint does not perform well out of sample. we instead find that ω < 1. this shows that the marginal impact of one more sick person diminishes as more and more people are sick. there are several reasons why this may be expected, with the greatest reason being that contagious individuals may end up endangering many of the same group of unexposed individuals. one might expect this to be the case if people often have the same or overlapping groups of friends or acquaintances. we see some of this directly when, for example, cases are clustered within households, nursing homes, or places of work. in the accompanying supplemental appendix we present a networking model and show that we would get ω < 1 if people have interconnected networks of contact. in order to better understand the variation of the rate of contagion, we allow r i,t to vary according to a number of factors instead of treating it as a constant parameter. thus, we model this specification implies that transmission rates can differ across counties (the county fixed effects α i reflect different population densities and also different demographic compositions), time periods (date fixed effects β t are included mostly to accommodate different rates of testing and also the different rates of reporting that happen on weekdays vs weekends), levels of social distancing d i,t , and different temperatures, h i,t , the impact of which has been debated 2 , 3 , 4 . the social distancing measure, d i,t , is based on cellphone gps location data that are provided by safegraph, and are available for free to researchers studying covid-19. we measure social distancing as the fraction of phones that stay exclusively at home during a given day. the ε i,t term is our statistical error term. equation (1) is estimated by taking the logarithm of both sides, with the details in the appendix. note that the social distancing level by individuals, as well as social distancing regulations, are not determined in a vacuum. rather, we observe that people social distance more in areas that are harder hit by covid-19. thus, the ε i,t term may be correlated with the social distancing measures, causing a biased, underestimated impact of social distancing on slowing the spread of the disease. we control for this endogeneity bias by estimating the model using an instrumental variables (iv) technique, where we use the amount of rain as an instrument for social distancing. specifically, we assume that rain directly shifts the level of social distancing, but is not correlated with ε i,t . to estimate the model parameters, we use county-level officially confirmed covid-19 daily case data of 2,704 us counties or county-equivalents from february 3 to april 28. we omit the data from new york, new jersey and connecticut, as explained in the technical appendix. we append the data with daily county-level weather data as well as cellphone mobility data provided by safegraph. the results are presented in table 1 . we find that social distancing indeed decreases the growth rate of covid-19: moving from the observed mean pre-covid level of social distancing (0.25) to the post-covid peak level (0.40), the magnitude of r is reduced by 56%. we also find that the exponent on the number of contagious people, ω, is 0.47. it is significantly lower than 1, the exponent assumed in a standard sir model. this shows that there is a strongly concave relationship between the number of infected people and the rate at which the disease spreads. this level of concavity also implies that while initial outbreaks of covid-19 will expand exponentially, they will quickly turn to a slower rate of growth. the growth looks linear or even plateauing when plotted cumulatively, although the disease will persist for a long period of time and continue building a substantial number of cases. this may explain why the recent growth rate of covid-19 cases has slowed considerably after a quick take-off, and yet this growth has persisted. we also find that higher temperatures may slow the spread of the virus, but with a much smaller impact. most of the variation of the contagion rates r i,t is captured by our county-level fixed effects, α i , in equation (2) . in order to understand the drivers of the contagion rates, we run a regression of the county fixed effects α i against county-level demographics. it has been shown in 5 that when the number of fixed effects is large, these coefficients can be treated as data for the purpose of statistical inference. the results are reported in table 2 . we find that population density is a crucial factor influencing the spread of the disease. in fact, covid-19 would be expected to never flare up beyond a very small base level in 2/5 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 29, 2020. some areas with sufficiently low population densities, as we discuss below. we also observe that greater concentrations of black and hispanic residents and public transit commuters are associated with higher contagion rates. interestingly, higher median incomes are related to greater contagion. we are uncertain what drives this result: one possibility is that people with higher incomes may interact more with nearby cities that have more outbreaks. we also include the share of seniors (age 70+) and children (below age 18) in the population. seniors are marginally more likely to spread the disease, but children show no sign of having a lower rate of infecting people, confirming the finding in 6 . using our model, we simulate future cases beyond our sample period. first, to examine how our model performs, we predict the out-of-sample case numbers from the end of our data period up to may 16, 2020, under different social distancing assumptions. we start by forecasting the cumulative covid-19 cases if each county continued the social distancing at the levels observed at the end of april. assuming the observed february level as normalcy, the end-of-april level is at the 50% between the peak lock-down level and normalcy (so we say that such a level is at 50% of normalcy). we also implement the same exercise under 3/5 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . several other social distancing benchmarks. these benchmarks are defined specifically in the supplemental appendix. the results appear in figure 1 . we observe that our model forecasts the pattern of disease contagion well if the level of social distancing in early may remained at the level seen at the end of april (50% return to normalcy). finally, we forecast how the disease will evolve up to september 30, 2020 under different reopening strategies. the cumulative and daily cases appear in figures 2 and 3 , respectively. the cyclicality observed in figure 3 reflects the variation we observe in the weekly data, which may reflect different reporting delays or different social-distancing behavior due to the day-of-the-week effect. the forecast shows that social distancing matters, but that the impact of increased mobility becomes higher as we move closer to normalcy. if social distancing is eliminated, we observe that the largest effects will be felt in the first two months. this occurs because of the shrinkage of the uninfected population in each county. note that cases will be elevated to almost double the daily rate that we would observe under a 50%-return-to-normalcy even into the september of 2020, when cases are likely to reach an almost steady weekly level. our estimates suggest that it will be difficult to return to school and normalcy in the fall of 2020 without sparking a large outburst of covid-19. ultimately, moving from the 50%-return-to-normalcy to a full return to normalcy will lead to 4 million additional confirmed cases. if we assume that confirmed cases are only 10% of actual cases, and that covid-19 has an 0.75% infection-fatality rate (ifr), as we justify in the supplemental appendix, we would expect 300,000 to 600,000 deaths by the end of september 2020 if the social distancing occurs at 50% to 100% levels of normalcy, respectively. we note, however, based on our forecast that 44% of the counties in the sample (1, 196) could completely reopen and still experience a confirmed case rate lower than 0.1% from june to august, 2020. these counties are less populated and account for less than 15% of the population in our sample. we also note that our analyses do not consider the positive effects of alternative preventive protocols such as wearing facial masks and better hand washing. such protocols may help slow the contagion process. taken together, we demonstrate that the rate of spread of covid-19 in the united states is concave in the number of contagious individuals. this explains why the growth rate of covid-19 cases has been slower than expected given the initial exponential growth, above and beyond the effect from social distancing. we empirically identify the substantial impact of social-distancing on combating the pandemic. we also forecast how covid-19 will evolve in the future, and the timing over which different parts of the country will reach their peaks and how the patterns may affect our reopening strategies. 16may2020 01jun2020 01jul2020 01aug2020 01sep2020 30sep2020 date 16may2020 01jun2020 01jul2020 01aug2020 01sep2020 30sep2020 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . https://doi.org/10.1101/2020.05.26.20113993 doi: medrxiv preprint in this online appendix, we first present our data. we then discuss our model and assumptions. finally, we present our assumptions for the simulations. our data come from a multitude of sources. we lay out the sources for each of these in turn. data of positive cases are based on the covid-19 data published by the new york times (https://github.c om/nytimes/covid-19-data, accessed on may 17, 2020). the data contain the daily confirmed case counts in the u.s. at the county or county-equivalents level. we exclude cases in the states of new york, new jersey, and connecticut due to the large outbreak there and the complicated relationship between new york city (which is the seat of 5 counties) and the surrounding counties. we drop 3 of the remaining counties because we do not have social distancing data for 2 of them, and we cannot match the demographic data for a third (oglala lakota county, sd). this reduces the number of counties to 2,778. in addition, we remove 74 counties that had no confirmed cases in the entire sample period. the remaining 2,704 counties and county-equivalents constitute our main sample. these counties account for 89.6% of the total u.s. population and 55.2% of the total u.s. confirmed cases as of april 23, 2020. there are a few days where there are negative cases that are reported. these are generally corrections to previous over-reporting. thus, we clean the negative numbers of cases by subtracting the absolute value of the negative cases from the proceeding day. in the event that that leads to a negative number of the proceeding day, we iterate again. covid-19 also has an incubation period of approximately 5 days 1 , 2 . because of this lag between when a person gets sick and when they are diagnosed with covid-19, we assume that the cases reported on a particular date actually measure the covid-19 infections from 5 days earlier. we also assume that the true number of cases is approximately 10 times the number of diagnosed cases. we get this number by assuming that the infection fatality rate (ifr) is 0.75% 3 . we also assume that any deaths that occur happen 14 days after the confirmed test result. on may 16, 2020, the last day of our confirmed case data, there were 88,660 deaths in the us. on may 2, 2020, there were 1,138,961 officially diagnosed cases. we hence obtain the factor as (88,660/0.0075)/1,138,961 = 10.4. we round this number to 10. our estimates are not sensitive to the specific factor we use. we use social distancing data from the company safegraph, which collects cellphone gps data from u.s. residents, and has made them available for free to academics studying covid-19. these data are collected through a series of pings that the company receives for all users who have installed a number of smartphone apps. the list of apps that collect this information is kept as a trade secret. for each county, we use the fraction of cellphones that stayed near home for the whole day as our measure of social distancing. the safegraph data are published at the census block group level. to accommodate other data sources which are available at a less granular level, we aggregate the this . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . variable to the county level by taking the weighted median, using the number of cellphones in each census block group as the weight. we obtain the demographic data from the census bureau's 2014-2018 american community survey (acs), which contains information of each county's profile of population, ethnicity, age, median income, and commuting pattern. the acs, however, does not report population densities. safegraph, the company who provides us with the social distancing data, also maintains a dataset of the land area of each census block group in the us. we aggregate the land areas to the county level. together with the county population information from the census bureau, we are able to construct the population density data of each county. we gathered historical daily rain and temperature data from national oceanic and atmospheric administration (noaa) (source: https://www.ncei.noaa.gov/metadata/geoportal/rest/metadata/item/ gov.noaa.ncdc:c00861/html, accessed on may 21, 2020). the raw weather data is at the weather station level and we match weather stations to the counties they are in. we use the average values across weather stations within the same county to construct the weather variables for that county. for a small number of counties where there are no associated weather stations, we use the daily state averages as proxies. we obtained county-level voting data from https://public.opendatasoft.com/explore/dataset/ usa-2016-presidential-election-by-county/table/?disjunctive.state (accessed may 20, 2020). this data is explained at https://github.com/deleetdk/usa.county.data (accessed may 20, 2020), and the election vote totals originally came from the new york times. shelter-in-place orders (sip) data are compiled by keystone, a strategy and economics consulting firm. the company collects and distributes the sip data (https://www.keystonestrategy.com/coronavirus-covid19 -intervention-dataset-model/, accessed on may 21, 2020.) for free to researchers studying covid-19. our sample is an unbalanced panel because counties start to have positive number of confirmed cases on different dates. the earliest date we observe in the sample is jan 29, 2020, and the last day is april 23, 2020. note that we construct actual cases using reported cases 5 days later, and thus the corresponding sample period based on reported cases is feb 3, 2020 to april 28, 2020. summary statistics of all of the variables we use in the estimation are presented in table a1 . note that our case data proceed past the dates used for estimating the model and are up to may 16, 2020. we use those data for validating the model. those data are publicly available, but we are happy to supply summary statistics for this hold-out sample upon request. in this subsection, we detail the assumptions we make and the estimation procedure. as noted in the main paper, the model we estimate is a modified version of the standard susceptible-infected-recovered (sir) model: where y i,t is the number of individuals who are infected in county i on day t, r i,t is the rate at which infectious individuals in the county transmit the disease, s i,t is the percentage of the population that has not yet had covid-19 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . and is thus susceptible to it, and y i,t is the number of cumulative individuals who have been infected up until day t. this model differs from the standard sir in two key ways. first, the standard sir model constrains ω = 1. we discuss in the paper that there are theoretical reasons to believe that the correct model of transmission involves ω < 1. as an example, we present a network model below that demonstrates that ω < 1 is possible even using the conventional transmission mechanism. second, the standard sir model does not specify a discrete time frame over which the infected individuals are contagious, but rather builds a stock of infected individuals and assumes that these individuals exit their infected period at a fixed rate. we view our model as an approximation of this process, which greatly eases our estimation and allows us to easily add important variables to explain the contagion process in our analysis. we use a 6-day infectious window and an assumed latent period of 2 days (y i,t−2 −y i,t−8 ). this gives a mean serial interval of 4.5 days, which is close to several estimates 4 . in this appendix, we also present the results where we use a 14-day window (y i,t−2 −y i,t−16 ), and show that results are similar. we assume that the rate of transmission, r i,t varies by a set of factors, which we model as r i,t = exp x i,t β + ε i,t . x i,t includes county-level fixed effects, date fixed effects, the measure of social distancing, and daily average temperature. the county fixed effects account for differences in demographics across counties, such as the demographics shown in table 2 of the main paper as well as other unobservable county-specific factors. the date fixed effects account for both day-of-the-week differences in the patterns of travel for people (e.g., the time away from the house to go to work or to go to the park, which may lead to different exposures to the disease) as well as differences in the rate of testing and reporting that occur across time. we assume that the errors ε i,t are uncorrelated across counties. we further assume that ε i,t is uncorrelated across time, although we cluster the standard errors by county. we estimate the model by taking logarithm of both sides. after rearranging we get: note that sometimes y i.t , the diagnosed case number, is 0 for some counties on some dates. therefore, we adjust this formula slightly by adding 1 to y i,t so the logarithmic values are always well-defined: in some counties, y i,t−2 −y i,t−8 is 0 for some periods. we do not use those observations for estimation. note that because this is a lagged variable, this is a selection based on independent variables and not based on dependent variables, and hence it does not bias our estimation. one concern that can arise in estimating this model is that the amount of social distancing is likely to be correlated with the error terms, ε i,t in the regression. we address this concern using an instrumental variable (iv) approach, which requires that we find a variable that affects social distancing but is not correlated with ε i,t . we use the amount of rain (measured in mm) as a shifter of social distancing that does not directly cause covid-19 to spread. we run a first stage regression of social distancing to test whether this instrument has much power. the f-statistic for this test is 435.43, indicating that this is a strong instrument. the main estimation results are presented in table 1 of the paper and replicated in column 1 of table a2 in this appendix. research on covid-19 is nascent, and there are different views of how long infected individuals stay contagious. suppose that such individuals are contagious for 14 days instead of 6 days. then the model becomes: we present the estimation results of this model in column 2 of table a2 . note that this regression has more observations because there are fewer instances where we observe no cases in a county for a 14-day window than for a 6-day window. the results are largely unchanged. the coefficient on social distancing levels are slightly lower, but well within one standard error of the corresponding coefficient in column 1. the exponent on the infectious individuals is 0.435. that is somewhat smaller (but statistically different) than the 0.470 we observe with the shorter 6-day window, but overall the curvature shape is similar to what we have observed with the 6-day window. the effect . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . of temperature is slightly smaller but similar. as we will discussion below in section simulation, both specifications give similar long-run forecasting results. all county-level demographic factors remain constant over time in our analysis. while our main regression gives many insights, impacts of these demographic factors on the spread of the virus are captured by the county fixed effects. in order to better understand how these factors affect the contagion rate, we next regress the county fixed effects on several demographic variables. the coefficients from this regression should be thought of as the impact of these demographics on the rate of contagion. the results from the model are reported in column 1 of table a3 (replication of table 2 in the paper). in column 2 we present the results we would obtain if we instead modeled the contagious period to be 14 days. similar to table a2 , the results are again very similar under this alternative specification. the only statistically significant difference is that the coefficient on log(population density) is slightly smaller, although the effect is of a very similar magnitude. we also observe that the r 2 of the 14-day contagious period model is slightly lower than the r 2 of the 6-day contagious period model. while the focus of this paper is on measuring the contagion effect, we consider an exploratory regression to see what is correlated with the rate at which individuals choose to social distance. the results are shown in table a4 . the results indicate that the levels of social distancing are driven much more by the number of national cases than the number of local cases. the results also indicate that the fraction of voters who voted for trump in the 2016 presidential election explains much of the observed levels of social distancing. going from a solidly clinton-voting to a solidly trump-voting county (i.e., going from the minimum to the maximum of trump vote shares) represents a swing of almost 9 percentage-points of social distancing. to put the change into prospective, the social distance measures range from 11% to 65%, a 54 percentage-point variation. demographically, higher population densities lead to lower social distancing, but increased compliance with sheltering-in-place orders. more affluent counties have less social distancing. counties with a higher fraction of black population are less likely to social distance, perhaps due to work demands. a greater hispanic population and a greater public transportation population are both associated with more social distancing. supporting the idea that much of the traveling is for work, we see that people are less likely to practice social distancing on weekdays than on weekends. in terms of policy, the closing of schools is the policy that is most-indicative of how much social distancing will occur. we observe that closing public venues is correlated with reduced social distancing, and that limiting gatherings (we treat all gathering size limits of 500-people or less the same, but most of these limits are for far smaller groups than 500 people) has a statistically significant effect on the amount of social distancing. after estimating our model, we forecast the number of cases that would emerge under different social distancing regimes. for this exercise, we first divide our model's predicted numbers of "true" cases by 10, which gives us the prediction of diagnosed cases (as described in section positive cases). next, because the 2,704 counties in our sample are a subset of the whole nation, on a given date the predicted diagnosed case number is a fraction of the total cases in the us. while this fraction changes on a daily basis, we use an approximation by taking the median of the daily ratios between diagnosed cases observed in our sample and in the whole nation during our sample period. the median of the daily ratios is 0.63. accordingly, we divide our predicted diagnosed cases by 0.63 to obtain the national number of diagnosed cases. the first step of our simulation involves validating the model: we predict how many cases would emerge in the weeks after our data (dates: april 29 to may 16, 2020) in order to validate our model. the results are shown in figure 1 in the original paper. we observe that we are well able to predict the number of observed cases if the social distancing in early may represented a 50% return to normalcy, which is defined as stayed home in county i in february, and fractionstayathome i,t represents the fraction of devices staying home in county i on date t. we compute the different levels of social distancing accordingly: for example, a 25% towards normalcy represents social distancing at the level of 0.75×(minimum social distancing) + 0.25×(maximum social distancing). we find that the levels of social distancing at the end of april were approximately at the 50% return to normalcy levels. overall, our model predicts the national cases well. we next forecast the cumulative and daily cases of covid-19 through the end of september at different levels of social distancing. those forecasts appear in figures 2 and 3 in the original paper. in figure a1 below, we replicate figure 2 in the paper but further add the confidence intervals. to avoid cluttering, we only depict 50% and 100% return-to-normalcy levels in a1. as a robustness check regarding the 6-day contagion window specification, we also consider forecasting us daily cases under the specification where the contagion window is 14 days. figure a2 shows the evolutions of daily cases till september 30, 2020 under 50% and 100% return-to-normalcy regimes. we overlay the forecasts of both 14-day and 6-day specifications for easy comparison. from the figure, we may see the forecasts of 14-day and 6-day contagion window specifications are fairly close under the 50% return-to-normalcy level. for the 100% normalcy level, the two specifications differ at the beginning but converge quickly. in the long run, the two specifications give similar forecasts for daily cases. one unique feature of our model is that we estimate an exponent on the number of contagious cases. we include this flexibility because such a model fits the data much better, and also leads to forecasts that have more limited growth after an initial take-off of covid-19 cases, as is commonly observed. to demonstrate our model's better fit, we compare the prediction of our model against an alternative model with the exponent fixed at 1 as the standard sir model in figure a3 . at the 50%-return-to-normalcy level that is observed at the end of april, the standard sir model would predict much higher cumulative case numbers than the actual numbers. in contrast, our model's prediction fits much better. we next illustrate that the concave relationship we estimate for the number of contagious individuals on the number of new cases can come from social networks between people. we seek to demonstrate the theoretical feasibility of our results rather than the necessary or sufficient conditions under which the nonlinearity will arise. thus, we simulate a very simplified model of networks and disease process. to do this, we simulate a network with the following process: we take 10,000 individuals. we create a network by first randomly assigning that any two individuals will be joined with a common node with probability 0.001999 (corresponding to each person getting almost 20 friends on average). call these connection "round-1 friends." we then expand this network by assigning each node to have an edge with each of the round-1 friends of their friends with a probability of 0.6. we assume that the disease spreads with the following process. we seed 4 individuals to have the disease in period 0. then in each period we assume that any connected individual will get sick with probability 7/(number of connections), where the number of connections is specific to the individual, and varies due to the random assignment of the people who are connected (this probability is capped at 1 in case someone is randomly assigned fewer than 7 connections, which is very unlikely). after simulating this process, we then regress (ln (y t ) − ln (s t )) = c + ω ln (y t−1 ) + ε t . we run this simulation 10 times. the mean value forω = 0.51, with a range of (0.44, 0.62). this shows the plausibility of network effects leading to an estimate in the range that we have estimated in our main model. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 29, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 29, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 29, 2020. . https://doi.org/10.1101/2020.05.26.20113993 doi: medrxiv preprint 16may2020 01jun2020 01jul2020 01aug2020 01sep2020 30sep2020 date actual cum. cases 50% return-to-norm. 50% return-to-norm. cis 100% return-to-norm. 16may2020 01jun2020 01jul2020 01aug2020 01sep2020 30sep2020 date . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 29, 2020. . https://doi.org/10.1101/2020.05.26.20113993 doi: medrxiv preprint serial interval of novel coronavirus (covid-19 ) infections high temperature and high humidity reduce the transmission of covid-19 role of temperature and humidity in the modulation of the doubling time of covid-19 cases temperature significant change covid-19 transmission in 429 cities generalized least squares inference in panel and multilevel models with serial correlation and fixed effects cluster of coronavirus disease 2019 (covid-19) in the french alps the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. the new engl a systematic review and meta-analysis of published research data on covid-19 infection-fatality rates serial interval of novel coronavirus (covid-19 ) infections key: cord-280394-v2pwvst7 authors: ma, lin-lu; li, bui-hui; jin, ying-hui; deng, tong; ren, xue-qun; zeng, xian-tao title: developments, evolution, and implications of national diagnostic criteria for covid-19 in china date: 2020-05-15 journal: front med (lausanne) doi: 10.3389/fmed.2020.00242 sha: doc_id: 280394 cord_uid: v2pwvst7 recently who has characterized covid-19 as a pandemic. diagnosing the disease accurately and decreasing misdiagnoses and missed diagnoses is very important for management. therefore, we have analyzed the seven versions of china's national guidelines to examine how the diagnostic criteria roadmap has developed and evolved, in order to share our experience worldwide. in this article, we present the developments from the first to seventh versions, involving changes of case classification, changes to “suspected case,” changes in “confirmed case,” changes in clinical classifications, changes in “severe case,” and unchanged criteria. we have also discussed the reasons and implications for these changes and are looking forward to providing suggestions for worldwide understanding and management of this pandemic. a nucleic acid test is currently accepted as the gold standard method to confirm diagnosis. in addition, imaging examination and epidemiological history should also be considered as auxiliary diagnosis methods. since december 2019, a new disease caused by 2019 novel coronavirus (2019-ncov) has resulted in a worldwide outbreak (1) (2) (3) . the disease has been named coronavirus disease 2019 (covid-19) and the virus has been named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (4). it is vital, both for individuals and governments, to have accurate diagnostic methods for this disease and to minimize misdiagnosis and missed diagnoses in order to facilitate prevention and treatment. because covid-19 is a new disease, our awareness and knowledge are gradually increasing based on ongoing research findings and clinical practice experience; hence, the diagnostic criteria are also evolving. the outbreak has continued to increase worldwide and on 11 march 2020, who characterized covid-19 as a pandemic (5) . therefore, we aim to share our experience with the rest of the world based on an analysis of the evolving changes in the diagnostic criteria incorporated in the different versions of china's national guidelines for covid-19. we searched for all versions of the diagnosis and treatment guidelines for covid-19, which have been issued by the national health committee of the people's republic of china (http://www.nhc. gov.cn/) up to 5 march 2020. the first to seventh versions were included and the data necessary to estimate the changes in diagnosis of covid-19 were extracted. two authors independently reviewed the full text. discrepancies were resolved by discussion and consensus. besides extracting all the context of diagnostic criteria, the issued data, clinical classification, and definition of severe disease were also collected. all changes have been compared and presented in a separated table for suspected cases, confirmed cases, and severe cases. other changed and unchanged items have also been described. all analyses used microsoft excel and powerpoint 2019. the first version was issued on 16 january 2020, and the seventh version on 3 march 2020. table 1 presents the changes in these seven guidelines. the criteria for case classification developed considerably over time. in the first edition, three types were described: observed case, confirmed case, and critical case; however, from the second edition onwards, the term "observed case" has been changed to "suspected case, " and the criteria for "severe case" has been added. in the third edition, more clinical manifestations were added, and were classified as moderate, severe, and critical according to the severity of clinical symptoms. in the fifth edition, the form "mild" was added. hence, in the seventh edition, the types were suspected case or confirmed case, with four clinical levels of severity: mild, moderate, severe, and critical cases. in the fifth edition, the term "clinically diagnosed cases" was defined for hubei province. in hubei province, the suspected patients who had imaging features of pneumonia were designated as clinically diagnosed cases. however, this type was merged into "suspected case" in the sixth and seventh editions. the term "observed case" used in the first edition was changed to "suspected case" in the second edition. for a patient to be diagnosed as suspected this had to be based on the epidemiological history and clinical manifestations. from the second to fourth editions, it had to include any two epidemiological history features in addition to clinical manifestations. however, from the fifth to seventh editions, a comprehensive analysis was required which included epidemiological history along with clinical manifestations; cases without clear epidemiological histories were added as a criterion judged by the presence of clinical features. epidemiological history initially included direct or indirect contact with related markets in wuhan, which was mentioned in the first edition. however, this item was deleted from the second edition onwards, but "a history of contact with patients with fever or respiratory symptoms from wuhan city within the last 14 days before symptom onset, or with a cluster of confirmed cases" was added. then in the fourth edition, the contact scope was enlarged to include other places where covid-19 had spread and also included an epidemiological relationship with 2019-ncov infected cases. for clinical manifestation, "symptoms without obvious improvement or with progressive severity after 3 days of standardized antimicrobial therapy" was mentioned in the first edition. however, this was deleted from the second edition onwards, and it only included fever, imaging features of covid-19, and total white blood cell counts showing normal, decreased, or reduced lymphocyte count. in the fifth edition, the fever symptom was expanded and respiratory symptoms added; the criterion of "suspected case" was divided into inside hubei province and outside hubei province. the suspected patients having imaging features of pneumonia were designated as clinically diagnosed cases. in the first edition, to define the case as "confirmed, " it was necessary to collect the respiratory tract sample for viral whole genome sequencing, and this needed to show high homogeneity to the known novel coronaviruses. the real-time pcr test for nucleic acid in the respiratory tract or blood samples was added in the second and third editions. the pathogenic detection via blood samples was added in the fourth and fifth editions; the serological evidence was added in the seventh edition. in the seventh edition, if "the specific igm antibody and igg antibody of 2019-ncov are reported in serum as positive, " or "the 2019-ncov specific igg antibody in serum changes from negative to positive, or rises ≥ 4 times in the recovery phase above that in the acute phase, " this is also diagnosed as covid-19. there was no clear clinical classification in the first three editions. in the first edition, it specified a clear criterion for "critical case, " and the criterion for "severe case" was added in the second and third editions. the clinical stages were added in the fourth edition, which divided confirmed cases into moderate severe and critical stages; the mild stage was then added in the fifth to seventh editions. the mild stage was defined as persons having slight clinical symptoms without any imaging features of pneumonia. the criterion for severe case has evolved since the second edition. compared with this edition, "oxygen saturation during inhalation ≤ 95%" was modified to "oxygen saturation during inhalation ≤ 93%" in the third to seventh editions; additionally, the descriptions of rapid sequential organ failure assessment (qsofa) score, curb-65 score, and coalescent pneumothorax were also deleted. the "other combined clinical conditions that necessitate hospitalization" in the second and third editions was also deleted in the latter four editions. the description "pulmonary imaging shows leafy lesions or progressive lesions > 50% in 48 h" was deleted in the fourth and fifth editions but was reincorporated in the sixth and seventh editions. in the sixth edition, "the necessity to adjust the arterial partial pressure of oxygen (pao 2 )/fraction of inspired oxygen (fio 2 ) for patients from high attitude areas" was added. in the seventh edition, "severe cases" were defined and divided into children and adults. the criteria for critical case were almost unchanged in these seven editions, except that in the third to seventh editions, "respiratory failure" was expanded to "respiratory failure occurs and mechanical ventilation is required, " and "septic shock" was revised to "shows symptoms of shock." we believe there are two major reasons for these changes: our increasing knowledge and awareness of covid-19 and the feedback from clinical practice. because covid-19 is a new infectious disease, we are still learning its etiology, source of infection, transmission route, capacity for transmission, clinical manifestation, diagnostic criteria, treatment, and other relevant information. in the early stages, the clinical symptoms of covid-19 are similar to common flu or pneumonia, which only present as fever and/or a cough (3) . hence, only observation and empirical antimicrobial therapy were given for mild cases. when the 2019-ncov was extracted from patient samples, the antimicrobial therapy was then deleted from the treatment criteria of suspected cases. in response to the rapid increase in the number of cases which were submitted by wuhan city, many government departments reacted rapidly (6) by establishing a joint prevention and control mechanism, increasing the information about covid-19, raising public awareness of the outbreak, revising "observed case" to "suspected case, " and providing centralized isolation for suspected cases. then, with reports of increasing numbers of confirmed and suspected cases in many regions, especially when human-human transmission was confirmed, and many cases were presenting with no history of living in or contact with wuhan (7), the designation of suspected case was not limited to a person's epidemiological history, and isolated cases whose clinical manifestations met the criteria of suspected case were included. according to the whole genome sequencing result of 2019-ncov and compared with other novel coronavirus (8), etiological detection become the main piece of evidence and the core criterion for diagnosis of covid-19. relevant research also continued to search for an optimal nucleic acid detection kit for rapid diagnosis, and the rapid real-time pcr test was established (9) . when sampling included blood, as well as samples from the respiratory tract, this increased the availability of different specimens (10) . then serological evidence was included in the etiological evidence category based on relevant studies, and this supported bringing the specific antibody positive result into the confirmed criteria. the frontline doctors and nurses also accumulated more and more clinical experience of seeing and treating covid-19. hence, the clinical severity stages were designated according to patients' symptoms from the third edition of the guideline, to facilitate individualized treatment and surveillance of patients. we found some patients' rt-pcr test became positive again after recovery from covid-19 (11) . moreover, due to the nucleic acid testing being too slow to meet clinical requirements, this resulted in many people not being correctly diagnosed. hence, in the fifth edition of the guideline, suspected patients who had imaging features of pneumonia in hubei province were considered as clinically diagnosed cases, and then given standardized treatment. this "clinically diagnosed case" was canceled when the capacity of nucleic acid detection improved. we can see that the diagnostic criteria of "severe case" became more detailed and specific. oxygen saturation during inhalation was reduced from ≤ 95 to ≤ 93%, and qsofa score, curb-65 score, and description of coalescent pneumothorax were removed. the qsofa score and curb-65 score were usually used to assess the severity and prognosis of community-acquired pneumonia (12) , but many studies believed they do not have an ideal correlation with prognosis of covid-19, the reason being covid-19 is more dangerous (13) . hence, they were deleted from the diagnostic criteria for severe cases. when confirmed cases came from the high-altitude areas having a low concentration of oxygen, the necessity to adjust pao 2 /fio 2 values for cases from these areas was added in the sixth edition. also, along with defining suspected and confirmed cases and the accumulation of clinical experiences in children, the "severe cases" definition for children was added in the seventh edition. some diagnostic criteria were significantly changed over the seven versions of the guidelines. there were many descriptions added relating to epidemiological histories of suspected cases, this indicates that relevant epidemiological studies should be performed as quickly as possible after an outbreak in order to detect the routes of infection and provide evidence for diagnosis and control. for the diagnostic criteria of covid-19, from nucleic acid tests to clinical imaging features, from etiological evidence to serological evidence, diagnostic means and methods continue to increase (14) . however, we must acknowledge that every diagnostic method has its own strengths and weaknesses. for example, a nucleic acid test may produce false-negative results and has a longer detection time, while imaging tests have a short detection time but accurate results are dependent on the radiologists' skill level. besides, some diagnostic criteria remain little changed, such as the criteria for severe case which is almost unchanged. this is because when covid-19 patients enter the critical phase it is often accompanied by organ failure and shock; these changes develop rapidly and have a high fatality rate. hence, the standard has been maintained for better clinical monitoring and judgment. when the case definition was gradually broadened as knowledge increased, it had a substantial influence on the proportion of infections being detected as cases. when cases are classified in detail, different types of patients are treated with customized interventions, and medical resources can be properly allocated. a study has found through modeling evaluation that from the first version to the second version, the proportion of infections being detected as cases increased by 7.1 times (95% confidence interval [ci], 4.8-10.9), from the second version to the fourth version it increased by 2.8 times (95% ci, 1.9-4.2), and from the fourth version to the fifth version by 4.2 times (95% ci, 2.6-7.3) (15) . after adding the category of "clinically diagnosed cases" in the fifth edition, 13,332 new clinically diagnosed cases were added (16) . many highly suspected patients who did not receive virological testing due to insufficient detection capabilities were able to be isolated in designated hospitals in a timely manner, with priority given to virus detection and treatment, so as to reduce the potential infection rate and mortality. after all clinically suspected cases were able to be tested by the laboratory, the "clinically diagnosed cases" were deleted in the sixth version. although the use of radiological evidence to confirm viral pneumonia may be an important alternative to the diagnosis and monitoring of covid-19, it also brought some problems. this procedure may include some patients with common pneumonia; hence criteria for clinically diagnosed patients also needs to include the nucleic acid results at a later stage to correct the actual number of cases. in addition, with the surge of patients, it was necessary to have enough hospital beds quickly to ensure the early diagnosis and treatment of patients. this requires more funds, equipment, and medical staff input, which poses a challenge to the country's regulatory and economic capabilities (17) . the nucleic acid test is currently used as a confirmed diagnosis method. in addition, imaging examination and epidemiological history should also be considered as auxiliary diagnosis methods. we suggest approaching covid-19 diagnosis with caution, doing as much as we can to reduce misdiagnosis and missed diagnoses, exploring and combining different methods, and actively seeking new methods, especially for screening asymptomatic patients and also identifying people who retest as positive again after recovery. all datasets generated for this study are included in the article/supplementary material. x-tz and x-qr: conceptualization. b-hl, td, and l-lm: writing-original draft preparation. x-tz, y-hj, and x-qr: reviewing and editing. all authors have read and agreed to the published version of the manuscript. this study was supported, in part, by the national key research and development program of china (2020yfc0845500) and the special project for emergency science and technology of hubei province (2020fca008). we express our gratitude to jean glover from tianjin golden framework consulting company for english editing. we also express our gratitude to the experts of all versions of the diagnosis and treatment guidelines for covid-19 by the national health committee of the people's republic of china. a novel coronavirus outbreak of global health concern early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) genetic diversity and evolution of sars-cov-2 world health organization. who characterizes covid-19 as a pandemic -11 transmission of 2019-ncov infection from an asymptomatic contact in germany a new coronavirus associated with human respiratory disease in china detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr detection of sars-cov-2 in different types of clinical specimens positive rt-pcr test results in patients recovered from covid-19 qsofa is a poor predictor of short-term mortality in all patients: a systematic review of 410,000 patients improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modeling study national health commission of the people's republic of china awareness, attitudes, and actions related to covid-19 among adults with chronic conditions at the onset of the u.s. outbreak: a crosssectional survey key: cord-288389-z0sz1msj authors: fanoy, ewout b; van der sande, marianne ab; kraaij-dirkzwager, marleen; dirksen, kees; jonges, marcel; van der hoek, wim; koopmans, marion pg; van der werf, douwe; sonder, gerard; van der weijden, charlie; van der heuvel, jet; gelinck, luc; bouwhuis, jolande w; van gageldonk-lafeber, arianne b title: travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia, may 2014 date: 2014-10-17 journal: emerg themes epidemiol doi: 10.1186/1742-7622-11-16 sha: doc_id: 288389 cord_uid: z0sz1msj background: in may 2014, middle east respiratory syndrome coronavirus (mers-cov) infection, with closely related viral genomes, was diagnosed in two dutch residents, returning from a pilgrimage to medina and mecca, kingdom of saudi arabia (ksa). these patients travelled with a group of 29 other dutch travellers. we conducted an epidemiological assessment of the travel group to identify likely source(s) of infection and presence of potential risk factors. methods: all travellers, including the two cases, completed a questionnaire focussing on potential human, animal and food exposures to mers-cov. the questionnaire was modified from the who mers-cov questionnaire, taking into account the specific route and activities of the travel group. results: twelve non-cases drank unpasteurized camel milk and had contact with camels. most travellers, including one of the two patients (case 1), visited local markets, where six of them consumed fruits. two travellers, including case 1, were exposed to coughing patients when visiting a hospital in medina. four travellers, including case 1, visited two hospitals in mecca. all travellers had been in contact with case 1 while he was sick, with initially non-respiratory complaints. the cases were found to be older than the other travellers and both had co-morbidities. conclusions: this epidemiological study revealed the complexity of mers-cov outbreak investigations with multiple potential exposures to mers-cov reported such as healthcare visits, camel exposure, and exposure to untreated food products. exposure to mers-cov during a hospital visit is considered a likely source of infection for case 1 but not for case 2. for case 2, the most likely source could not be determined. exposure to mers-cov via direct contact with animals or dairy products seems unlikely for the two dutch cases. furthermore, exposure to a common but still unidentified source cannot be ruled out. more comprehensive research into sources of infection in the arabian peninsula is needed to strengthen and specify the prevention of mers-cov infections. in 2012, the middle east respiratory syndrome coronavirus (mers-cov) was isolated for the first time from the sputum of a 60-year-old man who presented with an acute pneumonia in kingdom of saudi arabia (ksa) [1] . as of june 24 2014, 707 laboratory-confirmed cases of mers-cov infection have been reported to the who including 252 (36%) fatal cases [2] . following the first upsurge of cases in spring 2013, a second wave occurred in 2014, mainly in ksa [3] . most cases so far either resided in or travelled to the arabian peninsula and neighbouring countries or were close contacts of these cases. furthermore, travel related cases have been reported from the united states of america (usa), the united kingdom (uk), germany, france, tunisia, italy, greece, algeria, egypt, the philippines and malaysia [3] [4] [5] [6] [7] . so far, mainly sporadic cases are reported as well as a few hospital outbreaks, which is in line with a low reproduction rate (r0) of mers-cov, estimated to range from 0.4 to 1.5 [8] [9] [10] . the median incubation period is estimated to be 5.2 days (95% ci 1.9-14.7 days) [8] . for most non healthcare associated cases no clear source of infection could be identified, although camels are considered a reservoir as the virus and antibodies against mers-cov have been identified in camels (camelus dromedarius) and in their milk [9, 10] . the exact modes of transmission of the virus to humans remain unclear [11] [12] [13] [14] , leaving the possibility for other yet unidentified sources of infection. to stop or reduce transmission of mers-cov and prevent new human infections, it is important to identify all potential sources of infections as well as risk factors and route(s) of transmission. in may 2014, mers-cov infection was diagnosed in two dutch residents. these cases travelled with a group of 29 other people, returning to the netherlands from pilgrimage to medina and mecca, ksa [15] . we conducted a comprehensive epidemiological assessment of the travel group aiming to identify the likely source(s) of infection and presence of potential risk factors for these two cases. we compiled a questionnaire based on sample questionnaires originating from two who-protocols aimed to asses risk factors and investigate contacts of patients with mers-cov infection [16, 17] . we adapted the questionnaire to the specific route and activities of the dutch travellers. our questionnaire covered human, animal and food exposures to mers-cov during the whole trip to ksa. the questionnaires consisted of 28 open and closed questions, and it took approximately 20 minutes to complete the face-to-face interview with the travellers (additional file 1). nurses of the public health services administered the questionnaires approximately 3 weeks after return to the netherlands. for the two cases the questionnaire was completed with data on the travel route and stay in ksa collected by the national coordination centre for communicable disease control. the patients were a 70-year-old man (case 1) and his 73-year-old sister (case 2), both having underlying cardiovascular co-morbidities and diabetes mellitus. the remaining travel group included 29 persons (41% male) aged between 10 and 70 years (median age: 55 years). seventeen (59%) of the non-cases did not report any comorbidities (table 1 ). upon identification of case 1, throat swabs of all contacts were tested by pcr to assess other mers-cov cases. apart from case 2, who had reported mild respiratory complaints starting on the 5 th of may (t = day 9), all other 29 travellers tested negative and did not develop symptoms. the group made a pilgrimage to medina and mecca, ksa ( figure 1 ). they arrived on the 26th of april 2014 (day 0) in medina, left by private bus to mecca on the 4 th of may (day 8) and returned to amsterdam on the 10 th of may (day 14). case 1 started to feel feverish and had diarrhoea on the 1 st of may (day 5). respiratory complaints started on the 10 th of may (day 14). mers-cov was identified in case 1 on the 13 th of may (day 17) and in case 2 on the 14 th of may (day 18). detailed case reports of the two patients are described by kraaij et al. [15] . during their pilgrimage, the whole group stayed in the same hotels and had breakfasts together. there was no fixed collective travel programme, but they had some joint visits to several mosques. the travellers also spent time alone or in smaller subgroups, visiting different mosques, markets and restaurants. the exposures for both the cases and 29 asymptomatic travellers during their visit to saudi arabia are summarized in table 2 . on the 3 rd of may (day 7), 12 travellers (excluding the two cases) made a trip to the touristic valley wadi-e-jinn, and stopped when they came across a herd of camels laying behind an improvised razor fence. four travellers reported direct contact with the camels. eight travellers mentioned indirect contact, for example via the fences surrounding the animals. eleven travellers consumed unpasteurized camel milk, offered to them by the caretakers of the camels. besides contact with camels, two travellers reported that they spotted many pigeons in the cities and fed them. no other direct contact with animals was mentioned. page 2 of 6 http://www.ete-online.com/content/11/1/16 as mentioned above, eleven travellers, excluding the two cases, consumed unpasteurized camel milk. furthermore, 22 travellers, including case 1, visited one or more local markets in medina. seven of them, excluding case 1, bought and consumed a range of fruits such as prunes and dates. furthermore, 14 travellers bought souvenirs, such as dates, perfume and clothing on these markets. case 1 and his son visited two hospitals in medina on the 29 th of april (day 3) because of eye complaints of the son. as mentioned before, they spent 45 minutes in a crowded waiting room of a general hospital where they were exposed to coughing patients. subsequently, the son was referred to a specialized eye clinic where they spent no time in a waiting room and did not notice any coughing patients. on the 5 th of may (day 9), case 1, accompanied by his son, visited an emergency department of a general hospital in mecca because of acute malaise, weakness, nausea and diarrhoea (without respiratory complaints). these symptoms started on may 1 (day 5). case 1 and his son spend 30-45 minutes in the waiting room, though they had no specific exposure to coughing people. on the 7 th of may (day 11) case 1 returned to another hospital in mecca, accompanied by his son, where he only briefly stayed in the waiting room, received antibiotic treatment and was observed for approximately three hours. four other travellers, including his son, accompanied him. two of them did not notice any coughing persons, while the other two did not answer this question. case 2 did not visit a hospital during the pilgrimage or prior to her diagnosis in the netherlands. two travellers (case 1 and his son) waited 45 minutes in a crowded waiting room of a general hospital in medina (29th of april, day 3) where they were exposed to coughing patients. one other traveller, who did not enter a hospital, reported coughing persons outdoors. the duration and intensity of this contact was not clarified in more detail. case 1 and 2 had been in close contact with each other during the entire trip, as they shared hotel rooms. all 29 travellers had been in contact with both cases during the trip. seventeen travellers had minimal social contact with the two cases, for example only brief eye contact in the hotel lobby or in the bus. twelve travellers reported daily contact, such as visits to the hotel room of case 1 while he was sick, or accompanying him while shopping. the son of case 1 had the most intensive contact sharing a hotel room with both cases. nevertheless, his throat swabs tested negative to mers-cov by pcr during the follow-up after the identification of case 1. we found no indications that the two cases were infected via contact with specific animals or dairy products. infection during a hospital visit could be a source for case 1. sequence analysis of parts of the viral genome collected from both patients had shown that the strains were nearly identical, suggesting that the cases have infected each other, or that they were exposed to a common source. in the latter case, exposure during a hospital visit is unlikely because case 2 did not visit a hospital before onset of disease. the possibility of asymptomatic infections and transmission among the accompanying travellers cannot be ruled out, but seems unlikely in view of the negative pcr results. subsequently, although considered unlikely, we cannot exclude the possibility of asymptomatic travellers being a source of infection for case 1 and/or 2. no other common sources were identified although we cannot exclude the possibility of lack of accurate recall or indirect exposure to animal excreta. it might be possible that one or both cases were infected during non-specific contacts with the local population or the environment. if initial zoonotic introductions of mers-cov from camels have been followed by low level presence among the (asymptomatic) population or in the environment at large, this might result in an ongoing community-based outbreak, which, unlike sars, cannot be controlled by rigorous hospital hygiene alone. both patients were of older age and had comorbidities. in general, older age and comorbidity are considered risk factors for symptomatic mers-cov infections, although this was not the case in all outbreaks [18, 19] . in this study, the distribution of symptoms towards higher age and comorbidity fits this assumption. ongoing serological analysis upon travellers and people exposed to the case in the netherlands may reveal to what extent asymptomatic infection has occurred. the remainder of the travellers did not develop symptomatic mers-cov and did not have positive pcr-test results despite exposure to camels, dairy products and the two cases. this could point to a low transmissibility of the virus, which is also illustrated by the limited number of secondary infections among contacts of import cases described elsewhere, with the reservation that asymptomatic infections among the remaining travellers cannot be excluded fully as final serologic results are not available [5, 6] . currently, there is only limited epidemiological information published considering potential sources and transmission dynamics of mers-cov infection within the arabian peninsula. descriptive studies among a well-defined group of travellers can therefore provide relevant epidemiologic information, even when the number of travellers is too small to draw definite conclusions. more in depth epidemiological investigation of comparable travel groups will add to the body of evidence, although the diversity of activities during a relatively short trip makes accurate recall of potential exposure challenging. alternatively, prospective cohort studies among travellers to affected areas, whereby travellers are asked to keep a daily diary, are likely to be more informative and complete. due to the uncertainty about the source, the detailed questions of the who-questionnaire were a very useful guide to decide on the most relevant exposures to be explored, adapted to the specific route of travel. the exact source of infection remains difficult to identify. case 1 could have been infected during his visit to a page 4 of 6 http://www.ete-online.com/content/11/1/16 hospital and subsequently have infected case 2. however, exposure to a common source for both case 1 and 2 cannot be ruled out. besides, case 1 may have been exposed to a yet unidentified source and subsequently have infected case 2, or vice versa. more research to relevant sources in the arabian peninsula is needed. the suggested role of underlying disease to develop mers-cov infection is in line with the age and comorbidity of the two dutch cases and the absence of symptomatic mers-cov infections among the younger and healthier travellers. additional file 1: the questionnaire. isolation of a novel coronavirus from a man with pneumonia in saudi arabia organization wh: coronavirus infections global alert and repsonse (gar) first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a case of imported middle east respiratory syndrome coronavirus infection and public health response clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands world health organization: seroepidemiological investigation of contacts of middle east respiratory syndrome coronavirus (mers-cov) patients world health organization: case-control study to assess potential risk factors related to human illness caused by middle east respiratory syndrome coronavirus (mers-cov) al raiy b: clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description submit your next manuscript to biomed central and take full advantage of: â�¢ convenient online submission â�¢ thorough peer review â�¢ no space constraints or color figure charges â�¢ immediate publication on acceptance â�¢ inclusion in pubmed, cas, scopus and google scholar â�¢ research which is freely available for redistribution 11 medical centre haaglanden, the hague, the netherlands. 12 isalaklinieken, zwolle, the netherlands. 13 national institute for public health and the environment, epidemiology and surveillance unit, antonie van leeuwenhoeklaan 9, bilthoven, ma 3721, the netherlands. the authors declare that they have no competing interests. all readers have read and approved the manuscript.authors' contributions ef compiled the questionnaires, analysed the data and drafted the manuscript. ms is in charge of the epidemiology department; she designed and coordinated the study and reviewed the manuscript. mk assessed travel data and reviewed the manuscript. kd coordinated interviews with travellers and reviewed the manuscript. mj was responsible for laboratory testing and reviewed the manuscript. wh is in charge of respiratory epidemiology department and reviewed the manuscript. mpgk is involved in laboratory testing and reviewed the manuscript. dw coordinated interviews with travellers and reviewed the manuscript. gs coordinated interviews with travellers and reviewed the manuscript. cw coordinated interviews with travellers and reviewed the manuscript. jh coordinated interviews with travellers and reviewed the manuscript. lg was involved in clinical care and interviewed a case. jb was involved in clinical care and interviewed a case. ag was leading the study, including the study design and analysis and she reviewed the manuscript. all authors read and approved the final manuscript. key: cord-122594-0y34yxlb authors: teles, pedro title: predicting the evolution of sars-covid-2 in portugal using an adapted sir model previously used in south korea for the mers outbreak date: 2020-03-23 journal: nan doi: nan sha: doc_id: 122594 cord_uid: 0y34yxlb the new coronavirus covid-19 has spread very quickly worldwide, leading the world health organization (who) to declare a state of pandemic. one of the questions many policy makers, and governments are asking themselves is how the spread is going to evolve in time. in this study, i applied an adapted sir model previously used in south korea to model the mers outbreak, which is also caused by a coronavirus, to estimate the evolution of the curve of active cases in the case of the portuguese situation, using italian dara. i then construct five different scenarios for the evolution of covid-19 in portugal. in the out of control scenario, the number of active cases could reach as much as ~40,000 people on the 5th of april (out-of-control scenario). if the self-protective and control are taken in the same level as what was considered for the south korean model, this number could have be reduced to about 800 cases (scenario 1). considering that this scenario is now unrealistic, three other scenarios were devised. in all these scenarios, the government measures had a 50% effectiveness when compared to the measures in korea. but in scenario 2 the transmission rate $beta$ was effectively reduced to 50%, in this scenario active cases could reach circa 7,000 people. in scenario 3, the transmission rate $beta$ was reduced to 70% of its initial value, in which the number of cases would reach a peak of ~11,000 people. and finally in scenario 4, $beta$ was reduced to 80%. in this scenario, the peak would be reached at about ~13,000 cases. this study shows the importance of control and self-protecting measure to bring down the number of affected people by following the recommendations of the who and health authorities. with the appropriate measures, this number can be brought down to ~7,000-13,000 people. hopefully that will be the case not just in portugal, but in the rest of the world. there is already abundant information on the new coronavirus and its spread worldwide [1, 2, 3] , as well as the risks it poses to the population. the person infected with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [4] may develop the disease called covid-19, which manifests with mild to severe symptoms of fever, cough, shortness of breath and other signs [5] . in the most severe cases, the infection can lead to the development of acute respiratory distress syndrome (ards) causing respiratory failure, septic shock, or multi-organ failure, and even death [6] . there is yet a lot about this new coronavirus that is still unknown. studies suggest that the case fatality rate of the virus is of about 3.5% in mainland china, but the authors suggest considering the range 0.2-3.0% [7] . however, this value seems to be much higher in italy [8] , suggesting its strong dependence on demographics. in fact, the chinese center for disease control, released an analysis that showed that the case fatality rate ranged from 0% in children aged 0-9 years old to 14.8% in those over 80 [9] , which implies that countries with a high number of elderly citizens, as is the case of italy and portugal, are more prone to having higher death rates. the quick spread of the virus in europe has led the who to declare europe as the new epicentre of the disease on the 13th of march of 2020 [10] . the rapid growth of the number of active cases presenting severe symptoms has saturated the health services in most countries in the continent, especially in italy [11] . it is yet unknown when the epidemic will reach an end, but governments throughout europe have implemented harsh measures to prevent and mitigate the spread of the virus. yet, as of today there are as many as 77,394 confirmed cases on the european continent, in 46 territories, resulting in 3,457 deaths, and rising [2, 3] . in portugal, the two first confirmed cases in the portuguese territory were announced on the 2nd of march, which puts the country slightly behind the curve in the outbreak in the european continent. as of today, there are 642 confirmed cases, 2 deaths, and 3 recovered [12] . also, the country is on lockdown since the 13th of march 2020, and the portuguese parliament declared a state of emergency on the 18th of march 2020. one of the questions many policy makers, and governments are asking themselves is how the spread is going to evolve in time. a timely idea of the amount of cases that will exist in a near future can allow governments and policy makers to act accordingly. in this study, i adapted a model of the sir type used in south-korea to model the evolution of the active cases of the mers outbreak in the country back in 2015 [13] . although there are other models being used [14, 15] , this model seemed most appropriate given that the mers and the sars-cov-2 are related illnesses, both provoked by a coronavirus strand, and with similar symptoms, although mers was much deadlier. in order to fit the parameters and given that there is still very little data in the case of portugal, i used the data of italy instead. this allowed me to fit the curve of current active cases in portugal with a model, which i then use, by implementing the control measure parameters predicted in the model to predict the future number of cases, in five different scenarios (the out-of-control scenario, a scenario in which measures were the same as in the original model (scenario 1), a scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 50% (scenario 2), a similar scenario but in this case the transmission rate was only reduced to 70% (scenario 3), and a fourth scenario, similar to the two previous ones but in which the transmission rate was reduced to 80%. the model used is the model described in xia et al [13] , which can be understood by the flow diagram shown in figure 1 (which was taken from the paper, page 4). in this model, s corresponds to the number of susceptible individuals; e, the number of exposed individuals; a, the number of asymptomatic infected cases; i, the total number of mild-to-severe infected patients. h, the number of hospitalized cases; r, the number of removed cases, and finally n, the total population of portugal. β 1 is the transmission coefficient of the asymptomatic infected cases, β 2 is the transmission coefficient of the symptomatic infected cases (mild infected person and severe patients) to the susceptible, β 3 is the transmission coefficient of the hospitalized cases to the susceptible, σ −1 is the mean incubation period, λ −1 is the mean time between symptom onset to hospitalization, k −1 1 is the mean infectious period of asymptomatic infected person for survivors, k −1 2 is the mean duration for hospitalized cases for survivors, δ −1 is the mean time from hospitalization to death, γ is the clinical outbreak rate in all the infected cases. the time unit is 1 day. the set of differential equations can then be written as: given that the available data for covid-19 is still very preliminary, the model was further adapted. the only parameters that were considered "known" were the mean incubation time, which was taken as σ −1 =5.1 days from the literature (here using the median as equal to the mean) [16] , and the mean infectious period of asymptomatic people, where a conservative estimate of k −1 1 = 14 days was used. all other parameters were taken to be "unknown". furthermore, and because the fitting of the model was difficult to achieve, i took β 1 = β 2 = β 3 = β as a first approximation, meaning that the mean transmission coefficient is the same independently of type and setting of the transmission. finally, unlike xia et al [13] , i consider that the epidemic starts with only one person infected (i0=1), being the rest of the values 0, except for s 0 which is simply n-1. see table 1 for a breakdown of the different parameters of this model. the solving of the system of differential equations was performed using the mathematica code [17] , which also allows for the use of fittable parameters, using the function "nonlinearmodelfit" [18] . given that there is still very little data for portugal, the data for italy was used as proxy. this seems like the most reasonable approach (instead of using data from china or another asian country), given the cultural and health system proximity between the two countries. in order to determine the parameters λ and δ, i used the number of deaths, as reported by the italian ministry of health and available online [19] . for the estimation of the value of k 2 , the number of recovered was used. all data was taken until the 19th of march 2020. after determining the values for these parameters, they were used to fit the curve of infected cases for portugal, where the values of β 1 = β 2 = β 3 = β and γ were fitted to obtain a model. this model was then used to estimate the evolution of future cases in portugal, according to five different scenarios: • out-of-control scenario (nothing done, the virus is free to spread (out-of-control scenario); • scenario in which the government takes severe mitigating measures and the population adheres to selfprotecting measures equivalent to the original model (scenario 1) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 50% (scenario 2) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 70% (scenario 3) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 80% (scenario 4) 3.1 fitting of parameters λ and δ (italy). the number of deaths in italy was taken from [19] , and the set of differential equations described in eq. 1 fitted to obtain the best possible fit for parameters λ and δ. an 3.2 fitting of parameter k 2 (italy). now, using the values for λ and δ found in 3.1, parameter k 2 was obtained with a new fit of eq. 1, but now to the number of recovered cases in italy, which again was taken from [19] . the best possible fit for k 2 gave an r 2 value of 0.86178. the rest of the parameters are presented in table 3. as can be seen, in the case of recovered cases, the fit has not the same quality as the fit in 3.1. this is further evidenced by figure 3 , where a graphical representation of the fit is shown. after the two previous procedures, we now have all the parameters we need to fit eq. 1 to the number of portuguese active cases until the 19th of march, in order to determine the value of β and γ. the number of portuguese active cases was taken from [20], which is updated daily. the values previously obtained of parameters λ, δ, and k 2 were used. as explained previously, because the fitting was difficult to obtain, as a first approximation, we consideredβ 1 = β 2 = β 3 = β. the obtained values for β and γ are given in table 5. a graphical depiction of the fitted model is given in figure 4 . in this scenario, the out-of-control scenario, in which the corona virus would be "free" to spread within the country would lead to a peak of 40,000 infected around the 5th of april 2020. a scenario that will very likely be avoided given the strong containment measures implemented by the government. 3.4 discussion of the obtained values for λ, δ, k 2 , β, and γ the fitting of these parameters was difficult to obtain. there is enough systematic error and lack of statistics to make the present results very unreliable. in order to obtain the parameters for λ, δ, and k 2 , an ansatz value for γ had to be given. in this model, γ is originally defined as "the clinical outbreak rate in all the infected cases". in the cases of the fits of λ, δ, and k 2 , the ersatz value used for γ had a very strong impact in the results of the other parameters. in fact, as it comes multiplied with σ in the transfer from compartment e(t) to compartments a(t) and s(t), i think the high variation in this value could very well be due to the huge uncertainty there is at the moment for the appropriate [21] , although this number is probably an outlier. also, a case of an incubation period of 18 days seems to have been confirmed by a recent study [22] . the other parameters are somewhat less elusive. the obtained value for δ = 0.0168 is 50% the value obtained by xia et al [13] of 0.0348, s 24% the value obtained by xia et al [13] of 0.0660, which shows the difference in case fatality rates between the sars-covid-2 (7.2% currently for italy), and mers (estimated at 34%) which is 20%. this seems reasonable, although the case fatality rate of the sars-covid-2 is still difficult to ascertain. finally, from the obtained values it is also possible to determine a value for the basic reproduction number r 0 , which here i take as being simply the value r 0 = 2 β k1+k2 = 14.5 which demonstrates how strong the capacity for spreading of this particular virus is. in table 5, the final values of the obtained parameters are given. the paper by xia et al [13] further presents an extra 7 parameters to take into account control measures in time evolution of the epidemic. these parameters are meant to take into account two things: • isolation and monitoring measures taken by the government (parameters d 1 , d 2 , d 3 , and d 4 ); • self-protection measures taken by the population (parameters l 1 , l 2 , and l 3 ). the way these parameters are introduced in the model are as follows: the l 1 , l 2 , and l 3 parameters are basically multiplication factors for the transmission parameter β, so in our model we only consider one value for it as we did in the previous sections. the d 1 , d 2 , d 3 , and d 4 parameters are summed to the parameters of spread between the infected, asymptomatic and hospitalized cases, further stressing their connection to implemented government measures. the values provided by xia et al [13] , when applied to the portuguese case, seem to provide a very unrealistic scenario, in which the values would immediately start to diminish. perhaps this made sense in the case of the mers epidemic, given that it was much more localised that the one provoked by this virus. as such, and for the lack of a better model, i devised four different scenarios in addition to the "out-of-control" scenario described in the previous section: • scenario in which the government takes severe mitigating measures and the population adheres to selfprotecting measures equivalent to the original model (scenario 1) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 50% (scenario 2) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 70% (scenario 3) • scenario where government measures are 50% as effective as those in south korea, and self-protective measures reduce the transmission rate by 80% (scenario 4) [13] . in the second scenario i keep the 50% reduction in the transmission rate but diminish the efforts of the government by half. in the third and fourth scenarios i use 70% and 80% of the transmission rate respectively. the values for these scenarios are given in table 6. the day considered for the initiation of these measures is day 33 corresponding to the 18th of march 2020 which is 5 days after the government implemented the measures, so giving the virus another cycle of infection before the measures take effect. results are shown in figure 5 . results show that measures can be effective in "flattening the curve", as it has come to be called this effect in recent times. in the first scenario the effectiveness of the control measures seems to be at a maximum, and a maximum of just about 800 cases would be reached. this has proven to be an unrealistic scenarion given that as of today there are 1600 active cases in the country. in scenario 2, the maximum is shifted to the 20th of april, reaching a maximum number of 7,000 active cases in the country. in this scenario, the transmission rate has been cut to a half, but the government's measures effectiveness has been cut to a half of the value from the original model used in korea. in scenarios 3 and 4, the measures of the government's effectiveness are kept at the same value, but the transmission rate is only cut to 70% and 80%, respectively. in scenario 3 the number of infected woulf reach a peak of about 11,000 people, on the 12th of april, and finally in scenario 4, the peak would be reached at 13,000 cases, on the 9th of april. in the out-of-control scenario, the peak would be reached at about 40,000 people on the 5th of april. given the huge uncertainties concerning these numbers, and the current lack of knowledge of the ongoing epidemic, it is very hard to make sense of its evolution, even using well-established sir methods. the current work makes a series of assumptions which may or may not be proven correct in the future, including the incubation period of the virus, which is still not well-known. this proved to be a major source of uncertainty to fit the results, because it had a great influence in the other parameters, especially parameter γ, which seems to describe a probability of the person being exposed to the virus being symptomatic or not. instead this parameter seems to behave in this study as an indicator of the huge uncertainty of the value for the incubation period. finally, epidemiologists and pathologists will tell you that these parameters, unlike what is considered in this study, not only are not constant, but dependent on time, location, culture, and other factors. it is therefore particularly hard to model the current epidemic of covid-19 at this stage. also, this study takes into account the particular situation for one country (portugal), and we know that in globalised times, the likelihood that new foci of infections arise on a multi-country scale is also very high. although currently most government have implemented serious travel restrictions, it is now well-known exactly how this parameter could affect this model, which does not take that into account. in fact, one of the major issues with the sars-covid-2 virus is that, unlike the other sars, it seems to be much less contained locally, as is evident by the amount of countries afflicted. the amount of actual active cases is another clear source of uncertainty. this model takes the values of active cases and compares them with official confirmed numbers, which not only may be smaller than the actual cases, as are released at an administratively defined time, which probably does not correspond to something realistic. the author believes that the values obtained in this study should be taken qualitatively rather than quantitatively. one can think of the scenarios as defining two limits of a likelihood interval, and as such, the actual figure of maximum number of active cases in portugal will likely be in the interval 7,000-13,000 between the 9th-20th of april, if the government and the people of portugal manage to keep implementing self-protective and control measures. this can perhaps serve to advise the competent authorities in taking the appropriate measures. it also serves to show the importance of control and self-protecting measures. it is possible to bring down the number of affected people by following the recommendations of the who and health authorities. without them, this model predicts that the figures of active cases would reach a staggering 40,000 people. it also shows that with the appropriate measures this can be indeed brought down, thus flattening the curve. hopefully that will be the case not just in portugal, but in the rest of the world. novel coronavirus (2019-ncov) situation reports -world health organization coronavirus disease (covid-19) -research and statistics the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study covid-19: what is next for public health emerg infect dis why deaths from coronavirus are so high in italy, rachael rettner, livescience on march 10 novel coronavirus pneumonia emergency response epidemiology teamexternal icon who director-general's opening remarks at the media briefing on covid-19 -13 the extraordinary decisions facing italian doctors, yascha mounk, the atlantic modeling the transmission of middle east respirator syndrome corona virus in the republic of korea xia a time-dependent sir model for covid-19 risk assessment of novel coronavirus covid-19 outbreaks outside china the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application mathematica, version 12 dipartimento della protezione civile covid-19 italia -monitoraggio della situazione coronavirus incubation could be as long as 27 days, chinese provincial government says -reuters presumed asymptomatic carrier transmission of covid-19 key: cord-279539-s2zv7hr4 authors: narayanan, c. s. title: modeling the covid-19 outbreak in the united states date: 2020-05-05 journal: nan doi: 10.1101/2020.04.30.20086884 sha: doc_id: 279539 cord_uid: s2zv7hr4 the covid-19 contagion has developed at an alarming rate in the us and as of april 24, 2020, tens of thousands of people have already died from the disease. in the event of an outbreak like such, forecasting the extent of the mortality that will occur is crucial to aid the implementation of effective interventions. mortality depends on two factors: the case fatality rate and the case incidence. we combine a cohort-based model that determines case fatality rates along with a modified logistic model that evaluates the case incidence to determine the number of deaths in all the us states over time; the model is also able to include the impact of interventions. both models yield exceptional goodness-of-fit. the model predicted a range of death outcomes (79k to 246k) all of which are considerably greater than the figures presented in mainstream media. this model can be used more effectively than current models to estimate the number of deaths during an outbreak, allowing for better planning. the first case of coronavirus disease 2019, or covid-19, a respiratory infection caused 2 by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first identified 3 in wuhan, china in late 2019 [1] . subsequently, the outbreak has spread to 212 [2] 4 countries, including the united states, where the first case of covid-19 was detected 5 in washington state on january 20, 2020 [3] . as of april 24, 2020, the u.s has 6 reported 830053 cases and 42311 deaths [4] . 7 as the pandemic progresses, determining its prognosis is essential to inform the 8 adoption of adequate mitigation efforts. many have attempted to forecast the trajectory 9 of the epidemic in the united states, and at the forefront is the white house 11 dr. fauci suggested that the us would likely face 100,000 to 200,000 deaths, with 12 millions of cases [7] . however, on april 9 he said the estimate had been revised down 13 to 60,000 [8] . moreover, a model by the university of washington, closely followed by 14 the white house, projects 67,000 deaths as of april 24, 2020 [9] , in line with dr. 15 fauci's statement [8] . 16 it is well-known that there is a wide variance in the sizes of outbreaks between states 17 as well as the resulting incidence of deaths. the primary reason for the variance is the 18 uncertainty in the prediction of infections and the fatality rate of infected individuals. improved models to determine both the cumulative case incidence and the case fatality 21 rate. we calculated the case fatality rate (cfr) for each state using a cohort-based 22 approach, which has demonstrated greater accuracy than traditional methods [10] . 23 additionally, the number of cumulative cases in each state is predicted using a modified 24 logistic model. combining the two, we are able to forecast the number of cumulative 25 deaths by state. additionally, we analyze the drivers of deaths and discuss implications 26 on policy formation. 27 data sources 29 the primary data for this study is publicly available and was obtained from the center 30 for systems science and engineering (csse) at johns hopkins university [11] . we 31 obtained the data pertaining to daily new cases/deaths and cumulative cases/deaths for 32 the period of january 22, 2020, to april 24, 2020. in addition, we obtained us state 33 population data from the 2010 united states census bureau [12] . the number of 34 deaths is a product of the case fatality rate (cfr) and the population confirmed to 35 have been infected [13] . therefore, in order to determine cumulative fatalities due to 36 covid-19, it is necessary to first predict the cfr and the number of cases. cfr, case 37 incidence, and deaths were evaluated for all fifty states as well as the district of 38 columbia. calculating cfr there are three principal measures of disease lethality: the case fatality rate (cfr), 41 infection fatality rate (ifr) and mortality rate (mr). the mortality rate is represented 42 by the proportion of cumulative deaths to the total at-risk population. this is 43 ultimately indicative of the probability of any individual's mortality among the total 44 population. the cfr uses the same numerator (cumulative deaths) but instead divides 45 it by the number of cumulative confirmed cases. the case fatality [14] rate is the 46 proportion of individuals who die from a disease among all individuals diagnosed with 47 the disease within a specified timeframe [15] . that is, it reveals the percentage of 48 individuals that die among all individuals who test positive for the disease. the ifr is 49 similar to the cfr, except it represents the ratio of deaths to the total number of 50 people who are infected; it accounts for all infected individuals regardless of whether 51 their disease is reported or not. in an ideal scenario, where zero individuals with 52 covid-19 went unnoticed, and surveillance was faultless, the ifr and cfr would be 53 equivalent. however, this is not truly plausible; testing is limited, asymptomatic 54 infections commonly are not surveilled, and not all instances of the disease are 55 accounted for in reality. as a result, the cfr that we calculate will be much higher 56 than the ifr and the mortality rate. in this paper, we use a logistical function [10] to describe the exponential growth 58 and subsequent flattening of covid-19 cfr. the cfr depends on three parameters: 59 the final cfr (l), the cfr growth rate (k), and the onset-to-death interval (t 0 ) and is 60 expressed as: using this model, we calculate the number of deaths each day for each cohort or 62 group of individuals infected on the same day. next, we build an objective function that 63 april 30, 2020 2/10 minimizes the root mean square error between the actual and predicted values of 64 cumulative deaths. we ran 125,000 simulations, using numerous values of the 65 onset-to-death interval, the cfr, and the cfr growth rate. the cfr was kept in the 66 range of 0.5% to 20%, the slope was kept in the range of 0.005 and 0.7, and the 67 onset-to-death interval bounded between 0 and 60 days. we assigned these bounds 68 because after in-depth explorations of the model, we were convinced the solutions would 69 be within these parameters. we then identify the model parameters that best fit the 70 data (top 1% of the best-fit rmse). with a kernel density distribution of case fatality 71 rates, we determined the low cfr (the lowest value regardless of its frequency); the 72 mode cfr (the most probable cfr); and the high cfr (the highest value regardless of 73 its frequency). methods is the incorporation of the growth rate. in the sir model, this is r 0 , the 81 transmission rate-given that the population lacks immunity and there are no deliberate 82 interventions to impede disease transmission. the number of infections will continuously 83 rise in a population if r 0 > 1, will remain steady if r 0 = 1, and will decrease if r 0 < 1. 84 to explicitly account for the impact of mitigation efforts, models must support gradual 85 changes in the shape of the case growth rate. the logistic model forecasts the slow initial rise, exponential growth, and eventual 87 decay of cumulative cases, but cannot account for the changes that result from parameters: the terminal number of cumulative cases (c), the cfr growth rate (r), and 94 the days to the inflection point (t i ). the inflection point indicates the day at which the 95 number of daily cases reaches its maximum. this function that describes the change in 96 case incidencei(t) over time can be expressed as: the modified logistic model has five parameters; however, the terminal number of 98 cumulative cases (c) and inflection point (t0) remain unchanged. the set of equations 99 that describe the incidence using the modified logistic model are: april 30, 2020 3/10 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 5, 2020. . the parameter r m is the modified growth function that changes over time, and f is a 101 smoothing function that determines how quickly the rate diverges around the point of 102 inflection, as well as the magnitude of the transformation. using this model, we 103 calculated the number of cases for each state. 104 next, we built an objective function that minimizes the root mean square error 105 between the actual and predicted values of cumulative cases, and ran numerous 106 simulations by varying the four parameters. we held p constant at 0.1 in all our 107 simulations. we ran 375,000 simulations, using numerous values of the days to 108 inflection: t0, the terminal number of cases, c, the growth rate, r, and the growth rate 109 multiplier, k. the terminal number of cases was kept in the range of 0.01% to 3% of the 110 each state's penetration, the growth rate was kept in the range of 0.01 and 0.3, and the 111 number of days to inflection was bounded between 10 and 50 days. the growth rate 112 multiplier was kept in the range between 0 and 40%. the cumulative mortality is the product of the case fatality rate and the cumulative 120 case incidence. with the low, mode, and high values of both cfr and cumulative case 121 incidence, we evaluate the nine possible death tolls for each state by finding the product 122 of each cfr value and each value of the cumulative case incidence. to determine 123 cumulative mortality on the national scale, we add up the respective cells for all states. 124 predicting case incidence 126 we calculated the case incidence for each jurisdiction. fig 1 shows the goodness-of-fit 127 between the forecasted cases and the true number of cases for new york. the two sets 128 of figures show the cumulative case incidence and new case incidence. it demonstrates 129 that there is an excellent fit for both new and cumulative cases. we calculated the r 2 130 for all the states and the fit was excellent (greater than 98% for all the states) for all 131 states indicating that the modified logistical function does a great job of modeling the 132 transition after the intervention. more information regarding the model's goodness-of-fit 133 for the states is provided in the supplemental information (see s1 table) . s2 table) . however, new york is a substantial outlier: the model 137 predicts 500,000 cases for the mode case. all the numbers we will quote in the rest of 138 the document will be the mode case (unless otherwise specified), as it has the highest 139 likelihood. even if the best-case scenario transpires, its case incidence will probably 140 exceed the incidence of any other state by over 200000. next, we evaluated the forecasted case incidence for the entire united states (fig 3) . 142 the total number of cases predicted is above 1.2m, and the number of new daily cases 143 april 30, 2020 4/10 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 5, 2020. forecasted case incidence for the top us states. the range of forecasted total case incidence for the top states that contribute 90% of all the deaths. the low, mode, and high cases are displayed. the low and high cases are determined as the low and high end of the 95% confidence interval. peaks at more than 35000. as new york and new jersey contribute significantly toward 144 the overall case incidence, the united states peak daily cases is strongly dependent on 145 the peak of these two states. in order to understand whether the different states have a case incidence 147 proportionate to their populations, we calculated the discrepancy between the projected 148 cases per capita for individual states and u.s average projected cumulative cases per 149 capita (fig 4) . new york, new jersey, massachusetts, connecticut, and louisiana have 150 much higher case incidence per capita. this means that the disease affected these areas 151 disproportionately. in contrast, the states of california, texas, florida, and ohio did 152 much better in controlling the spread of the infection. difference in forecasted case incidence between states. difference in forecasted case incidence between the top states that contributed 90% of all the deaths. this was calculated by subtracting the difference between projected cases per capita for individual states and the u.s average projected cumulative cases per capita. we previously calculated the case fatality rates for hubei province and showed that the 155 goodness-of-fit was excellent [10] . we used the same methodology and calculated the 156 cfr for all the states. the model is able to fit the data extremely well showing that 157 both the model and the methodology are sound. we provide the r 2 value for all the 158 states in the supplemental information (see s1 table) . 159 we calculated the range of final case fatality rates for each state (fig 5) . the cfr 160 for most states is between 5% and 10%. compared to the case incidence, case fatality 161 rates have far less variability. massachusetts, connecticut, new york, and maryland 162 have relatively higher cfr's. in contrast, texas, california and georgia have much 163 lower cfr's. we also provide supplemental information for all state (see s3 table) . in order to measure how well states are faring relative to each other, we calculate the 165 difference between projected cfr's for each state and the average cfr for the us 166 (fig 6) . positive (negative) values indicate that the cfr is worse (better). it clearly 167 shows that there is a wide disparity between states' case fatality rates. furthermore, the 168 difference in cfr closely corresponds with projected cumulative deaths. this shows 169 even more dramatically how much greater new york's and connecticut's outbreaks are 170 compared to other states. april 30, 2020 5/10 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 5, 2020. . fig 6. difference in forecasted case fatality rates between states. difference in forecasted case fatality rates between the top states that contributed 90% of all the deaths. this was calculated by subtracting the difference between projected cfr for individual states and the u.s average cfr. considering that cfr and case incidence are the factors of death, it is fitting now to 173 discuss the projected incidence of deaths (fig 7) . we calculated the number of deaths 174 for each of the top 15 states that contribute 90% of the deaths. this reveals the 175 tremendous disparity between the size of outbreaks in different states (see also s4 176 table) . the majority of states will experience less than 10,000 deaths. new york is once 177 again a significant outlier; the model returns a minimum of 40,000 deaths and a mode of 178 65,000 deaths, 45% of the u.s. total and more than the next five jurisdictions combined. 179 this can be traced back to the state's relatively high projected case fatality rate and indicative of differences in cfr and case incidence. sources of variation include the 185 chronology and success of mitigation efforts, the prevalence of testing, and the 186 distribution of age [19] and comorbidities [20] within populations at risk of infection. 187 fig 7. forecasted deaths for the top us states. the range of forecasted deaths for the top states that contribute 90% of all the deaths. the low, mode, and high cases are displayed. the low and high cases are determined as the low and high end of the 95% confidence interval. table 1 summarizes the various possible death tolls under each of the 9 conditions. 188 there is a 95% likelihood that any of these results are possible. the lowest cumulative 189 deaths the u.s. could experience is 78,000, considerably higher than both fauci's 245904 predicted deaths tolls for the nine different scenarios. these scenarios are constructed using the low, mode, and high cases for cfr and cases. the low and high cases are determined as the low and high end of the 95% confidence interval. for the best-fit case in all the states, we calculated the number of deaths per day 193 and the cumulative number of deaths (fig 8) . this shows that the number of deaths 194 will reach an asymptote in the end of june and the number of daily deaths will peak in 195 early may. we used a cohort analysis approach to estimate cfr and a modified logistic model 198 (that explicitly accounts for the impact of mitigation efforts) to forecast case incidence 199 on the state level, and afterwards calculated mortality on the state and national levels. 200 our model showed a wide range of mortality, with 79,000 deaths on the low end and a 201 maximum of 245,000 deaths. every possibility predicted by the model exceeds the 202 prognostications produced by both the white house and the university of washington 203 model. our model also revealed the deep disparity in deaths among different states, 204 which is attributable to differences in case fatality rate and case incidence. we postulate 205 reasons for these variations. many states in the us northeast, including new york, new jersey, massachusetts, 207 and connecticut are disproportionately represented in the cumulative death toll. this 208 disparity is primarily because these states have much worse case incidence and case 209 fatality rates. new york is forecasted to experience the largest outbreak, the greatest 210 cfr, and the highest mortality of any state by far. one explanation for its high case 211 fatality rate is the strain the epidemic has placed on its healthcare system. as a result 212 of its high case incidence, more hospitalizations will be required, overwhelming the 213 medical care system. this could result in diminished quality of medical care, resulting 214 in a high case fatality rate. additionally, the disease has disproportionately impacted 215 low-income, more vulnerable areas. [21] 216 the reason for the high case incidence itself is more perplexing. numerous factors 217 are likely at play, such as the popularity of public transportation [22] and the high 218 population density of the new york city metropolitan area [23] ] (where the vast 219 majority of cases have been reported [24]). however, it is difficult to find conclusive 220 evidence that any of these factors are directly accountable for the outbreak. it is very 221 likely that luck played a large role in determining where clusters appeared. there is 222 ample evidence that super-spreading events, or sse's, can cause sizable outbreaks [25] . 223 for instance, officials in new york stated that as many as fifty infections could be 224 traced back to a single man in westchester county [26] . 225 we propose two principal explanations for the discrepancy between the death tolls 226 forecasted by the university of washington model and that of our model: the 227 differences in both the procedure for calculating cfr and the procedure for calculating 228 mortality. our cohort-based method to determining cfr's predicts case fatality rates 229 more accurately at every stage of the outbreak than other models because it explicitly 230 accounts for the onset-to-death interval. further, we forecast cumulative mortality by 231 independently evaluating cfr and case incidence. in contrast, the university of 232 washington model directly predicts deaths; this method is prone to greater errors. while both the cfr and case incidence models fit the data extremely well, there are 234 several challenges with estimating the number of deaths accurately. our model assumes 235 the scale and methods of surveillance do not significantly change between today and the 236 future. any changes to the testing process will affect the number of confirmed cases. breakthroughs in leveraging telemedicine, for instance, would result in increased 238 detection of infected individuals. in this case, the model's current forecasts for case 239 incidence would be underestimates [27] . additionally, if the shelter-in-place order is 240 withdrawn from states too early, there will likely be an increase in both the case 241 incidence and the mortality. the model itself has limitations; if our assumptions do not 242 hold true, then our analysis will not hold true either. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 5, 2020. i would like to thank chandra narayanan for all his guidance on building the best case 254 incidence model. first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa who coronavirus disease first case of 2019 novel coronavirus in the united states who. coronavirus disease 2019 (covid-19) situation report -95 npr fauci estimates that 100,000 to 200,000 americans could die from the coronavirus npr the coronavirus crisis -fauci says u.s. coronavirus deaths may be 'more like 60 institute for health metrics and evaluation. covid-19 projections a novel cohort analysis approach to determining the case fatality rate of covid-19 and other infectious diseases jhu 2019 novel coronavirus covid-19 (2019-ncov) data repository by johns hopkins csse united states census bureau state population totals methods for estimating the case fatality ratio for a novel, emerging infectious disease how testing completely skews coronavirus case fatality rates case fatality rate the mathematics of infectious diseases estimating epidemic exponential growth rate and basic reproduction number estimation of the final size of the covid-19 epidemic clinical characteristics of coronavirus disease 2019 in china comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis data suggests many new york city neighborhoods hardest hit by covid-19 are also low income areas land area, and population density by county identifying and interrupting superspreading events-implications for control of severe acute respiratory syndrome coronavirus 2. emerg infect dis new york officials traced more than 50 coronavirus cases back to one attorney virtually perfect? telemedicine for covid-19 key: cord-288052-qfjet2sa authors: paparini, sara; green, judith; papoutsi, chrysanthi; murdoch, jamie; petticrew, mark; greenhalgh, trish; hanckel, benjamin; shaw, sara title: case study research for better evaluations of complex interventions: rationale and challenges date: 2020-11-10 journal: bmc med doi: 10.1186/s12916-020-01777-6 sha: doc_id: 288052 cord_uid: qfjet2sa background: the need for better methods for evaluation in health research has been widely recognised. the ‘complexity turn’ has drawn attention to the limitations of relying on causal inference from randomised controlled trials alone for understanding whether, and under which conditions, interventions in complex systems improve health services or the public health, and what mechanisms might link interventions and outcomes. we argue that case study research—currently denigrated as poor evidence—is an under-utilised resource for not only providing evidence about context and transferability, but also for helping strengthen causal inferences when pathways between intervention and effects are likely to be non-linear. main body: case study research, as an overall approach, is based on in-depth explorations of complex phenomena in their natural, or real-life, settings. empirical case studies typically enable dynamic understanding of complex challenges and provide evidence about causal mechanisms and the necessary and sufficient conditions (contexts) for intervention implementation and effects. this is essential evidence not just for researchers concerned about internal and external validity, but also research users in policy and practice who need to know what the likely effects of complex programmes or interventions will be in their settings. the health sciences have much to learn from scholarship on case study methodology in the social sciences. however, there are multiple challenges in fully exploiting the potential learning from case study research. first are misconceptions that case study research can only provide exploratory or descriptive evidence. second, there is little consensus about what a case study is, and considerable diversity in how empirical case studies are conducted and reported. finally, as case study researchers typically (and appropriately) focus on thick description (that captures contextual detail), it can be challenging to identify the key messages related to intervention evaluation from case study reports. conclusion: whilst the diversity of published case studies in health services and public health research is rich and productive, we recommend further clarity and specific methodological guidance for those reporting case study research for evaluation audiences. the need for methodological development to address the most urgent challenges in health research has been welldocumented. many of the most pressing questions for public health research, where the focus is on system-level determinants [1, 2] , and for health services research, where provisions typically vary across sites and are provided through interlocking networks of services [3] , require methodological approaches that can attend to complexity. the need for methodological advance has arisen, in part, as a result of the diminishing returns from randomised controlled trials (rcts) where they have been used to answer questions about the effects of interventions in complex systems [4] [5] [6] . in conditions of complexity, there is limited value in maintaining the current orientation to experimental trial designs in the health sciences as providing 'gold standard' evidence of effect. there are increasing calls for methodological pluralism [7, 8] , with the recognition that complex intervention and context are not easily or usefully separated (as is often the situation when using trial design), and that system interruptions may have effects that are not reducible to linear causal pathways between intervention and outcome. these calls are reflected in a shifting and contested discourse of trial design, seen with the emergence of realist [9] , adaptive and hybrid (types 1, 2 and 3) [10, 11] trials that blend studies of effectiveness with a close consideration of the contexts of implementation. similarly, process evaluation has now become a core component of complex healthcare intervention trials, reflected in mrc guidance on how to explore implementation, causal mechanisms and context [12] . evidence about the context of an intervention is crucial for questions of external validity. as woolcock [4] notes, even if rct designs are accepted as robust for maximising internal validity, questions of transferability (how well the intervention works in different contexts) and generalisability (how well the intervention can be scaled up) remain unanswered [5, 13] . for research evidence to have impact on policy and systems organisation, and thus to improve population and patient health, there is an urgent need for better methods for strengthening external validity, including a better understanding of the relationship between intervention and context [14] . policymakers, healthcare commissioners and other research users require credible evidence of relevance to their settings and populations [15] , to perform what rosengarten and savransky [16] call 'careful abstraction' to the locales that matter for them. they also require robust evidence for understanding complex causal pathways. case study research, currently under-utilised in public health and health services evaluation, can offer considerable potential for strengthening faith in both external and internal validity. for example, in an empirical case study of how the policy of free bus travel had specific health effects in london, uk, a quasi-experimental evaluation (led by jg) identified how important aspects of context (a good public transport system) and intervention (that it was universal) were necessary conditions for the observed effects, thus providing useful, actionable evidence for decisionmakers in other contexts [17] . the overall approach of case study research is based on the in-depth exploration of complex phenomena in their natural, or 'real-life', settings. empirical case studies typically enable dynamic understanding of complex challenges rather than restricting the focus on narrow problem delineations and simple fixes. case study research is a diverse and somewhat contested field, with multiple definitions and perspectives grounded in different ways of viewing the world, and involving different combinations of methods. in this paper, we raise awareness of such plurality and highlight the contribution that case study research can make to the evaluation of complex system-level interventions. we review some of the challenges in exploiting the current evidence base from empirical case studies and conclude by recommending that further guidance and minimum reporting criteria for evaluation using case studies, appropriate for audiences in the health sciences, can enhance the take-up of evidence from case study research. case study research offers evidence about context, causal inference in complex systems and implementation well-conducted and described empirical case studies provide evidence on context, complexity and mechanisms for understanding how, where and why interventions have their observed effects. recognition of the importance of context for understanding the relationships between interventions and outcomes is hardly new. in 1943, canguilhem berated an over-reliance on experimental designs for determining universal physiological laws: 'as if one could determine a phenomenon's essence apart from its conditions! as if conditions were a mask or frame which changed neither the face nor the picture!' ( [18] p126). more recently, a concern with context has been expressed in health systems and public health research as part of what has been called the 'complexity turn' [1] : a recognition that many of the most enduring challenges for developing an evidence base require a consideration of system-level effects [1] and the conceptualisation of interventions as interruptions in systems [19] . the case study approach is widely recognised as offering an invaluable resource for understanding the dynamic and evolving influence of context on complex, system-level interventions [20] [21] [22] [23] . empirically, case studies can directly inform assessments of where, when, how and for whom interventions might be successfully implemented, by helping to specify the necessary and sufficient conditions under which interventions might have effects and to consolidate learning on how interdependencies, emergence and unpredictability can be managed to achieve and sustain desired effects. case study research has the potential to address four objectives for improving research and reporting of context recently set out by guidance on taking account of context in population health research [24] , that is to (1) improve the appropriateness of intervention development for specific contexts, (2) improve understanding of 'how' interventions work, (3) better understand how and why impacts vary across contexts and (4) ensure reports of intervention studies are most useful for decision-makers and researchers. however, evaluations of complex healthcare interventions have arguably not exploited the full potential of case study research and can learn much from other disciplines. for evaluative research, exploratory case studies have had a traditional role of providing data on 'process', or initial 'hypothesis-generating' scoping, but might also have an increasing salience for explanatory aims. across the social and political sciences, different kinds of case studies are undertaken to meet diverse aims (description, exploration or explanation) and across different scales (from small n qualitative studies that aim to elucidate processes, or provide thick description, to more systematic techniques designed for medium-to-large n cases). case studies with explanatory aims vary in terms of their positioning within mixed-methods projects, with designs including (but not restricted to) (1) single n of 1 studies of interventions in specific contexts, where the overall design is a case study that may incorporate one or more (randomised or not) comparisons over time and between variables within the case; (2) a series of cases conducted or synthesised to provide explanation from variations between cases; and (3) case studies of particular settings within rct or quasi-experimental designs to explore variation in effects or implementation. detailed qualitative research (typically done as 'case studies' within process evaluations) provides evidence for the plausibility of mechanisms [25] , offering theoretical generalisations for how interventions may function under different conditions. although rct designs reduce many threats to internal validity, the mechanisms of effect remain opaque, particularly when the causal pathways between 'intervention' and 'effect' are long and potentially non-linear: case study research has a more fundamental role here, in providing detailed observational evidence for causal claims [26] as well as producing a rich, nuanced picture of tensions and multiple perspectives [8] . longitudinal or cross-case analysis may be best suited for evidence generation in system-level evaluative research. turner [27] , for instance, reflecting on the complex processes in major system change, has argued for the need for methods that integrate learning across cases, to develop theoretical knowledge that would enable inferences beyond the single case, and to develop generalisable theory about organisational and structural change in health systems. qualitative comparative analysis (qca) [28] is one such formal method for deriving causal claims, using set theory mathematics to integrate data from empirical case studies to answer questions about the configurations of causal pathways linking conditions to outcomes [29, 30] . nonetheless, the single n case study, too, provides opportunities for theoretical development [31] , and theoretical generalisation or analytical refinement [32] . how 'the case' and 'context' are conceptualised is crucial here. findings from the single case may seem to be confined to its intrinsic particularities in a specific and distinct context [33] . however, if such context is viewed as exemplifying wider social and political forces, the single case can be 'telling', rather than 'typical', and offer insight into a wider issue [34] . internal comparisons within the case can offer rich possibilities for logical inferences about causation [17] . further, case studies of any size can be used for theory testing through refutation [22] . the potential lies, then, in utilising the strengths and plurality of case study to support theorydriven research within different methodological paradigms. evaluation research in health has much to learn from a range of social sciences where case study methodology has been used to develop various kinds of causal inference. for instance, gerring [35] expands on the within-case variations utilised to make causal claims. for gerring [35] , case studies come into their own with regard to invariant or strong causal claims (such as x is a necessary and/or sufficient condition for y) rather than for probabilistic causal claims. for the latter (where experimental methods might have an advantage in estimating effect sizes), case studies offer evidence on mechanisms: from observations of x affecting y, from process tracing or from pattern matching. case studies also support the study of emergent causation, that is, the multiple interacting properties that account for particular and unexpected outcomes in complex systems, such as in healthcare [8] . finally, efficacy (or beliefs about efficacy) is not the only contributor to intervention uptake, with a range of organisational and policy contingencies affecting whether an intervention is likely to be rolled out in practice. case study research is, therefore, invaluable for learning about contextual contingencies and identifying the conditions necessary for interventions to become normalised (i.e. implemented routinely) in practice [36] . at present, there are significant challenges in exploiting the benefits of case study research in evaluative health research, which relate to status, definition and reporting. case study research has been marginalised at the bottom of an evidence hierarchy, seen to offer little by way of explanatory power, if nonetheless useful for adding descriptive data on process or providing useful illustrations for policymakers [37] . this is an opportune moment to revisit this low status. as health researchers are increasingly charged with evaluating 'natural experiments'-the use of face masks in the response to the covid-19 pandemic being a recent example [38] -rather than interventions that take place in settings that can be controlled, research approaches using methods to strengthen causal inference that does not require randomisation become more relevant. a second challenge for improving the use of case study evidence in evaluative health research is that, as we have seen, what is meant by 'case study' varies widely, not only across but also within disciplines. there is indeed little consensus amongst methodologists as to how to define 'a case study'. definitions focus, variously, on small sample size or lack of control over the intervention (e.g. [39] p194), on in-depth study and context [40, 41] , on the logic of inference used [35] or on distinct research strategies which incorporate a number of methods to address questions of 'how' and 'why' [42] . moreover, definitions developed for specific disciplines do not capture the range of ways in which case study research is carried out across disciplines. multiple definitions of case study reflect the richness and diversity of the approach. however, evidence suggests that a lack of consensus across methodologists results in some of the limitations of published reports of empirical case studies [43, 44] . hyett and colleagues [43] , for instance, reviewing reports in qualitative journals, found little match between methodological definitions of case study research and how authors used the term. this raises the third challenge we identify that case study reports are typically not written in ways that are accessible or useful for the evaluation research community and policymakers. case studies may not appear in journals widely read by those in the health sciences, either because space constraints preclude the reporting of rich, thick descriptions, or because of the reported lack of willingness of some biomedical journals to publish research that uses qualitative methods [45] , signalling the persistence of the aforementioned evidence hierarchy. where they do, however, the term 'case study' is used to indicate, interchangeably, a qualitative study, an n of 1 sample, or a multi-method, in-depth analysis of one example from a population of phenomena. definitions of what constitutes the 'case' are frequently lacking and appear to be used as a synonym for the settings in which the research is conducted. despite offering insights for evaluation, the primary aims may not have been evaluative, so the implications may not be explicitly drawn out. indeed, some case study reports might properly be aiming for thick description without necessarily seeking to inform about context or causality. we recognise that definitional and methodological plurality is not only inevitable, but also a necessary and creative reflection of the very different epistemological and disciplinary origins of health researchers, and the aims they have in doing and reporting case study research. indeed, to provide some clarity, thomas [46] has suggested a typology of subject/purpose/approach/process for classifying aims (e.g. evaluative or exploratory), sample rationale and selection and methods for data generation of case studies. we also recognise that the diversity of methods used in case study research, and the necessary focus on narrative reporting, does not lend itself to straightforward development of formal quality or reporting criteria. existing checklists for reporting case study research from the social sciences-for example lincoln and guba's [47] and stake's [33] -are primarily orientated to the quality of narrative produced, and the extent to which they encapsulate thick description, rather than the more pragmatic issues of implications for intervention effects. those designed for clinical settings, such as the care (case reports) guidelines, provide specific reporting guidelines for medical case reports about single, or small groups of patients [48] , not for case study research. the design of case study research in health care (descarte) model [44] suggests a series of questions to be asked of a case study researcher (including clarity about the philosophy underpinning their research), study design (with a focus on case definition) and analysis (to improve process). the model resembles toolkits for enhancing the quality and robustness of qualitative and mixed-methods research reporting, and it is usefully open-ended and non-prescriptive. however, even if it does include some reflections on context, the model does not fully address aspects of context, logic and causal inference that are perhaps most relevant for evaluative research in health. hence, for evaluative research where the aim is to report empirical findings in ways that are intended to be pragmatically useful for health policy and practice, this may be an opportune time to consider how to best navigate plurality around what is (minimally) important to report when publishing empirical case studies, especially with regards to the complex relationships between context and interventions, information that case study research is well placed to provide. the conventional scientific quest for certainty, predictability and linear causality (maximised in rct designs) has to be augmented by the study of uncertainty, unpredictability and emergent causality [8] in complex systems. this will require methodological pluralism, and openness to broadening the evidence base to better understand both causality in and the transferability of system change intervention [14, 20, 23, 25] . case study research evidence is essential, yet is currently under exploited in the health sciences. if evaluative health research is to move beyond the current impasse on methods for understanding interventions as interruptions in complex systems, we need to consider in more detail how researchers can conduct and report empirical case studies which do aim to elucidate the contextual factors which interact with interventions to produce particular effects. to this end, supported by the uk's medical research council, we are embracing the challenge to develop guidance for case study researchers studying complex interventions. following a meta-narrative review of the literature, we are planning a delphi study to inform guidance that will, at minimum, cover the value of case study research for evaluating the interrelationship between context and complex system-level interventions; for situating and defining 'the case', and generalising from case studies; as well as provide specific guidance on conducting, analysing and reporting case study research. our hope is that such guidance can support researchers evaluating interventions in complex systems to better exploit the diversity and richness of case study research. abbreviations qca: qualitative comparative analysis; qed: 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publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable authors' contributions jg, mp, sp, jm, tg, cp and ss drafted the initial paper; all authors contributed to the drafting of the final version, and read and approved the final manuscript. this work was funded by the medical research council -mrc award mr/ s014632/1 hcs: case study, context and complex interventions (triple c). sp was additionally funded by the university of oxford's higher education innovation fund (heif). not applicable (article based on existing available academic publications)ethics approval and consent to participate not applicable the authors declare that they have no competing interests. 1 key: cord-276254-q04hqra2 authors: paul, kishor kumar; salje, henrik; rahman, muhammad w.; rahman, mahmudur; gurley, emily s. title: comparing insights from clinic-based versus community-based outbreak investigations: a case study of chikungunya in bangladesh date: 2020-06-02 journal: int j infect dis doi: 10.1016/j.ijid.2020.05.111 sha: doc_id: 276254 cord_uid: q04hqra2 abstract background outbreak investigations typically focus their efforts on identifying cases that present at healthcare facilities. however, these cases rarely represent all cases in the wider community. in this context, community-based investigations may provide additional insight into key risk factors for infection, however, the benefits of these more laborious data collection strategies remains unclear. methods we used different subsets of the data from a comprehensive outbreak investigation to compare the inferences we make in alternative investigation strategies. results the outbreak investigation team interviewed 1,933 individuals from 460 homes. 364 (18%) of individuals had symptoms consistent with chikungunya. a theoretical clinic-based study would have identified 26% of the cases. adding in community-based cases provided an overall estimate of the attack rate in the community. comparison with controls from the same household revealed that those with at least secondary education had a reduced risk. finally, enrolling residents from households across the community allowed us to characterize spatial heterogeneity of risk and identify the type of clothing usually worn and travel history as risk factors. this also revealed that household-level use of mosquito control was not associated with infection. conclusions these findings highlight that while clinic-based studies may be easier to conduct, they only provide limited insight into the burden and risk factors for disease. enrolling people who escaped from infection, both in the household and in the community allows a step change in our understanding of the spread of a pathogen and maximizes opportunities for control. infectious disease outbreaks have the potential to place a significant burden on public health resources. understanding who is at risk of becoming infected is critical for the focused targeting of interventions. due to relative ease of access and limited cost requirements, outbreak investigations typically focus on cases that present at formal healthcare centers such as hospitals or community clinics. for example, data collection performed as part of epidemiological investigations during the recent epidemics of ebola, zika and mers focused on quantifying the number of cases and their characteristics. (al-abdallat et al., 2014 , lu et al., 2015 , teixeira et al., 2016 these case-counting exercises provided key insights into fundamental epidemiological parameters such as the basic reproductive number and case fatality rates, and allowed the projection of the future course of the epidemic. (aylward et al., 2014 , lessler et al., 2014 , lewnard et al., 2014 , yamin et al., 2015 however, without information on the underlying population, and especially characteristics of individuals who avoid infection, these approaches limit our ability to make mechanistic insights, quantify burden of disease, and identify risk factors for infection, hampering efforts to develop targeted control strategies. cases that present at healthcare centers may only represent a small minority of all cases. in addition, some individuals are more likely to visit formal healthcare providers than others, including those with more severe illness, and differences in healthcare seeking can vary by age, gender and socioeconomic status. (chowdhury et al., 2007 , nikolay et al., 2017 , pandey et al., 2002 household-based outbreak investigations, where investigation teams visit affected j o u r n a l p r e -p r o o f communities, permit a more comprehensive understanding of pathogen spread that limits the impact of healthcare seeking patterns. (france et al., 2010) however, these investigations are usually still focused on identifying individuals that got sick. (boore et al., 2013 , france et al., 2010 without also understanding who is avoiding infection in a community, it is difficult to identify the key risk factors for infection, limiting potential inferences. the possible insights from alternative investigation strategies have not previously been systematically compared. here, we use the results of a detailed chikungunya outbreak investigation from bangladesh as an example to consider the inferences made under different investigation scenarios. chikungunya virus is a mosquito-borne alphavirus transmitted to humans by aedes mosquitoes causing acute fever, joint pain, and skin rash. (aubry et al., 2015) chikungunya fever was first recognized in 1952 in tanzania. (lumsden, 1955) since then, outbreaks of chikungunya have been regularly identified across the tropics and sub-tropics. the first chikungunya outbreak in bangladesh was identified in 2008 in two northwestern districts bordering india.(icddr) since then regular outbreaks have been detected. (khatun et al., 2015 , salje et al., 2016b here we use the results from a detailed investigation of an outbreak of chikungunya virus in a village in tangail, bangladesh where the outbreak team visited every household in the community and interviewed all members in each household. the comprehensive household investigation captured both those who did get infected and those that escaped from infection. the objective of this study was to compare our approach, in terms of the inferences about the outbreak, to more limited investigation strategies. in late november 2012, a local health official of gopalpur sub-district in tangail households in the village consented to being enrolled in the study. questionnaires were administered in all households to identify suspected cases, identify demographic characteristics, and travel histories of individuals within households. suspect cases were defined as residents with acute onset of fever with rash or joint pain within 6 months prior to beginning the investigation. study staff administered questionnaires to household heads about household members' demographic data and history of illness, water source, construction materials, and mosquito control measures in the household. potential mosquito breeding containers in and around the participating households with stored water were inspected for presence of larvae. suspected cases were asked about their symptoms with onset date and specifics about their treatment seeking behavior. the gps location of all homes was also recorded. determining the etiology of the outbreak all household members, irrespective of their suspected case status, were asked to provide a single 5 ml blood specimen for laboratory testing. blood specimens were spun in the field to separate serum, which were then stored on ice and transported to the virology laboratory of iedcr. the serum samples were tested for igm antibodies against chikungunya by enzyme linked immunosorbant assay (elisa) (standard diagnostics, inc., south korea). suspected casepatients who had igm antibodies against chikungunya in their serum were termed laboratory confirmed cases. we created four different datasets that allowed us to consider different outbreak investigation strategies: this dataset consisted of all suspect cases that reported that they visited a formal healthcare setting (defined as government or non-government primary healthcare center/clinic/hospital) following the onset of symptoms. this dataset consisted of all suspect cases, irrespective of their healthcare seeking behaviors. this dataset consisted of all suspect cases plus controls consisting of household members of these cases. this dataset consisted of all members of all households in the community, regardless of symptoms. the epidemic curve was constructed using symptom onset date of chikungunya cases. gps locations of households with and without chikungunya cases were used to prepare spatial distribution maps. for the case-only datasets (datasets a and b), we compared the age and sex distribution of the cases with that for the district from the 2011 census (bangladesh bureau of statistics, 2011) . for the datasets with information on individuals who escaped from infection (datasets c and d), we initially used simple logistic regression to compare the demographics, typical apparel worn, travel history within the last six months, and household characteristics of cases with non-cases. we then built multivariable logistic regression models to identify adjusted risk factors for chikungunya fever. we initially placed all variables with a p-value of <0.05 in the unadjusted analysis into a multivariable model. we then used backward stepwise selection using the akaike information criterion (aic) (sauerbrei et al., 2007) to identify the best model. not all individuals who get infected will present with symptoms. we attempted to capture these individuals by asking for blood samples from all community members. to assess the impact of j o u r n a l p r e -p r o o f misclassifying asymptomatically infected individuals as controls in datasets c and d, we conducted sensitivity analyses where these individuals were reclassified as cases. all participants provided written informed consent prior to interviews and blood specimen collection and the ministry of health and family welfare, government of bangladesh reviewed and approved the outbreak investigation plan. the ninety-five suspect cases reported visiting a formal healthcare facility for symptoms consistent with chikungunya between july and november, with the peak number of cases occurring in j o u r n a l p r e -p r o o f october (figure 1 : panel a). cases sought care in three different centers: 25 sought care in a government run community clinic, 8 in a government run sub-district health complex and 62 in a private clinic. the median age was 28 years (interquartile range (iqr) = 14-45 years) and the majority (60%) were female (table 1 ). if we used the age and sex distribution of the district from the 2011 national census, we find that there is an increased risk of disease in those between the ages of 30-49 compared to those aged below 10 years (or 2.51, 95% 1.24-5.51) and that females were at increased risk of infection compared to males (or of 1.43, 95% ci: 0.94-2.23) ( table 2) . an additional 269 suspect cases were identified in the community who did not seek care in formal healthcare facilities. of these, 246 individuals visited a local pharmacy and 21 individuals visited the informal sector (unlicensed medical practitioner, traditional healer, and homeopath). the distribution of dates of symptom onset for all cases was nearly identical to the distribution for those that visited clinics (spearman correlation of 0.95) ( figure 1a ). the proportion of suspect cases visiting a clinic varied between 18% in 51-55 years age group and 44% in ≤5 years age group ( figure 1b) . the conclusions about age, sex, educational levels, use of mosquito controls and clinical presentation of suspect cases were similar when using datasets of all cases or only those that sought care in clinics (table 1) , however, those who presented to clinics were more likely to travel outside the district (41% vs 28%, p-value 0.001). cases who attended formal healthcare settings also appeared to come from similar parts of the community as cases who did not (figure 2a-b) . similar to the analysis using clinical cases only, using data from the national census identified increasing risk among females for being a case (table 2) . inference from community cases plus controls from same household j o u r n a l p r e -p r o o f incorporating controls from the households where cases reside allowed us to assess additional potential risk factors for being a case. consistent with inferences using census data, logistic regression models that used household controls also identified increased risk among females (aor 1.75, 95% ci 1.31-2.36) ( table 2 ). in addition, this analysis showed that cases were significantly less likely to have secondary (aor 0.64, 95% ci 0.42-0.98) or more formal (higher secondary) education (aor 0.28, 95% ci 0.17-0.47) compared to the household controls. incorporating data from the entire community showed that the chikungunya outbreak was largely constrained to the center of the village, with few households affected on the east and west borders but virtually all households affected in the center ( figure 2c ). this is despite the entire community only being a few hundred meters wide. the expanded dataset also allowed us to understand the risk factors for infection in the wider community. as with the previous analyses, females had an increased risk of being a case (or: 1.62, 95% ci 1.29-2.03) ( table 2 ), although the difference by sex was concentrated in adults with no difference among children ( figure 1c ). further individuals who reported usually wearing clothing that exposed both limbs had 1.80 the odds of being a case compared to individuals wearing clothing that exposed upper limbs only (95% ci 1.30-2.50). those who had travelled outside tangail district within the last six months also had increased odds of being a case (aor 1.47, 95% ci 1.06-2.03). individuals who had higher secondary or more formal education (aor 0.38, 95% ci 0.24-0.62) were less likely to be a case than individuals without formal education. we did not identify any household characteristics that were associated with being a case, including presence of mosquito larvae (aor 0.87, 95% ci: 0.66-1.16), daily use of anti-mosquito coil (aor 1.03, 95% ci: 0.82-1.31), number of j o u r n a l p r e -p r o o f household members (aor 1.00, 95% ci: 0.79-1.26), and number of rooms in the household (aor 1.11, 95% ci: 0.82-1.51). fifty-two individuals without symptoms tested positive for chikv. we found no significant demographic differences between symptomatic suspected cases and igm-confirmed asymptomatic cases in those who gave blood (table s1 ). in sensitivity analysis, we removed these individuals from the 'control' population and included them in the 'case' populations. risk factors for being a case identified in the previous analysis remained similar in both scenarios where we considered household contacts as controls and individuals from all community households as controls (supplementary information, table s4 ). however, we found important differences in the probability of providing blood. those with symptoms were 6.1 times more likely to provide blood than those without symptoms. further, among asymptomatic individuals, only 2% of children 0-9 years provided a sample compared to 15% among those 30-49 (table s2 ). there were also significant differences by sex (14% of asymptomatic males gave blood compared to 8% of females, p-value <0.001) and educational level with more educated people less likely to provide samples (table s2 ). outbreak investigations are central to informed responses to public health emergencies caused by the emergence of an infectious pathogen. however, outbreak investigations currently largely revolve around case-counting exercises that limit our ability to identify who is at risk for j o u r n a l p r e -p r o o f infection and who is not. here, by using the results of a comprehensive outbreak investigation, we have been able to explicitly explore the impact of different investigation strategies in the same outbreak. we found that a clinic-based study that used data from all the formal healthcare settings would have identified a quarter of all cases and, using census data, have correctly identified female sex as an important risk factor for disease. however, it is only through the recruitment of people who did not get sick that we could identify the importance of travel history, educational level and apparel usage in determining who gets sick. controls from the wider community were also required to demonstrate which household-level characteristics were important for risk, showing that the use of mosquito coils was not protective, and to map spatial heterogeneity in risk, key to intervention development and deployment. this study highlights the significant heterogeneity in healthcare seeking. even in a small community such as this, cases visited nine different sources of healthcare, three of which could be considered formal healthcare settings. infectious disease surveillance activities are unlikely to be able to collate datasets from this diverse range of healthcare sources, even among only those within formal sector, suggesting that outbreak investigations that rely on cases that seek healthcare likely substantially underestimate the magnitude of outbreaks. using the results of our study, we provide our assessment of the ability of different investigation strategies to capture key characteristics of an outbreak (table 3 ). in practice, the decision to expand outbreak investigations beyond information available from healthcare systems will depend on the resources available. where outbreak teams are already performing communitybased case-investigations, the additional time and effort to also collect data on those without symptomsboth from case-households as well as neighboring householdsmay be marginal. this comprehensive outbreak investigation employed ten field-based investigators and took seven days to complete. an investigation strategy only focused on cases in the community would have taken only marginally less person-time as finding cases in the community anyway typically requires comprehensive door-to-door surveys. our findings highlight how this additional data collection effort can help reveal the drivers of transmission, allowing mechanistic insight into pathogen spread and maximizing opportunities to control, many of which would not be possible from case-based investigations (table 3 )..where it is collected, an additional major benefit of the comprehensive dataset is that it can inform mathematical models that reconstruct entire outbreaks, allowing us to estimate the mean transmission distance (previously estimated here at 95 meters) (salje et al., 2016b) . travelling outside tangail district within the six months before the outbreak was associated with increased chikungunya fever risk. human movement can introduce chikv into new areas, causing epidemics (chretien and linthicum, 2007) . no other areas of bangladesh were reporting outbreaks of chikv at this time, though outbreaks may have been missed due to poor surveillance. although individuals of all ages were affected by chikungunya in this outbreak, incidence increased with age among females, potentially linked to increased time women spend at home compared to males, increasing their risk of being bitten by the largely home-dwelling aedes mosquito (salje et al., 2016b) . in this outbreak, household use of mosquito coils was not protective against chikungunya, which is consistent with the findings from a recent meta-analysis on household level risk factors for dengue, which is also spread through aedes mosquitoes (bowman et al., 2016) . serum samples have the potential to provide important information about the level of asymptomatic infection during an outbreak, as has previously been shown during previous chikv outbreaks (salje et al., 2016a , sissoko et al., 2008 . in addition, this outbreak investigation was carried out six months after the outbreak began and community members may have been unable to reliably recall their symptoms or the date their symptoms started, particularly for milder illnesses, which may have led to an underestimation of suspected cases. serological confirmation could help detect any missing infections. however, our study highlights how some caution needs to be taken when interpreting serological data. firstly, while we sought to obtain blood samples from all participants, only one in five individuals agreed. we found that the probability of agreeing to provide blood depended strongly on having had chikungunya symptoms (individuals who had symptoms were more likely to provide samples). children, women and those with a high educational level were less likely to give blood. secondly, the sensitivity of the commercial assay we used has been estimated to be <40% in individuals where igm is still circulating (johnson et al., 2016) and is likely to be even lower here, as the blood draw occurred after igm antibodies would have waned to undetectable levels for many infected individuals (kam et al., 2012) . future studies should consider underlying biases in who is providing blood as well as considering the use of complementary igg assays to help improve the interpretability of serological findings. this investigation suggests that chikungunya virus has become an emerging public health problem in bangladesh, and outbreak investigations of emerging infections often have the objective of estimating attack rates of diseases and identifying the risk factors that lead to infection. our analysis suggests that the optimal strategy for attaining these objectives during an outbreak is to conduct case finding, testing, and data collection in communities. many recent j o u r n a l p r e -p r o o f outbreaks of emerging infections have suffered due to a lack of detailed information about attack rates and risk for infection, due to their limited investigation strategies (ahmed et al., 2015 , ballera et al., 2015 , khatun et al., 2015 . future investigations of emerging infection outbreaks should consider using these more intensive strategies, at least in a subset of investigations, to improve our understanding of these infections and our public health response. according to institutional data policy of the international centre for diarrhoeal disease research, bangladesh (icddr,b), summary of data can be publicly displayed or can be made publicly accessible. to protect intellectual property rights of primary data, icddr,b cannot make primary data publicly available. however, upon request, institutional data access committee of icddr,b can provide access to primary data to any individual, upon reviewing the nature and potential use of the data. requests for data can be forwarded to: this work was supported by centers for disease control and prevention (cdc), atlanta, usa [cooperative agreement no: 5u01ci000628]. in addition, the government of bangladesh, canada, sweden and the uk provided core/unrestricted funding support for this work. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. chikungunya virus outbreak hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description chikungunya outbreak ebola virus disease in west africa--the first 9 months of the epidemic and forward projections investigation of chikungunya fever outbreak in laguna bangladesh population and housing census 2011 added value of a 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epidemic of virus disease in southern province, tanganyika territory general description and epidemiology evaluating hospital-based surveillance for outbreak detection in bangladesh: analysis of healthcare utilization data gender differences in healthcare-seeking during common illnesses in a rural community of west bengal reconstruction of 60 years of chikungunya epidemiology in the philippines demonstrates episodic and focal transmission how social structures, space, and behaviors shape the spread of infectious diseases using chikungunya as a case study selection of important variables and determination of functional form for continuous predictors in multivariable model building seroprevalence and risk factors of chikungunya virus infection in mayotte the epidemic of zika virus-related microcephaly in brazil: detection, control, etiology, and future scenarios effect of ebola progression on transmission and control in liberia the authors have no competing interests to declare. j o u r n a l p r e -p r o o f key: cord-286477-0euaaspo authors: li, xiaochen; xu, shuyun; yu, muqing; wang, ke; tao, yu; zhou, ying; shi, jing; zhou, min; wu, bo; yang, zhenyu; zhang, cong; yue, junqing; zhang, zhiguo; renz, harald; liu, xiansheng; xie, jungang; xie, min; zhao, jianping title: risk factors for severity and mortality in adult covid-19 inpatients in wuhan date: 2020-04-12 journal: j allergy clin immunol doi: 10.1016/j.jaci.2020.04.006 sha: doc_id: 286477 cord_uid: 0euaaspo abstract background in december 2019, covid-19 outbreak occurred in wuhan. data on the clinical characteristics and outcomes of patients with severe covid-19 are limited. objective the severity on admission, complications, treatment, and outcomes of covid-19 patients were evaluated. methods patients with covid-19 admitted to tongji hospital from january 26, 2020 to february 5, 2020 were retrospectively enrolled and followed-up until march 3, 2020. potential risk factors for severe covid-19 were analyzed by a multivariable binary logistic model. cox proportional hazard regression model was used for survival analysis in severe patients. results we identified 269 (49.1%) of 548 patients as severe cases on admission. elder age, underlying hypertension, high cytokine levels (il-2r, il-6, il-10, and tnf-a), and high ldh level were significantly associated with severe covid-19 on admission. the prevalence of asthma in covid-19 patients was 0.9%, markedly lower than that in the adult population of wuhan. the estimated mortality was 1.1% in nonsevere patients and 32.5% in severe cases during the average 32 days of follow-up period. survival analysis revealed that male, elder age, leukocytosis, high ldh level, cardiac injury, hyperglycemia, and high-dose corticosteroid use were associated with death in patients with severe covid-19. conclusions patients with elder age, hypertension, and high ldh level need careful observation and early intervention to prevent the potential development of severe covid-19. severe male patients with heart injury, hyperglycemia, and high-dose corticosteroid use may have high risk of death. the sars-cov-2 virus, as a betacoronavirus, shares 88% of two bat-derived sars-like 133 coronaviruses and distances from sars-cov (around 79%) and middle east respiratory 134 syndrome coronavirus (mers-cov, around 50%). 1 sars and mers epidemics posed threats to 135 global health due to high mortality rates of 9.6% for sars-cov and 34.4% for mers-cov 136 globally. 2, 3 epidemiological data released by the chinese center for disease control and 137 prevention showed that 50,005 confirmed cases have been identified in wuhan and 31,513 in 138 mainland china except wuhan as of march 22, 2020. the mortality rate of covid-19 patients 139 was 5.0% in wuhan, which was close to that in the world (4.2%) and much higher than that in 140 mainland china except wuhan (2.4%). this study aims to describe and compare the 141 epidemiological, demographic, clinical, laboratory and radiological characteristics as well as the 142 complications, treatment and outcomes of hospitalized patients with nonsevere and severe 143 covid-19. potential risk factors for severe covid-19 and factors associated with death in severe 144 cases were analyzed to provide scientific data for relieve severe cases and reduce mortality. 145 this study was an ambispective cohort study of consecutive hospitalized patients with covid-19 159 enrolled at sino-french new city branch of tongji hospital, huazhong university of science and 160 technology in wuhan from january 26, 2020 to february 5, 2020. the final date of follow-up was 161 march 3, 2020. sino-french new city branch of tongji hospital is one of the major nationally 162 designated hospitals only providing medical care for adult covid-19 patients in wuhan. all 163 cases with covid-19 enrolled in this study were diagnosed based on the who interim guidance 4 164 and the diagnostic and treatment guideline for covid-19 issued by chinese national health 165 committee (version 5). detection of sars-cov-2 nucleic acids was shown in text in the online 166 repository. 5 this study was approved by institutional review board of tongji hospital, 167 huazhong university of science and technology. written informed consent was waived in light 168 of the urgent need to collect data. 169 the epidemiological and demographic data were obtained by face-to-face or telephone interview. 170 clinical symptoms, laboratory, and radiological findings on admission as well as the 171 complications, treatment and outcomes during hospitalization were extracted from electronic 172 medical records. serum cytokines (il-1β, il-2r, il-6, il-8, il-10 and tnf-α) were measured on 173 admission. patient data were cross-checked for consistency before final data entry and then 174 entered into a computerized database. 175 the presence of underlying comorbidities was identified based on the international classification 176 of diseases and injuries-10 diagnostic codes. the complications of covid-19 after admission 177 were assessed and the definitions were shown in text in the online repository. cardiac injury was 178 one of the complications, which was defined as a serum hypersensitive cardiac troponin i level 179 higher than 15.6 pg/ml without acute coronary symptoms or abnormal electrocardiogram. the 180 clinical outcomes were classified into discharge from hospital, in-hospitalization, and death. 181 severe covid-19 was defined according to 2019 clinical practice guideline from infectious 182 diseases society of america and american thoracic society for diagnosis and treatment of adults 183 with community-acquired pneumonia. 6 based on whether or not requiring ventilatory support on admission, severe cases upon admission were divided into two cohorts, severely ill and critically ill cases. 186 the descriptive statistics are median and interquartile range (iqr) for continuous data. a total of 549 patients with covid-19 were enrolled, of whom 548 cases were included in the 219 study. one case not meeting inclusion criteria was excluded due to inclusion criteria. almost half 220 of the patients (49.1%, 269 of 548) were identified as severe cases and 50.9% (279 of 548) were 221 nonsevere cases on admission. 68.7% (347 of 505) of cases were positive for sars-cov-2 222 nucleic acid test pre-admission. comparison of findings between nonsevere and severe cases in 223 the patients with positive viral nucleic acid test pre-admission showed essentially the similar 224 differences to that in the total patients (see table e1 in the online repository). 225 the epidemiological and demographic characteristics are shown in table 1 . 52 (9.5%) of 546 226 patients got the infection in hospital. 45 (8.2%) of 547 patients were health-care workers and 67 227 (12.2%) patients were family members of health-care workers. nonsevere cases had a higher 228 proportion of health-care workers and family members than severe cases (p<0.001). the date of 229 onset of the first reported case with covid-19 was december 1, 2019. 7 the median time from 230 december 1, 2019 to the onset of covid-19 was 54 days, ranging from 19 days to 63 days. 231 the median age of study population was 60 years (iqr 48-69), ranging from 18 years to 95 years, 232 of whom 210 (38.3%) were aged 65 years or older. the patients aged 65 years or older in severe 233 cases were almost twice as nonsevere cases of the same age (50.2% vs. 26.9%, p<0.001). slightly 234 more than half (50.9%) of all patients were male and the proportion of males in severe cases is 235 higher than in nonsevere cases (56.9% vs. 45.2%, p=0.006). 236 19.2% of patients with severe covid-19 were smokers (table 1) asthma (0.9%) were identified in total population. 42 (7.7%) of 545 patients regularly took 242 angiotensin-converting enzyme inhibitors or angiotensin ii receptor blockers; no significant 243 difference was found between nonsevere and severe cases. sore throat (5.1%), cough (75.5%), chest pain (7.5%), dyspnea (56.6%), chest tightness (38.1%), 246 dizziness (10.2%), confusion (3.1%), headache (11.3%), myalgia (20.3%), vomiting (8.2%), 247 diarrhea (32.7%), abdominal pain (2.9%). 6 patients were asymptomatic and diagnosed by ct 248 screening. duration of fever pre-admission was significantly longer among severe cases compared 249 with that in nonsevere cases (p=0.031). severe cases experienced longer duration from onset to 250 outpatient visit and longer duration from onset to hospitalization compared with nonsevere cases 251 (p=0.018 and p=0.035, respectively). 64 (11.9%) of 540 patients were treated with corticosteroids 252 delivered by oral or intravenous pre-admission. 304 (56.5%) of 538 patients had received at least 253 one of the following antiviral medications: umifenovir (32.9%), oseltamivir (35.1%), 254 lopinavir/ritonavir (2.4%), and ribavirin (1.5%). 255 ct scans for 461 patients were evaluated pre-admission, and showed multilobar pulmonary 257 infiltrates in 436 patients ( table 2 ). the median time from onset to pneumonia diagnosed by ct 258 scan was 4 days. on admission, oxygen saturation (spo2) less than 93.1% on room air presented 259 in 33.3% of all patients, of whom 163 (89%) were severe cases. 90.2% of all patients experienced 260 lymphopenia (<1500 cells per mm 3 ) and 29.1% of all patients had thrombocytopenia (<150,000 261 cells per mm 3 ). compared with nonsevere cases, inflammation-related marker levels (hscrp, esr, 262 and ferritin) were significantly higher in severe cases the levels of procalcitonin, globulin, lactate 263 dehydrogenase (ldh), nt-prob-type natriuretic peptide (nt-probnp), d-dimer, alanine 264 aminotransferase, aspartate aminotransferase, total bilirubin, conjugated bilirubin, blood urea 265 nitrogen, and creatinine were elevated in 9.5%, 40.4%, 73.6%, 27.5%, 67.4%, 23.1%, 33.1%, 266 4.4%, 9.2%, 15.8%, and 27.1% of all patients, respectively. serum cytokine levels of il-2r, il-6, 267 il-10, and tnf-α were significantly higher in severe patients than those in nonsevere patients (all of the 269 severe cases, 46 were classified as critically ill for requiring respiratory support. 271 compared with severely ill cases, the time from december 1, 2019 to onset was shorter and the 272 time from onset to outpatient visit was longer in critically ill cases (see table e2 in the online high procalcitonin, high nt-probnp, high ldh, high d-dimer, low albumin, and high creatinine) 276 were observed in critically ill cases compared with severely ill cases (all p<0.05). 34.1% of 277 critically ill patients received systemic corticosteroids pre-admission, the proportion of whom was 278 significantly higher than that in severely ill cases (12.2%, p<0.001). 279 compared with nonsevere cases, systemic corticosteroid use pre-admission was more common in 280 severe cases with larger cumulative dose and longer duration (p=0.007; p<0.001; p<0.001; 281 respectively). stratification of patients by corticosteroid exposure is presented in table e3 in the 282 online repository. severe patients treated with corticosteroids had higher ldh level compared 283 with severe patients without corticosteroid use pre-admission (p<0.05). 284 nonsevere cases were more likely to receive antiviral drugs pre-admission, including umifenovir 285 and oseltamivir (p<0.001 and p=0.005; respectively). in severe case subgroup, the patients 286 receiving umifenovir were younger than those without umifenovir use (p<0.05). a comparison of 287 baseline demographic and clinical characteristics between patients with and without antiviral drug 288 use revealed no marked difference in spo2 or laboratory findings in both nonsevere and severe 289 case subgroups (see table e4 and e5 in the online repository). 290 in the final logistic regression model, variables such as age 65 or older (or 2.2; 95% ci 1.5-3.5), 292 hypertension (or 2.0; 95% ci 1.3-3.2), ldh >445 u/l (or 4.4; 95% ci 2.6-7.6), and 293 d-dimer >1 mg/l (or 2.2; 95% ci 1.4-3.3) were significantly associated with severe cases with 294 covid-19 ( figure 1) . 295 in the follow-up period, the complications of covid-19 were assessed , including acute 297 respiratory distress syndrome (ards) (38.3%), cardiac injury (21.7%), liver dysfunction (19.3%), 298 acute kidney injury (17.3%), bacteremia (7.7%), diffuse intravascular coagulation (7.7%), and 299 hyperglycemia (33.2%) ( this study provided a comprehensive data on the epidemiological, demographic, clinical, 338 laboratory, and radiological characteristics as well as the complications, treatment, and outcomes 339 of hospitalized patients with nonsevere and severe covid-19 in wuhan. almost half of the 340 patients in this study were identified as severe cases, which may differ from the results of the 341 previous studies. 8 the proportion of patients aged 65 years or older were higher in our study than 342 in nanshan zhong's study (38.8% vs. 15.1%, respectively). 9 the time from december 1, 2019 to 343 the onset of most patients was longer than 50 days. during mid-january to early february, wuhan 344 experienced the highest peak of covid-19 outbreak, with a family cluster and high prevalence of 345 covid-19 in older adults. longer wait for access to medical care was observed in severe cases 346 compared with that in nonsevere cases. more than half of the patients experienced at least two 347 hospital visits, which may increase the risk of nosocomial transmission events. diagnosis and 348 treatment may be delayed due to the long wait for access to medical care. severe covid-19 349 patients would likely develop ards and died of respiratory failure. although there are currently 350 no effective antiviral drugs for sars-cov-2, prompt identification and early respiratory support 351 would relieve severe cases and reduce mortality. the severity of disease in patients with initial 352 positive nucleic acid test was similar to that of all covid-19 patients. we thus propose that 353 urgent timely diagnosis is crucial, and that early intervention should not be delayed based on 354 nucleic acid test. proinflammatory cytokine levels (inf-γ, il-1β, il-6, il-8, il-12, and tnf-α) and were 362 associated with the development of ards. 11-13 in our study, severe covid-19 patients had (0.9%) in our study was markedly lower than that reported in the adult population of wuhan 366 (6.4%). 1416 we thus speculate that th2 immune response in asthmatic patients may counter the 367 inflammation process induced by sars-cov-2 infection. further studies are required to 368 characterize the immune response and inflammation features of the majority of severe patients showed rapid progression and multiple organ dysfunction. the to onset in critically ill cases than that in severely ill cases may reflect a higher virulence of 378 sars-cov-2, or earlier onset of the risk factors for severity identified in this study included age, high ldh level, and high 380 d-dimer level, consistent with those in previous reports. 15, 20 however, different from the findings 381 of previous studies 21 , hypertension was the only comorbidity associated with the severity of 382 covid-19 after adjustment for age, sex and smoking status. the distinct features of pneumonia 383 and high severity in patients with covid-19 in this study may lead to this difference from 384 previous reports. ace2, a gateway to sars, was reported to be a protective factor against 385 sars-cov-induced lung injury. 22, 23 the association between ace2 expression and hypertension 386 was confirmed in a previous study. 24 this fact may partly explain the high prevalence of severe 387 covid-19 in patients with hypertension. ldh has been recognized as a marker for severe 388 prognosis in various diseases, including cancer and infection. 25 the high ldh level in covid-19 389 in severe cases suggested that ldh may be associated with lung injury and tissue damage, 390 warranting an investigation for the potential mechanism. 391 this study evaluated pre-admission medications for patients with severe covid-19. although the 392 proportion of nonsevere cases in patients receiving oseltamivir was higher than that in patients 393 without oseltamivir use, stratification analysis showed that there was no significant difference in case subgroup. therefore, oseltamivir use may just be an indicator of disease severity. the 396 patients receiving umifenovir were younger than those without umifenovir use, indicating that 397 younger patients may have easier access to drugs or prefer umifenovir. 398 older age, leukocytosis and high ldh level were reported to be risk factors associated with 399 in-hospital death in previous studies. 20, 26, 27 the present study also revealed that hyperglycemia 400 was related with increased mortality in covid-19 patients. the prevalence of hyperglycemia may 401 be associated with underlying diabetes and corticosteroid therapy. however, the localization of 402 ace2 expression in the pancreas in sars was reported to damage islets, resulting in 403 hyperglycemia 19 ; this finding suggested that hyperglycemia may also be an indicator of severe 404 this study indicated that corticosteroid use was more common in severe cases than in nonsevere 406 cases and that high-dose corticosteroid use was related to high risk of death in severe covid-19 407 patients. high-dose steroid use may be an indicator of disease severity rather than a predisposing 408 factor. in a previous study, treatment with methylprednisolone was shown to be beneficial for 409 covid-19 patients who developed ards. 20 however, critically ill cases had more signs of 410 infection and abnormal laboratory findings, including high leukocyte, high procalcitonin, high 411 d-dimer, low albumin, and high creatinine levels. high-dose corticosteroid use should be used 412 with cautious in critically ill patients to avoid aggravating complications. 413 a recent study by bin cao et al showed that lopinavir/ritonavir treatment offered no significant 414 benefit over the standard care for hospitalized adult covid-19 patients. 28 cao's study also 415 reported that lopinavir/ritonavir led to a shorter median time to clinical improvement than the 416 standard care (hr 1.39; 95% ci 1.00 to 1.91) in a modified intention-to-treat analysis. compared 417 with cao's study, the severity of patients was more serious and lopinavir/ritonavir treatment was 418 associated with a lower risk of death in severe covid-19 patients in this study. however, our 419 study was an observational study; thus, the benefit of lopinavir/ritonavir for severe covid-19 420 patients needs to be further confirmed. 421 there were limitations to the current study. firstly, epidemiological data were collected 422 respectively and recall bias might have occurred. secondly, missing data on some variables, such 423 as detailed information of ct scan, may cause bias in the estimation and reduce the representativeness of the samples. thirdly, laboratory findings were measured upon admission and may indicate the severity of covid-19. the causal relationship between abnormal laboratory 426 findings and severity could not be determined. fourthly, this study was an observational study 427 with limitations in terms of evaluating the efficacy of corticosteroids and antiviral drugs. finally, 428 the absence of comparative data from covid-19 patients not admitted or from other critically ill 429 patients were limitations of this study. 430 in conclusion, covid-19 outbreak caused widespread concern and has threatened the global clinical characteristics 523 of 140 patients infected with sars-cov-2 in wuhan an increased 525 prevalence of self-reported allergic rhinitis in major chinese cities from ace2 528 links amino acid malnutrition to microbial ecology and intestinal inflammation cholangiocytes may cause liver damage after 2019-ncov infection binding of sars coronavirus to its receptor damages 534 islets and causes acute diabetes risk factors associated with acute 536 respiratory distress syndrome and death in patients with coronavirus disease comorbidity and its 539 impact on 1590 patients with covid-19 in china: a nationwide analysis where n is the total number of patients with 638 available data. p values comparing nonsevere cases and severe cases are from χ² test, fisher's exact test, 639 or mann-whitney u test cov-2 nucleic acid test was performed pre-admission table 3. complications and treatment during hospitalization and clinical outcomes of covid-19 data are expressed as median (iqr), n (%), or n/n (%), where n is the total number of patients with 676 available data. p values comparing nonsevere cases and severe cases are from χ² test, fisher's exact test ards=acute respiratory distress syndrome key: cord-282125-3i2jhvwn authors: maitra, s.; biswas, m.; bhattacharjee, s. title: casefatality rate in covid19 patients: a meta-analysis of publicly accessible database date: 2020-04-14 journal: nan doi: 10.1101/2020.04.09.20059683 sha: doc_id: 282125 cord_uid: 3i2jhvwn a novel coronavirus was reported in wuhan, china in december 2019 to cause severe acute respiratory symptoms (covid19). in this meta-analysis, we estimated case fatality rate from covid19 infection by random effect meta-analysis model with country level data. publicly accessible web database worldometer (https://www.worldometers.info/coronavirus/) was accessed on 24th march 2020 gmt and reported total number of cases, total death, active cases and seriously ill/ critically ill patients were retrieved. primary outcome of this meta-analysis was case fatality rate defined by total number of deaths divided by total number of diagnosed cases. pooled case fatality rate (95% ci) was 1.78 (1.342.22) %. between country heterogeneity was 0.018 (p<0.0001). pooled estimate of composite poor outcome (95% ci) was 4.06 (3.244.88) % at that point of time after exclusion of countries reported small number of cases. pooled mortality rate (95% ci) was 33.97 (27.4440.49) % amongst closed cases (where patients have recovered or died) with. meta regression analysis identified statistically significant association between health expenditure and mortality amongst closed cases (p=0.037). a novel coronavirus was reported in wuhan, china in december 2019 to cause severe acute respiratory symptoms (covid-19) [1] . subsequently, this viral outbreak was reported in 197 countries and one international ship on 24 th march 2020. a wide range of mortality rate was reported from this viral illness and world health organization reported mortality rate of 3.4% on 3 rd march 2020 [2] . in this meta-analysis, we estimated 'case fatality rate' from covid-19 infection by random effect meta-analysis model with country level data. publicly accessible web database 'worldometer' (https://www.worldometers.info/coronavirus/) was accessed on 24 th march 2020 gmt and reported total number of cases, total death, active cases and seriously ill/ critically ill patients were retrieved. primary outcome of this meta-analysis was 'case fatality rate' defined by total number of deaths divided by total number of diagnosed cases. secondary outcomes were mortality rate amongst closed cases (where patients recovered or died) and proportion of composite poor outcome (defined by the number of patients died or critically ill). metaregression analysis was performed to identify association between population density, health expenditure (percentage of gdp) and percentages of patients over 65y age with mortality. between country heterogeneity (τ) was estimated by restricted maximum likelihood method and 95% prediction interval of pooled mortality were reported [3] . countries reporting small number of cases (<100 on 24 th march 2020) were excluded from analysis; however, sensitivity analysis was planned with including them. association between mortality and clinical characteristics of the patients were assessed by mixed effect meta-regression model. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 14, 2020. between country heterogeneity (τ) was 0.018 (p<0.0001). sensitivity analysis was performed by exclusion of one country at times and pooled estimates of crude fatality rate remained mostly similar. another sensitivity analysis was performed including countries where reported total number of cases were less than 100, and estimated case fatality rate (95% ci) was 1.77% (1.44-2.14) % with i 2 = 97.75%. significant amount of publication bias was identified by regression test both with (p<0.0001) and without (p=0.0017) including countries will small number of cases. pooled estimate of composite poor outcome (95% ci) was 4.06 we have found a pooled mortality rate of 1.77%, which was less than who reported death rate of 3.4% but similar to the first published report of mortality from wuhan, china. [1] our pooled estimate of composite poor outcome was around 4%, which was similar to the previous report from china. another important finding is that presence of a significant heterogeneity and publication biases in the pooled analysis. underreporting from different . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 14, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 14, 2020. proportion . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 14, 2020. clinical characteristics of coronavirus disease 2019 in china who director-general's opening remarks at the media briefing on covid-19 -3 march 2020 -world health organization cochrane handbook for systematic reviews of interventions version key: cord-262646-64ldtrjf authors: chuang, pei-hung; chuang, jen-hsiang; lin, i-feng title: a dynamic estimation of the daily cumulative cases during infectious disease surveillance: application to dengue fever date: 2010-05-27 journal: bmc infect dis doi: 10.1186/1471-2334-10-136 sha: doc_id: 262646 cord_uid: 64ldtrjf background: in infectious disease surveillance, when the laboratory confirmation of the cases is time-consuming, there is often a time lag between the number of suspect cases and the number of confirmed cases. this study proposes a dynamic statistical model to estimate the daily number of new cases and the daily cumulative number of infected cases, which was then applied to historic dengue fever data. methods: the duration between the date of disease onset and date of laboratory confirmation was assumed to follow a gamma distribution or a nonparametric distribution. a conditional probability of a case being a real case among the unconfirmed cases on a given date was then calculated. this probability along with the observed confirmed cases was integrated to estimate the daily number of new cases and the cumulative number of infected cases. results: the distribution of the onset-to-confirmation time for the positive cases was different from that of the negative cases. the daily new cases and cumulative epidemic curves estimated by the proposed method have a lower absolute relative bias than the values estimated solely based on the available daily-confirmed cases. conclusion: the proposed method provides a more accurate real-time estimation of the daily new cases and daily cumulative number of infected cases. the model makes use of the most recent "moving window" of information relative to suspect cases and dynamically updates the parameters. the proposed method will be useful for the real-time evaluation of a disease outbreak when case classification requires a time-consuming laboratory process to identify a confirmed case. timeliness and accuracy of case reporting are two of the most important performance measures when evaluating an infectious disease surveillance system [1] [2] [3] [4] [5] . laboratory confirmation is usually needed for case diagnosis in many infectious diseases. when laboratory confirmation of the diagnosis is time-consuming, however, there is often a time-lag between the onset date of symptoms and the diagnosis date [6] . for example, the median time for confirmation of the meningococcal disease is about 13 days based on the national notifiable diseases surveillance system (nndss) dataset for the united states from 1999 to 2001 [7] . time from disease onset to diagnosis has been also reported to account for most of the delay in case reporting in korea [8] . correct estimation of daily cases or daily-cumulative infected cases helps the implement of immediate disease control and allows prevention activities for infectious diseases to be instituted [6] . using a disease surveillance system, one is able to apply statistical methods, such as cumulative sum (cusum) [9, 10] or autoregressive integrated moving average (arima) [11] [12] [13] [14] [15] , in order to forecast an epidemic curve or to detect aberrations in disease spread. these estimations are based on having a correct daily number of cases or a daily-cumulative number of cases. an epidemic of dengue fever occurs every year in southern taiwan. in the year 2002 in particular, there was a large-scale epidemic with 5,388 confirmed cases out of totally 15,221 suspect cases [16] . this epidemic continued until march 2003. surveillance and the control of dengue fever have become one of the most important routine areas of work at the taiwan centers for disease control (taiwan cdc) in recent years. by 2008, taiwan cdc had defined a confirmed case of dengue fever as an acute febrile illness together with one of the following criteria: (1) isolation of dengue virus; (2) demonstration of positive results by real-time reverse transcription--polymerase chain reaction (real-time rt-pcr); (3) demonstration of positive seroconversion or a fourfold increase in dengue-specific igm or igg antibody titers in paired serum samples; or (4) demonstration of high-titer dengue-specific igm and igg antibodies in a single serum specimen [17] [18] [19] . when the dengue fever case classification only included confirmed cases by this protocol, the time needed for isolating the agent or measuring the titers for the acute-and convalescent-phase serum specimens was significant. the result was that there was a gap between the available daily cases or the daily-cumulative cases for given a day and the actual final confirmed cases for the same day given that all diagnosis had been completed on that given day. assuming that a time cost for laboratory confirmation of diagnosis is sometimes inevitable, daily numbers of infected cases and daily-cumulative number of infected cases may be underestimated during an epidemic. the objective of this study was to develop a new method to estimate the number of daily cumulative cases and that this method will be applied to dengue fever in taiwan, as an example. since there are almost no dengue fever cases occurred during the winter in taiwan, we chose may 1 as the beginning of the dengue epidemic season when estimating the cumulative epidemic curve. the data come from the dengue notification dataset containing suspect cases in taiwan whose date of onset was from may 1, 2005 to april 30, 2007. all serum samples from suspect cases were sent to the two reference laboratories at the taiwan cdc in order to further identify if they were positive (dengue fever infected) or negative cases. the reason we retrieved data based on the date of onset rather than the report date was to avoid the influence of lag reporting on the course of the disease. all imported cases of the disease were removed. the variables we used were the date of onset, the date of laboratory confirmation (diagnosis date), and the final confirmed status of each suspect case (a binary variable that is either positive or negative). in this article, we use confirmed dengue cases and positive cases interchangeably. no personal identification information was contained in the dataset. our proposed method estimates the real-time daily new number of cases and the daily cumulative number of dengue cases; specifically, these numbers of dengue cases are updated daily. let c be the "current" date when the number of dengue cases is to be estimated. in this study, the date c runs from may 1, 2006 to april 30, 2007. for the i th reported suspect case counting from the 1 st day of the epidemic season, that is may 1 in this study, we define the suspect case's onset date as o i and the laboratory confirmation date as d i if d i >c on date c, the case i does not have a confirmation result as of date c; on the other hand, if d i ≤ c, this case i is either confirmed to be a positive dengue case or has a negative result as of date c. let the final confirmation status for the i th suspect case be, where as a positive dengue case, and as a negative case. in the situation where there are unconfirmed suspect cases as of date c, we assigned a probability of being a dengue case, p(i), to those unconfirmed cases (d ≤ c). then for each suspect case i, the expected final confirmation status on date c, e i (c), can be written as the values of p i (c), and e i (c)are updated for each case i every day. without applying the proposed method, one would be only able to observe the case status from the upper part of e i (c)in equation (1) . after e i (c)is calculated for each suspect case, daily new cases are easily estimated by summing the e i (c)over all new suspect cases on date c, and cumulative cases can be obtained by summing e i (c)over all cases from i = 1 to the newest suspect cases on date c. is estimated for unconfirmed cases using information from the confirmed cases before date c within one year. let t i be the onset-to-confirmation time (oc-time), the time interval between the onset date and the lab-confirmation date. the oc-time for the i th suspect case as of date c, t i (c), is calculated as follows, the t i (c) is the oc-time for confirmed cases and the censored oc-time for unconfirmed cases on date c. by applying several steps of bayes' rules, the probability p i (c) is given by: to estimate p i (c) using the information as of date c, we applied the following steps. we first estimated p(y i = 1) by calculating the proportion of confirmed positive dengue cases out of the suspect cases using the data with onset date within 1 year before the date c. based on a parametric approach, we assumed the oc-time for a given case status, p(t|y i ), follow a gamma distribution. gamma distributions are frequently used to fit time-delay distributions or time event distributions when carrying out disease surveillance analysis [20, 21] . the probability density function of the gamma distribution is , where . the gamma distribution is denoted by with two parameters, the shape parameter α and the scale parameter β, and the mean and variance are αβ and αβ 2 , respectively. the values of α and β were estimated and solved by setting up the sample mean and the sample variance of the oc-time equal to αβ and αβ 2 , respectively. as mentioned in the previous section, the mean and standard deviation of the oc-time were different between positive and negative cases, we estimated different sets of α and β for the positive dengue cases (y = 1) and negative cases (y = 0) separately. a nonparametric approach was also performed in which the probability p(t >t i (c)|y i ) was simply replaced with the cumulative proportion of confirmed data given their final status. both the parametric and nonparametric models were based on the data within a 1-year "moving window" before date c. the p i (c) and e i (c) were also updated everyday. to evaluate the performance of the proposed method, we estimated the daily new cases and daily cumulative cases for each calendar date c from may 1, 2006 to april 30, 2007. four epidemic curves are presented. there are: (1) the final status curve, which is the number of dengue cases based on their final confirmation status ("real data", "gold standard"). (2) the daily confirmed curve, which is the number of dengue cases based on the confirmed cases as of date c. (3) the gamma-model curve, which is the number of dengue cases, estimated using the gamma distribution. (4) the nonparametric-model curve, which is the number of dengue cases, estimated using the nonparametric distribution. to summarize the magnitude of the bias, we defined the absolute relative bias (arb) at date c as: where are the cumulative cases estimated by the proposed methods or by the confirmed cases observed on date c without using the proposed methods, n c and are the cumulative confirmed cases based on the final status ("real data", "gold standard"). an arb closer to zero is a more accurate estimate. all analyses were performed using sas 9.1.3 software (sas institute, inc., cary, nc). special sas macros for estimating the cumulative cases and daily new cases, based on our proposed model, were developed. figure 2 compares the daily new cases estimated by the proposed models, the confirmed curve (confirmed new cases observed on date c without using the proposed models), and the final status curve (confirmed new cases based on final status; the "gold standard"). since the daily new cases estimated by the proposed methods or the daily new cases observed on date c were different when viewed on different dates, arbitrary view dates of august 1, 2006 (beginning of the epidemic), september 1, 2006 (rising stage, before the peak), october 1, 2006 (rising stage, before but closer the peak) and november 1, 2006 (around the peak), december 1, 2006 (downward stage, after the peak), and january 1, 2007 (end of the epidemic) were chosen to illustrate the results of the estimated daily new cases. each graph in figure 2 shows the epidemic curves three weeks before the view dates. when viewed on august 1, most of suspect cases had been lab-confirmed before july 15 thus all four curves were close to each others before that date. from july 19 to august 1, the estimated curves by the proposed methods (red dashed lines with triangle symbols by gamma distribution and blue dashed lines with cross symbols by the nonparametric method) were much closer to the final status curve (shaded area) than that by simply observing the dailyconfirmed new cases (purple dashed line). similar patterns were observed when the results are viewed on september 1, october 1, november 1, and december 1. the observed daily-confirmed cases usually underestimated the true daily new cases as would be expected, especially within the two weeks before the view date. the curves estimated by gamma distribution or the nonparametric approach were quite similar. however, the daily new cases, as estimated by the proposed method, did not give an accurate estimate towards the end stage of the epidemic, namely when viewed on january 1, 2007. the epidemic curves in terms of daily cumulative cases are shown on figure 3 . in this figure, the cumulative number of positive cases was updated every day. the two estimated daily-cumulative curves by the proposed methods are quite similar to the final status curve before january but again the proposed method does not work well during the end stage of the epidemic. table 1 compares the arb of the daily cumulative number of positive cases between the different methods. after the first confirmed positive case appeared on july 6, 2006, the estimates based on the gamma model results in an estimate closer to the real data than the other methods. for other two curves, the nonparametric method performs worst at the end of the epidemic after january 1 and there was about 20 cases higher than the final status curve. the daily confirmed curve was about 50 cases lower than the final status curve during the peak of epidemic. figure 4 showed the daily parameter estimates, α and β, of the gamma distributions used to dynamically calculate the daily number of positive cases. the parameter estimates varied from day to day and thus the probability of being a positive case changes. for the negative cases, the parameters had a jump during late september. as noted previously, timeliness and accuracy are the two of most important characteristics when we evaluate an infectious disease surveillance system. our results show that when an infectious disease required a time-consuming process for diagnosis, such as the dengue fever using the previously mentioned protocol, the actual daily number of infected cases and cumulative positive cases are potentially underestimated. the proposed method dynamically updates the parameters daily by making use of the most recently available information on suspect cases, and then performed estimates with a lower absolute relative bias than when using observed daily lab-confirmed cases only. as shown in table 1 , the proposed method performed a lower median absolute relative bias (abs range 1.7% ~ 8.5%) than those solely based on daily confirmed cases (abs range 4.7% ~ 67.7%) between july 6 and december 31. these dates covered the rising stage and around the peak stage which were of public health interest. the proposed method provides a more accurate estimate of the epidemic curves when applied to the dengue fever dataset for taiwan during the 2006-2007 season. based on these results, this approach can be used for the real-time evaluation of the severity of a disease outbreak when case classification requires that a confirmed case involves a time-consuming process. in this study, we first established the different distributions for the onset-to-confirmation time of the positive cases and negative cases. next, either a gamma distribution was assumed in order to estimate the probability of being a confirmed case given cases status in equation (1), or, alternatively, a nonparametric approach was used. we actually experimented with several types of distribution. the estimates using a log-normal distribution were numerically very similar to the results for the gamma distribution. the estimates using a weibull distribution did not perform as well as the gamma distribution applied in our dengue fever data. from figure 4 , we learn that the shape parameter α changed from 0.5 to 2 and therefore an exponential distribution may not be appropriate. for simplification, we have chosen to present only the results from the gamma distribution as one example of a parametric approach and compare this with a nonparametric approach. as shown in figure 2 for daily new cases, the differences in the estimates based on parametric approach with gamma distribution and those with nonparametric approaches were minor. the figure 3 and table 1 for cumulative cases showed that a gamma distribution is a more appropriate assumption for the onset-todiagnosis time when estimating the probability of being a positive case using the dengue fever example; nonetheless, the difference between the gamma and the nonparametric method is again only slight except towards the end stage of the epidemic after january 1. the reason that the nonparametric method did not work well after january 1 is because p(y i = 1), p(y i = 0), and p(t|y i ) had not changed substantially, resulting in a near constant estimate of the daily positive cases. in practice, any form of the probability of being a positive case can be assumed. it is also not restricted to certain distributions when the models are adapted to different types of infectious disease. when applying this approach to other diseases, researchers should investigate several distributions according to the shape of their data and choose an appropriate one based on some appropriate measures, for instance, those shown in table 1 . our method estimated the probability of being a positive case based on the data within a 1-year "moving window" before date c and updated p i (c) and e i (c) everyday. the epidemic profiles of dengue fever are different from one year to another in taiwan. choosing the data from most recent one year was done in order to insure that there was enough information to cover a whole epidemic season. the study shows that before the first positive case appeared on july 6, the proposed method did not work well and are not that useful (table 1 ). our method worked well after the first positive case appeared during the 2006-2007 season. indeed, it needed only four days to be able to consistently estimate the final status curve. in the 2006-2007 season, taiwan cdc activated a central command center for intensively dengue epidemic control on october 2. the task of this command center included expanded blood sample collection and it is likely that this resulted in more suspect cases for laboratory confirmation, which might have led to a lower proportion of positive cases. this would influence the estimation of probability of being a positive case over the following few days. as we can see on figure 4 , it also influenced the estimation of the parameters for negative cases. while our manuscript was being prepared, the taiwan cdc changed their laboratory protocol for dengue fever to one that requires only a single laboratory test for dengue surveillance and control. the result is a substantial reduction in the waiting time for laboratory confirmation. however, confirmation time can never be completely avoided with dengue fever. a situation where a large number of serum specimens are sent for diagnosis at the same time will result in overloading at the laboratory, which might increase the confirmation waiting time. as described previously, the estimation used information based on a "moving window" time period before the estimated date and the parameters of the model are updated everyday. since the observed confirmed cases counts on date c are always underestimated as long as there is a time lag, our method potentially can be applied while waiting for further investigation of the status of cases. there are some limitations to our method. firstly, the approach needs sufficient historical data to be available in order estimate the parameters of the model; therefore our model cannot be applied effectively to an emerging disease, such as sars or avian flu. secondly, we used confirmed cases, the dates of onset of which were within 1year before the date estimated and if a case needs more than 1-year for diagnosis such a case might never provide any information to the parameter estimation; in such a circumstance a different "moving window" needs to be chosen. thirdly, when missing diagnosis dates exist, the estimated curve using the nonparametric method cannot converge with the final status curve. there were 134 and 289 cases missing confirmation results for the 2005-2006 season and the 2006-2007 season, respectively, at the time that the manuscript was prepared. the nonparametric method estimates by plugging in the cumulative proportion of confirmed data given the final status. as we mention before, at the end stage of epidemic, the probabilities in equation (2) almost remained unchanged. in this study, we could only assume that the proportion of positive cases out of all suspect cases among the missing observations were similar to those having results, which basically assumes that the missing data were missing at random thus ignorable. when diagnosis of infectious diseases required laboratory confirmation, the time lag between onset and confirmation of a positive cases often exists and case counts are usually underestimated. this study has proposed a statistical method that more accurately estimates the real-time daily new cases and daily cumulative number of infected cases using a dengue fever epidemic as an example. the model makes use of the most recent "moving window" of information on suspect cases and dynamically updated the parameters of the assumed probability distributions. different parametric or nonparametric distributions of the onset-to-confirmation times can be specified for different infectious diseases. the results show that, after the first confirmed case occurred, the estimated daily new cases or the cumulative case count fit the real data well compared to the daily counts based only on the available confirmed cases; this was done by assuming a gamma distribution for the onset to confirmation times and involved the use of a dynamic one-year "moving window" of suspected cases when applied to dengue fever outbreaks in taiwan. this method can be used for the realtime evaluation of a disease outbreak when case diagnosis requires time-consuming laboratory process. how complete and accurate is meningococcal disease notification? public health surveillance of aids and hiv infections updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group cdc's national violent death reporting system: background and methodology an outbreak of leptospirosis, thailand--the importance of the laboratory evaluation of reporting timeliness of public health surveillance systems for infectious diseases syndromic surveillance systems: public health and biodefense timeliness of national notifiable diseases surveillance system in korea: a crosssectional study methods for monitoring influenza surveillance data the relative efficiency of the sets and the cusum techniques in monitoring the occurrence of a rare event time series analysis forecasting and control on the application of integer-valued time series models for the analysis of disease incidence use of time-series analysis in infectious disease surveillance dynamic linear model and sarima: a comparison of their forecasting performance in epidemiology a monitoring system for detecting aberrations in public health surveillance reports number of reported and confirmed cases -by month world health organization: dengue haemorrhagic fever: diagnosis, treatment and control laboratory-based dengue surveillance in taiwan, 2005: a molecular epidemiologic study current status of dengue diagnosis at the center for disease control epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong sars incubation and quarantine times: when is an exposed individual known to be disease free? this study was supported in part by the centers for disease control, department of health, taiwan, republic of china (doh94-dc-2036 and doh96-dc-2038) and was also supported by the aim for the top university plan of national yang-ming university. the authors would like to thank the reviewers' excellent comments for improving this manuscript. the authors declare that they have no competing interests. authors' contributions ifl designed and conducted the study, and finalized the manuscript. phc participated in the design of the study, performed the statistical analyses, and drafted the manuscript. jhc helped conceive the study, participated in the data collection, gave input to the manuscript, and provided medical advice from the public health perspective. all authors have read and approved the final manuscript. key: cord-290567-hyod58n2 authors: laxmipriya, s.; narayanan, r.m. title: covid-19 and its relationship to particulate matter pollution – case study from part of greater chennai, india date: 2020-10-09 journal: mater today proc doi: 10.1016/j.matpr.2020.09.768 sha: doc_id: 290567 cord_uid: hyod58n2 it is well known that atmospheric contamination, especially the particulate matter (pm), causes severe human diseases. yet, presently air pollution levels have dropped primarily attributable across the nation lockdown forced in the wake of the novel coronavirus outbreak. in this study, we have attempted to establish a conceivable relationship between covid 19 and pm10–2.5, obtained from eleven airquality monitoring stations in chennai city, india for both pre and during covid situations and its influence over covid positive cases. the observations of the materials (+ve cases, pm 10, pm 2.5) collected proved that during precovid regime less polluted areas are indicated with less than 5 infection cases reflecting the healthy people and they are less vulnerable to covid except the few occurrence of foreign source indicating no community spread whereus most polluted spots of precovid regimes are indicated with more than 90 % cases and indicated that people in pollution zones are succumbed to get infected quickly. however, during covid the lockdown has considerably reduced the particulate suspension and the results revealed that the +ve cases are of the nature of community spreading through primary and secondary contacts as reported from the media. if covid is a visible, brutally virulent, incredibly contagious pandemic that kills rapidly and mercilessly, air pollution is its unseen evil twin. under the radar, but even ruthlessly, if covid and pm paired together lead to murder without delay. this is a non-communicable disease (ncd) slow-motion pandemic, equivalent-if not exceeding-the catastrophic wrath of sars-cov-2. air pollution has emerged as an increasing cause of concern worldwide, especially in developing nations such as india [1] . india saw financial development, a quick extension of urban areas, industrialization, and quick-paced improvement of the foundation since progression during the 1990s. at the same time, the degree of air pollution in india has expanded to a significant wellbeing danger and reason for massive untimely mortality [2] . around one million individuals kicked the bucket in 2015 because of the encompassing particulate issue (pm) contamination alone in india [3] . indian urban communities have been continually making into the best 20 most contaminated urban areas of the world for as far back as hardly any years and surpassing the surrounding air quality gauges suggested by the world health organization and central pollution control board (cpcb). air pollution can exacerbate a broad scope of respiratory ailments, and coronavirus seems, by all accounts, to be no particular case. in the first four months, the outbreak of coronavirus disease 2019 (covid 19), initially identified in china's wuhan, has resulted in more than one million cases worldwide.the pandemic situation has brought about lockdown in numerous countries around the world. though india's first confirmed case was on january 30 th 2020, the cases were multipled to 10,12,442 as on today (18 th july 2020). from march 16 th , the country was cut off from all places of mass gatherings such as schools, shopping malls, and theatres through the lockdown. further, the lockdown imposes limitations and measures of self-isolation which diminish emanations from transport and businesses. the researchers, based at several universities across italy, propose the virus could be scattered all the more generally on air pollution particles [4] . harvard researchers suggest their results are especially important for vulnerable minority groups, which will be slowly exposed to air pollution in general, leading to the likelihood of coronavirus being genuinely sick. for every small increment in air pollution, there's a generous increase in death [5] . based on epha study comprising people's ability to fight off infection resulted with weakening of immune system by airpolloutants, [6] . one recent study found that even little increments in fine particulate matter of only 1 microgram for every cubic meter related to a 15% expansion in covid-19 passings [7] . further the relations between pm and communicable diseases were also recorded in more recent studies revealed inhalation may transmit pm concentrated into the lungs, and particle-attached viruses may directly conquer the inferior respiratory tract, thus raising infection induction [8] . if this is true that novel coronavirus remains active on various surfaces from a few hours to several days [9] , it is reasonable to conclude the same might appear when adsorbed or absorbed by the particulate matter in the atmosphere, which can also possibly bring the viruses into the respiratory epithelium a spatial distribution for pre-covid pm2.5 and positive corona cases between the 11 stations is geographically heterogeneous, and it is presented in figure 2 . and/or contact with contaminated surfaces [12] , the veracity of covid-19 occurrence (table 1. ) observed in most of the highly polluted areas withcompared to that of low, also suggested to consider possible route of transmission by airborne route. the pre covid particilate matter pollution zones revealed that less polluted areas are indicated with < 3 +ve cases with < 3.5 % of distribution and might be resulted from foreign source and indicating that there is no community spread however while most polluted areas has more than 13 +ve cases and max of 26 +ve cases with 90 percent distribution and started carried away through atmospheric medium by means of particulate suspension. the lockdown brought numerous anthropogenic (human-made) supporters of a halt. chennai, one of the most critically polluted cities, also reported a significant reduction in air pollution. the contamination levels of pm2.5 during covid was found to be 3 µg/m 3 larger particles (> 5 μm) appear to settle on the upper respiratory tract surfaces of nose breathers, and occur with deposition on the upper respiratory tract. exposure to pm10 is associated with increased bronchiolitis in infants with respiratory syncytial virus hospitalisations [13] . the pre covid particulate matter pollution zones revealed that less polluted areas are indicated with < 2 +ve cases with < 3 % of distribution and might be resulted from foreign source and indicating that there is no community spread however while most polluted areas has more than 9 +ve cases to the maximum of 38 +ve cases with 89.77 percent distribution and might be resulted from transportation of particulate suspension through atmospheric medium . the contamination levels of pm10 has significantly decreased during the lockdown period. there was about 80 to 90 % reduction rate. the variation of covid positive cases also showed significant change. from figure 2 .(d) it was observed that areas under the range (5 to 40 µg/m 3 ) has more positive cases( 98 percentage ) than other areas. during covid particulate matter pollution zones revealed that less polluted areas are indicated with < 87 +ve cases with < 98 % of distribution and might be resulted from community spread or pimary contacts however while most polluted areas has only 1 +ve case with < 2 percent distribution and has no significant role in spreading due to atmospheric medium by means of particulate suspension. the air pollution might be clearing in chennai due to lockdown; however, the harm has just been done to human wellbeing and individuals' capacity to ward off disease. governments ought to have handled ceaseless air contamination quite a while in the past. however, it has organized the economy over wellbeing. science articulates epidemics like covid-19 will occur with increasing rate of recurrence [14] . incredibly contagious pandemic that kills rapidly and mercilessly, air pollution is its unseen evil twin. under the radar, but even ruthlessly, if covid and pm paired together lead to murder without delay. this is a non-communicable disease (ncd) slow-motion pandemic, equivalent-if not exceeding-the catastrophic wrath of sars-cov-2.the hypothesis could exploit ways to connect atmospheric particulates is a challenging point which we believe deserves more, immediate and in-depth experimental work. it is to be expected that steps to clarify the dynamics involved in the current pandemic will be taken promptly. esri india ltd in preparation of gis maps. we also thank the er a.c.s. arunkumar president, dr m.g.r educational and research institute for providing the necessary facilities and for their constant motivations to carry air pollution monitoring network using low-cost sensors, a case study in hanoi the relationship between indoor particulate matter and home ventilation field calibration of low-cost air pollution sensors particulate matter air-pollution a familial cluster of covid-19 indicating virus can be transmitted by asymptomatic carriers familial cluster of covid-19 infection from an asymptomatic beijing smog chinese bicycl. body power chernobyl 25 years later mex. coral reefs battling deforestation honduras outbreak desertif. chile india's e-waste ecotourism phil novel coronavirus: how atmospheric particulate affects our environment and health covid-19 asymptomatic infection estimation impact of urban expansion on the air pollution landscape: a case study of hanoi recent increase of surface particulate matter concentrations in the seoul metropolitan area q&a: the novel coronavirus outbreak causing covid-19 a novel coronavirus (covid-19) outbreak covid-19 -a timeline of the coronavirus outbreak the authors are thankful to those who helped in placing the airsampler in the prescribed hotspots for obtaining the results during field observations and the technical support rendered by the observations of the materials (+ve cases, pm 10, pm 2.5) collected proved that during precovid regime less polluted areas are indicated with less than 5 infection cases reflecting the healthy people and they are less vulnerable to covid except the few occurrence of foreign source indicating no community spread whereus most polluted spots of precovid regimes are indicated with more than 90 % cases and indicated that people in pollution zones are succumbed to get infected quickly. however, during covid the lockdown has considerably reduced the particulate suspension and the results revealed that the +ve cases are of the nature of community spreading through primary and secondary contacts as reported from the media. if covid is a visible, brutally virulent, incredibly contagious pandemic that kills rapidly and mercilessly, air pollution is its unseen evil twin. under the radar, but even ruthlessly, if covid and pm paired together lead to murder without delay. this is a non-communicable disease (ncd) slow-motion pandemic, equivalent-if not exceeding-the catastrophic wrath of sars-cov-2. the hypothesis could exploit ways to connect atmospheric particulates is a challenging point which we believe deserves more, immediate and in-depth experimental work. it is to be expected that steps to clarify the dynamics involved in the current pandemic will be taken promptly. key: cord-287520-51kmd2ds authors: carneiro, arie; wroclawski, marcelo langer; nahar, bruno; soares, andrey; cardoso, ana paula; kim, nam jin; carvalho, fabricio torres title: impact of the covid-19 pandemic on the urologist’s clinical practice in brazil: a management guideline proposal for lowand middle-income countries during the crisis period date: 2020-05-20 journal: int braz j urol doi: 10.1590/s1677-5538.ibju.2020.04.03 sha: doc_id: 287520 cord_uid: 51kmd2ds this letter to the editor aims to provide suggestions and recommendations for the management of urological conditions in times of covid-19 crisis in brazil and other lowand middle-income countries. it is important to highlight that one of the main characteristics of this pandemic is the oversaturation of the health system capacity, mostly due to a high demand for personal protective equipment (ppe), hospital/icu beds, as well as ventilators. in places with limited resources and where the health care systems are already saturated, such consideration is even more worrisome. therefore, most worldwide authorities are recommending to avoid, as much as possible, patient’s elective visits to hospitals, as well as a judicious use of the operating room in order to mitigate the strain put on the health system. while efforts should be directed to the care of covid-19 patients, other conditions (especially urgencies and oncological cases) must continue to be assisted. thus, through a panel of experts, we have prepared a practical guide for urologists based on the recommendations from the main urologic associations, as well as data from the literature to support the suggested management. we will try to follow the standard guideline recommendations from the american urological association (aua) and european association of urology (eau), with the aim of pursuing the best outcomes possible. however, some recommendations were based on the consensus of the panel, taking into consideration the reality of developing countries and the unprecedented situation caused by the covid-19 crisis. most importantly, all recommendations on this manuscript are based on the expectancy of a maximum 3-month duration of the crisis. if this period shall extended, these recommendations will be revised and updated. the format of the text will be given through questions and answers. similar to other specialties, the pandemic has drastically changed the routine of the urologists. elective clinic visits are being canceled, postponed or, in some situations, replaced by remote care through telemedicine, recently regulated and temporarily authorized by the brazilian ministry of health (1) . we believe that tele-screening, test reviews and follow-up evaluations that do not require physical examination are the ideal situations for this type of care, especially when the patient is in the high-risk group and must be socially isolated (2) . in regard to surgeries, all postponable procedures must be rescheduled, in order to reduce the exposure of the surgical team and the patient to a potential contamination. furthermore, cancelation of surgeries collaborates with social isolation and save resources (such as ppes) for the care of patients with covid-19 infection. the main question is how to define which operations can really be postponed, especially in urologic oncology, without interfering with the patient's outcomes. the most important recommendation at this point is that elective surgical procedures should be postponed. the diagnostic, therapeutic and human resources of the health care facilities must be available to fight the pandemic (3) . some considerations should be made: a) we must consider all cases as suspect, until proven otherwise. ideally, every case should be tested by rrt-rna-crp for sars--cov-2 48 prior to surgery, but unfortunately this is not feasible in most developing countries. negative confirmed cases should be kept in a separate environments. b) surgeries for covid-19 negative patients should ideally be performed in a surgical center different from the location where patients with positive covid-19 are being treated. if it is not possible to separate an entire surgical block, we suggest designating specific rooms for the care of patients with covid-19 that will not be used for regular cases. c) a trained and dedicated multidisciplinary team should be available for the management of suspected and confirmed patients for covid-19. it is preferable that this team does not assist co-vid-19 negative cases. d) whenever possible, we should prioritize surgeries with local anesthesia or spinal blockade. e) always obtain a consent form, as recommended by the brazilian society of medical and bioethics law. patients are at risk of contracting covid-19 infection during their hospital stay and major surgeries in asymptomatic infected patients during the incubation period appear to predict worse outcomes, with a mortality rate up to 20% (3). f) after the procedure, covid-19 positive patients should be admitted to the designated areas for suspected and / or confirmed patients with covid-19, if the institution in question provides such area. -if available, we recommend testing all patients for rrt-rna-pcr for sars-cov-2 48 hours before performing the procedure. -if it is impossible to test everyone with the resources available, all cases should be considered suspect. for everyone in the room: caps, personal protective glasses, n95 mask (pff2 or pff3), protective gowns for contacts, procedure gloves and shoe covers. for those who will perform procedures: cap, personal protective glasses, face shield, n95 mask (pff2 or pff3), sterile waterproof apron, sterile gloves, shoe covers and waterproof disposable boots whenever secretions (when urine, stool or blood are expected, such as in endourological procedures) are expected. -in the setting of n95 masks rationing, the face shield is important in order to prevent soiling of the mask, that can be further reutilized. -increased care should be taken when handling patient's stool. studies have shown that even in patients with negative airway crp, the clearance of the the viral rna is longer in the stool (4). thus, surgeons should take extra precautions with surgeries that include bowel manipulation and trans-rectal prostate biopsies. a) on the corridor: -hand hygiene; -put on the n95 mask and goggles or face shield for anesthetists, in the case of intubation. -perform surgical hand antisepsis. b) in the lobby -put on a surgical gown. -put on sterile gloves (surgical team); -the anesthesiologist should use 2 pairs of gloves and after intubation the second pair should be changed as soon as possible. what is the correct order to remove the surgical ppe? a) inside the room: -remove gloves; -hand hygiene; -remove disposable gown; -hand hygiene. b) outside the room: (leave a side table with an oxivir® drum and procedure gloves). -remove glasses; -remove the n95 mask and place it in an identified plastic bag; -remove the cap; -hand hygiene; -put on procedure gloves and clean and disinfect the glasses and support surface (use disinfectant detergent -oxivir® or optigerm ®); -remove the gloves; -hand hygiene. after removing protective equipment, remember not to touch your hair or face before hand washing. some studies have suggested transmission of some pathogens (coryneobacterium, papillomavirus and hiv) by the pneumo peritoneum through the release of smoke generated by the laparoscopic electrocautery (5) (6) (7) . a parallel situation may be extrapolated to the coronavirus. therefore, additional care in these procedures must be performed: a) as mentioned earlier, it is important to test all patients before the procedure, if possible. b) additional protection in relation to aerosol dispersion: always keep materials clean, assistants must have additional care when placing and removing trocars, do not use trocars with air leakage, avoid using monopolar energy and give preference to bipolar, keep settings of the electrocautery to minimum in order to reduce smoke formation. c) handling of pneumoperitoneum: keep it as low as possible, minimizing the trendelenburg as much as possible. if possible, use devices that are able to aspirate and filtrate the smoke from the pneumoperitoneum. d) during disinflation, the co2 gas and smoke should be captured with an ultra-filtration system. a disinflation mode should be used on your insufflator if available. e) if available, use filters on vacuum cleaners, there are different models and brands. f) use drains only when extremely necessary because post-operative care in the presence of organic fluids demand extra-caution and additional ppe. g) favor the open approach in cases where minimally invasive surgery has not shown considerable benefit. when should we go into isolation? urologists, like other physicians, should be isolated only when they become suspected or confirmed cases of covid-19. in suspicious cases, the sars-cov-2 rt-rna-crp should be collected and physicians should be kept isolated until the result. physicians with the following symptoms should be considered highly suspicious for co-vid-19: fever, respiratory symptoms (cough, runny nose, nasal obstruction, sore throat, shortness of breath, loss of smell), in addition to body aches, fatigue, diarrhea and nausea (8) . healthcare professionals should always be tested when symptomatic. the duration of the quarantine is 14 days, starting on the day of onset of symptoms. individuals should be asymptomatic at the end of this period. otherwise, they should remain isolated until symptoms are resolved, and only return to activities 72 hours after the resolution of all symptoms. the center for disease control and prevention (cdc-usa) recommends the utilization of rt-rna-crp for control and only release physicians to work after a negative result. however, due to the lack of tests in most of the country, this is not mandatory by the brazilian ministry of health (9). in cases of patients with urinary lithiasis all surgeries to treat urolithiasis should be suspended, unless these are emergencies. in the presence of ureteral lithiasis associated with fever or other signs of infection, there is an absolute indication for antibiotic therapy and urinary drainage. preferably, we opted for the passage of a ureteral stent (i.e double j stent) under spinal anesthesia (or even with local anesthesia). as an alternative, bedside ultrasound percutaneous guided nephrostomy might be considered in centers with the necessary expertise (10) . in addition to lithiasis associated with urinary tract infection, ureteral obstruction in a solitary kidney or bilateral ureteral obstruction, acute impairment of renal function and pain refractory to clinical treatment should not be postponed. unlike other recommendations(11), our position is that, once the surgical procedure is indicated, we should be as resolutive as possible, in order to reduce the number of visits to the hospital for new surgeries to the emergency department. thus, instead of just draining the urinary tract, our tendency is to perform ureterolithotripsy whenever possible and safe, keeping the double j stent with a string externalized by the urethra to be removed on an outpatient basis. the remaining cases of renal colic should preferably be managed clinically, with medical expulsive therapy and pain control. however, it is important to note that, invariably, cases initially conducted in this way may evolve into emergency situations, such as those previously mentioned. patients who are already with a double j stent may remain with the stent for as long as possible. surgery may be indicated in cases of extreme ureteral stent discomfort. otherwise, clinicians must keep strict control of all cases in order to avoid the forgotten double j syndrome. patients with benign prostatic hyperplasia should not be operated at this time of a pandemic, unless they develop complication that will require hospitalization and possible surgery, such as massive hematuria and clot retention. in this scenario, we believe that the evacuation of clots and / or cauterization of the prostate should already be accompanied by resection, vaporization or endoscopic enucleation of the prostate (12) . in all other cases, even if there is urinary retention, we recommend postponing the procedure. if necessary, indwelling urinary catheter placement or percutaneous cystostomy with local anesthesia are indicated for preservation of renal function (13, 14) . the investigation of hematuria through imaging tests during covid-19 pandemic should be limited to cases of macroscopic hematuria, especially if there are clots or hemoglobin decrease. greater attention should be given to patients at higher risk for urothelial carcinoma, such as men, over 50 years of age and with a history of smoking and exposure to known carcinogenic agents. the gold standard test for investigation of the upper urinary tract is uro-tomography, but in times when we need to consider the use of resources, ultrasound could potentially be used since many imaging services are overloaded due to the frequent indication of thoracic cts for the diagnosis and follow-up of patients with sars-cov-2. these data are extrapolations from recent evidence that ultrasound could replace tomography in the investigation of microscopic hematuria (14) . however, once the outbreak is resolved or if the resource is available, uro-tomography should be performed. regarding the lower urinary tract, flexible cystoscopes are not widely avaliable in brazil. thus, diagnostic cystoscopies should initially be postponed avoiding hospitalization for cystoscopy in the or. ultrasound could also be used to evaluate the lower urinary tract during this time of pandemic. in addition to the procedures previously mentioned, non-urologic oncology conditions that deserve urgent treatment are testicular torsion, scrotal abscess and / or fournier's syndrome, infection of penile prosthesis or artificial sphincter, priapism and urological trauma. we should consider postponing surgical treatment for all other urological conditions, such as urinary incontinence, prolapses, urethral stenosis, prosthetic implants, infertility-related operations (including vasectomy) and genital procedures such as circumcision or hydrocele correction (15) . urodynamic study: we suggest that urodynamic study programs should be suspended during the crisis period. whenever possible, cystoscopies should be postponed. if indispensable, priority should be given to outpatient procedures, using a flexible cystoscope. it's well known that most programs in developing countries do not have flexible cystoscopy, so it is necessary to perform it in the operating room. this should be postponed whenever possible. as a rule, prostate biopsies should be postponed, since delaying the diagnosis of prostate cancer for 3-6 months will not interfere with survival outcomes in the vast majority of cases. it's worth discussing prostate biopsy in highly suspicious cases of patients with symptoms related to advanced / metastatic disease, such as bone pain or urinary retention. given that most medications for metastatic prostate cancer (such as androgen deprivation therapy) are not approved/released without histological confirmation of prostate adenocarcinoma, we recommend to biopsy the most easily accessible site, which may be the prostate (often the most quickly available resource) or some metastasis focus. however, if clinical signs of metastatic disease are evident, a shared decision should be made with the patient and additional efforts should be performed to expedite the release of these medications even without the biopsy. we believe this exception should be highly considered in selected patients, especially in times of crisis. if indicated, the biopsy should be performed under local prostatic block, avoiding sedation. as mentioned earlier, additional care must be taken, given the high prevalence of covid-19 in the stool of infected patients. -in case of small bladder tumors, consider a single-dose intravesical chemotherapy within 24 hours of turbt (not immunotherapy). the most commonly used agents are: mitomycin and gemcitabine, in brazil just gemcitabine isavaiable (16) . -in intermediate-risk and high-risk non--muscle-invasive bladder cancers: clinically fit patients with no major comorbidities should receive induction therapy followed by at least 1-year maintenance bcg. in selected cases we can consider postpone one dose during the maintenance. in the case of bcg shortage supply, gemcitabine can be used (16, 17) . patients with d'amico low-risk prostate cancer -treatment: active surveillance is recommended for all patients with grade group 1. -follow-up: follow-up tests as well as confirmatory and control biopsies should be postponed. -treatment: treatment of these patients should be postponed until the pandemic is over. we have robust evidence to support that postponing treatment in these patients for 3 months do not impact cancer-specific mortality (protect, pivot, spcg 4 trials). afterward, local treatment should follow the current recommendations in the guidelines (18) (19) (20) . -follow-up: patients who have already been treated should ideally be followed via telehealth. in-person consultations should only be carried out if they are really necessary. the first post-operative psa can be performed after 3 months, because no early adjuvant therapy would be initiated in this scenario. -treatment: we recommend initiation of hormone deprivation therapy immediately and, after 3 months, discuss the most appropriate local therapy. there is good evidence supporting this approach, particularly when associated with radiation therapy and, less common, as neoadjuvant for surgery. while no survival benefit was seen with neoadjuvant studies, pre-operative androgen deprivation therapy reduced positive margin rates as well as extra prostatic extension without compromising cancer control (21) . -follow-up: patients who have already been treated should undergo additional tests and visit after the pandemic. in-person consultations should only be carried out if they are really necessary. the first post-operative psa can be performed after 3 months, because no early adjuvant therapy would be initiated in this scenario. no adjuvant radiation therapy may be indicated during covid-19 pandemic. all cases, even in the presence of unfavorable features, can be managed later with salvage radiation therapy if necessary. we recommend use of adt in a 6-month formulation (22) in association with apalutamide 240mg vo daily or enzalutamide 160mg vo daily (23), when indicated. if apalutamide or enzalutamide is not available, an alternative option is abiraterone 1000mg vo daily associated with prednisone 5mg vo daily. the use of low dose prednisone should be considered since the impact of corticoid usage during the covid-19 pandemic are not well known (24) . chemotherapy associated with adt should be indicated only in extremely selected cases. when indicated, it can be postponed until 120 days after initiation of adt (25) . use of colony stimulating factor is recommended in cases of chemotherapy. we recommend the use of adt in semiannual formulation (22) associated with preferably 160mg of enzalutamide vo daily (if not previously received) (26) or alternatively abiraterone 1000mg vo daily associated with prednisone 5mg vo daily (if not previously received). alternatively, in patients with isolated bone metastases, the use of radium 223 adt may be considered (27) . alternatively, docetaxel with reduced dose every 3 weeks can be considered. it's important to emphasize that colony stimulating factor is highly recommended when chemotherapy is administered (28) . zoledronic acid should be used in patients with bone metastases every 3 months (29). asymptomatic ct1a patients should have their treatment postponed, unless there is a rare risk that a nephron-sparing procedure becomes not feasible with the delay of surgery. asymptomatic ct1b or ct2 patients, eligible to partial nephrectomy, should be operated to avoid losing the window of a nephron sparing surgery. if the indication is radical nephrectomy, it may be postponed. patients with ct3-4 disease and/or with symptoms such as gross hematuria should be operated, especially those with thrombus in the renal vein and / or vena cava. what is the role of cytoreductive nephrectomy in the current scenario? should we always try systemic treatment first? we should proceed with cytoreductive nephrectomy whenever this is the best short-and medium-term treatment option for symptoms control. asymptomatic patients with low and intermediate risk who can wait 3 months to start treatment should wait. patients with indications of immediate systemic treatment should start treatment despite nephrectomy, especially in cases of intermediate and poor risk (30) . regarding the choice of first line treatment, we favor, if available, the combination of a tyrosine kinase inhibitors (tki) with immunotherapy, such as axitinib with pembrolizumab (31, 32) . we also suggest adjusting the dose intervals to less frequent (pembrolizumab to 400mg every 6 weeks). if not available, the choice between the combination of immunotherapy with ipilimumab and nivolumab versus tki should take into account the risk of complications and potential readmissions (33) . it is relatively safe to start systemic therapy with tyrosine kinase inhibitors (tki) and immunotherapy, although it might have a hypothetical detrimental effect on the immunological response to covid-19. nonetheless, no reliable evidence regarding therapy with immunotherapy or tyrosine kinase inhibitors has been identified but the landscape is changing rapidly, and we should be attentive to any evidence that could show the opposite (30) . in general, turbt should be performed whenever possible. it is important to highlight that we recommend performing a cold-cup biopsy of the base of the lesion to ensure representation of the detrusor muscle and avoid the need for repeat turbt due to undersampling (16) . elderly patients with multiple comorbidities and asymptomatic patients with radiologically small and superficial tumors can have the procedure postponed. virads staging system may play an important role in selecting such cases. repeat turbt is the standard of care for non-muscle-invasive high-risk bladder tumors (34) . however, during the pandemic, some exceptions can be considered. if the initial procedure was performed by an experienced surgeon who is confident that the entire lesion was completely removed and there is presence of muscle layer in the pathology, the repeat turbt may be postponed. in patients with high-grade pta, repeat turbt can also be delayed, even if no muscle is represented. muscle invasive bladder cancer is an aggressive disease with great potential to be curable. in this scenario, surgical delays might represent a loss on the window of treatment. in general, we recommend performing neoadjuvant chemotherapy and, after finishing, assess the conditions of the hospital to decide on the need for a cystectomy vs bladder preservation protocol (if indicated). of note, neoadjuvant chemotherapy with cisplatin and gemcitabine before cystectomy can be delayed for up to 6-8 weeks from diagnosis (35) . cystectomy after neoadjuvant chemotherapy can also be safely delayed for up to 10 weeks post chemotherapy (36) without jeopardizing oncological outcomes (37) . patients who have undergone cystectomy should consider adjuvant chemotherapy with cisplatin and gemcitabine. this approach still offers survival benefits even 180 days after surgery (38) . we should always consider using colony stimulating factor when chemotherapy is recommended. for patients who wish to preserve the bladder, or who are not eligible for surgery, or the hospital does not have adequate resources, trimodally therapy should be considered. (tur-bt followed by hypo-fractionated radiotherapy associated with weekly chemotherapy with gemcitabine 100mg/m 2 ). for cisplatin eligible patients we recommend first line of treatment (35) : -cisplatin 35mg/m 2 on days 1 and 8 and gemcitabine 1000mg/m 2 on days 1 and 8 every 3 weeks (39). -consider colony stimulating factor in all patients. for cisplatin ineligible pd-l1 negative patients we recommend the first line of treatment: -carboplatin, auc 4.5-5 and gemcitabine 1,000mg/m² on day 1 and day 8, every 3 weeks (41). -consider colony stimulating factor in all patients. radical orchiectomy should be performed as soon as possible because it is an outpatient procedure and will guide further treatment. we recommend surveillance for most patients with stage i over any adjuvant treatment, despite unfavorable features (42) . -for low volume stage ii patients (iia and iib) we recommend radiotherapy instead of chemotherapy for seminomas (42) . -for high volume stage ii seminomas: 4 cycles of ep(etoposide 100mg/m 2 iv on days 1-5 and cisplatin 20mg/m 2 iv on days 1-5, every 21 days), considering using g-csf (42); -for non-seminoma stage iia with normal markers: retroperitoneal lymph node dissection might be considered to avoid use of chemotherapy (42) . -for non-seminoma stage iia with significant elevation of tumor markers and stage iii (for both seminoma and non-seminoma): in case of favorable risk, we recommend 4 cycles of ep. we do not recommend 3 cycles of bep. in case of intermediate or unfavorable risk, 4 cycles of vip (etoposide 75mg/m 2 iv on days 1-5; ifosfamide 1200mg/m 2 iv on days 1-5 with same protection and cisplatin 20mg/m 2 iv on days 1-5, every 21 days) are preferable or alternatively, 4 cycles of bep, (with strong recommendations of g-csf use), because of the bleomycin pulmonary toxicity (42) . portaria número 467 cancer guidelines during the covid-19 pandemic clinical characteristics and outcomes of patients undergoing surgeries during the incubation 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the effect on survival of time from diagnosis to neoadjuvant chemotherapy to cystectomy for muscle invasive bladder cancer delaying radical cystectomy after neoadjuvant chemotherapy for muscle-invasive bladder cancer is associated with adverse survival outcomes survival benefit persists with delayed initiation of adjuvant chemotherapy following radical cystectomy for locally advanced bladder cancer a phase i/ii study of gemcitabine and fractionated cisplatin in an outpatient setting using a 21-day schedule in patients with advanced and metastatic bladder cancer first-line pembrolizumab in cisplatinineligible patients with locally advanced and unresectable or metastatic urothelial cancer (keynote-052): a multicentre, single-arm, phase 2 study randomized phase ii/iii trial assessing gemcitabine/carboplatin and methotrexate/carboplatin/ vinblastine in patients with advanced urothelial cancer who are unfit for cisplatin-based chemotherapy: eortc study 30986 testicular cancer: european association of urology arie carneiro and marcelo langer wroclawski contributed similarly as first author. none declared. key: cord-296229-pkwxlydz authors: liu, yang; funk, sebastian; flasche, stefan title: the contribution of pre-symptomatic infection to the transmission dynamics of covid-2019 date: 2020-04-01 journal: wellcome open res doi: 10.12688/wellcomeopenres.15788.1 sha: doc_id: 296229 cord_uid: pkwxlydz background: pre-symptomatic transmission can be a key determinant of the effectiveness of containment and mitigation strategies for infectious diseases, particularly if interventions rely on syndromic case finding. for covid-19, infections in the absence of apparent symptoms have been reported frequently alongside circumstantial evidence for asymptomatic or pre-symptomatic transmission. we estimated the potential contribution of pre-symptomatic cases to covid-19 transmission. methods: using the probability for symptom onset on a given day inferred from the incubation period, we attributed the serial interval reported from shenzen, china, into likely pre-symptomatic and symptomatic transmission. we used the serial interval derived for cases isolated more than 6 days after symptom onset as the no active case finding scenario and the unrestricted serial interval as the active case finding scenario. we reported the estimate assuming no correlation between the incubation period and the serial interval alongside a range indicating alternative assumptions of positive and negative correlation. results: we estimated that 23% (range accounting for correlation: 12 – 28%) of transmissions in shenzen may have originated from pre-symptomatic infections. through accelerated case isolation following symptom onset, this percentage increased to 46% (21 – 46%), implying that about 35% of secondary infections among symptomatic cases have been prevented. these results were robust to using reported incubation periods and serial intervals from other settings. conclusions: pre-symptomatic transmission may be essential to consider for containment and mitigation strategies for covid-19. since its emergence in early december 2019, sars-cov-2 has spread rapidly despite multiple layers of interventions designed to prevent a pandemic 1 . in less than two months the covid-19 outbreak progressed from fewer than 300 cases across four countries to more than 100,000 cases in over 100 countries 2,3 . covid-19 infection in the absence of clinical symptoms has been frequently described [4] [5] [6] [7] alongside anecdotal evidence for potential asymptomatic or pre-symptomatic transmission or transmission while symptoms were suppressed by fever-reducing and prescription-free medication [8] [9] [10] [11] . in combination with highly non-specific and generally mild symptoms in most cases 12 , pre-symptomatic transmission of covid-19 would substantially hinder control and mitigation efforts, which typically focus on rapid syndromic identification and isolation of cases 13,14 . we thus estimated the possible extent of pre-symptomatic transmission to help inform infection control and mitigation plans. multiple estimates for the covid-19 incubation period and serial interval have been reported to date. while estimates for the incubation period are relatively similar, the serial interval is dependent on the local context, in particular local contact patterns and the presence of interventions, active case finding with subsequent isolation, in particular, that may right-censor the serial interval. as our base case, we used the estimates provided by bi et al. derived from 391 cases and their 1286 contacts as identified by the shenzen centre for disease control 15 . this study currently includes the most extensive investigation of the infector-infectee transmission dynamics in a single setting. the probability of pre-symptomatic transmission can be expressed as the probability of onward transmission during one's incubation period. ideally, this probability could be estimated from individual-level observations on exposure, onset of symptoms and the transmission to others. however, such data remains largely unavailable and is inherently difficult to generate. instead, we approximated this quantity from population estimates of the density distribution of the incubation period and the serial interval. we did so by first using the incubation period density distribution to calculate the probability of having developed symptoms on each day since infection and then using this to stratify the serial interval distribution, assumed to be a proxy for the generation time, into likely pre-symptomatic and symptomatic onward transmissions. the observations of the incubation period and the serial interval may be correlated. in other words, individuals with a long incubation period may not have a serial interval of typical length but may be exactly those who have a long serial interval, or alternatively, exactly those who have a short serial interval. to account for this, we also calculated the proportion of pre-symptomatic transmission by randomly drawing from the incubation period and serial interval distributions and pairing samples not randomly, but aligning them either both in ascending or one in ascending and the other in descending order (rank ordering), before calculating the proportion of draws with a shorter serial interval than the incubation period. we report the estimates assuming no correlation accompanied by a range indicating estimates that assume positive and negative correlations. we consider two scenarios: transmission with and transmission (largely) without active case finding to accelerate case isolation. for the no active case finding scenario we use the serial interval reported based on cases that were only isolated at least 6 days after their symptom onset; i.e. 8.0 days (95% empirical confidence interval (eci): 4.7 -11.3). for the active case finding scenario we used the overall serial interval; i.e. 6.3 days (95% eci: 0.9 -16.9) ( figure 1 ). the incubation period in this study was on average 6.0 days (95% eci: 1.3 -17.2). we also included sensitivity analyses. we used the reported mean incubation period (5.2 days (95% eci: 1.1 -14.3)) and serial interval 3)) from the first 425 cases in wuhan province as an alternative scenario for a setting with no or limited active case finding 16 . further, nishiura et al. provide estimates of the serial interval from published reports across all affected countries, mostly including attempted rapid case finding and isolation 17 . we use their mean serial interval estimates based on high certainty transmission pairs (4.8 days (95% eci: 1.1 -9.6)) in combination with either the incubation time reported by bi et al. 15 or li et al. 16 . we implemented all density distributions to match reported estimates and their distributions 18 . we then calculated the effectiveness of case isolation in reducing the number of secondary cases during the symptomatic period (φ) let x be the proportion of pre-symptomatic transmission in the absence of active case finding, and y the same proportion in the presence of active case finding. then, y can be expressed as a function of x while accounting for a reduction φ in the symptomatic cases: solving for φ: all analyses were done in r 3.6.3 19 ; codes and data are available online 18 . in the scenario of no active case finding and isolation before six days after symptom onset, and assuming uncorrelated serial interval and incubation period distributions, we estimate that 23% (range accounting for correlation: 12 -28%) of onward transmissions in shenzen have occurred during the pre-symptomatic period ( figure 2 and table 1 ). 6.0 (1.6 -14.0) * a summary table of the parameters used and the underlying assumptions regarding the probability distributions used is publicly available 18 . the uncertainty ranges above are those of the simulated distributions of serial interval and incubation periods (instead of the mean or median of these parameters), and thus may be different from what is reported in the original articles. note that serial intervals sampled for with and without active case finding scenarios were independent, i.e., based on separate sets of parameters. active case finding and subsequent accelerated isolation right censors the serial interval and thereby reduces the number of symptomatic onward transmissions. in shenzen, the presence of active case finding increased the percentage pre-symptomatic among all transmissions to 46% (21 -46%). therefore, case isolation in shenzen has on average reduced the number of secondary cases during the symptomatic period by 35% (42 -59%) and hence the total number of secondary cases by 27% (32 -45%). both the sensitivity analyses for the scenario of limited to no active case finding and for the scenario of real-world effects of active case finding and isolation during the containment phase of the outbreak response are similar to the estimates in our main analysis (table 1 ). we estimated that without active case finding to accelerate isolation in shenzen 12 to 28% of transmissions occurred during the pre-symptomatic period. we further estimated that active case finding reduced the number of secondary infections after symptom onset and thereby increased the percentage of pre-symptomatic transmissions to 21 to 46%. this implies that in shenzen accelerated case isolation has prevented 35 -60% of secondary cases among symptomatic infectees. these results were similar when alternative settings with and without accelerated case isolation were considered. in addition to the mostly-mild and non-specific symptoms of covid-19, a high contribution of pre-symptomatic cases to the transmission dynamics of covid-19 could be part of the reason why controlling covid-19 has been challenging and seems to have failed in many countries 13 . it implies that control and mitigation of covid-19 may be substantially improved if also targeting pre-symptomatic infections. this may be achieved via social distancing interventions that do not rely on the identification of symptoms. having 10 -30% pre-symptomatic transmission also suggests that immediate isolation at symptom onset can at best prevent 70-90% of cases. however, immediate case isolation on the day of symptoms onset is unlikely. we also find that, based on parameters estimated under no active case finding scenarios, case isolation on days 1, 3 or 7 after symptom onset reduces the proportion of or preventable onward transmissions by syndromic case finding and isolation to approximately 60%, 50% and less than 20%, respectively (figure 3 ). we find that in shenzhen syndromic identification and isolation has prevented 35 -60% of secondary cases from symptomatic infectees, which is in line with previous model-based estimates 14 . even for well-studied pathogens like influenza, there is a substantial amount of uncertainty in the contribution of asymptomatic and pre-symptomatic infections to the transmission dynamics. for influenza, 10 -20% of infections have been found to be asymptomatic but shedding, although at lower viral loads 20,21 . for sars, 7.5% of exposed and seroconverted health care workers were found to not have displayed symptoms although it is unclear if those were also effectively transmitting 22 . for covid-19 the proportion of pre-symptomatic or asymptomatic infections is likely much higher. during an outbreak aboard a cruise ship, 50% of passengers who tested positive were asymptomatic at sample collection and therefore may or may not have developed symptoms at some point later on 6 . whether asymptomatic carriers are also infectious has not yet been clearly established. our work is based on observations of eventually symptomatic cases and hence we implicitly assume that only those who will eventually become symptomatic can effectively transmit. if there is an additional role for asymptomatic individuals in the transmission of covid-19 this will further decrease the proportion of secondary infections attributable to symptomatic cases. hence, our estimate that among those coivd-19 cases who eventually will develop symptoms the pre-symptomatics substantially contribute to transmission is a conservative approximation for the contribution of individuals without symptoms to covid-19 transmission. a key limitation for this study is that we had to use populationlevel estimates rather than individual-level estimates for the incubation period and the serial interval. we address biases potentially arising from the correlation of the two estimates on the individual level by estimating the sensitivity of estimates to the assumption of positive or negative correlation. additionally, our estimates are in essence estimating the proportion of pre-symptomatic transmission from the amount of overlap of the incubation period distribution and the serial interval distribution. if the heterogeneity observed in either distribution was originated in measurement bias rather than in actual between individual heterogeneity of the infection process, then we would overestimate the role of pre-symptomatic transmission. in our sensitivity analyses, we find similar estimates for other settings indicating that the observed heterogeneity in shenzhen is unlikely to be purely an observation artefact. however, our study supports the world health organization's prioritisation for epidemiological studies, ideally individual-level observations, to further enhance the evidence base around the role of pre-symptomatic transmission for covid-19 23 . in summary, we find that pre-symptomatic transmission may play a key role in the epidemiology of covid-19. given the relevance of pre-symptomatic transmission for control and mitigation efforts, additional studies are urgently needed to reduce the uncertainty in the role of pre-symptomatic transmission. source data this project does not involve any primary data collection. all data used are based on references 15, 16, and 17. more specifically: i have only few remarks listed below. remarks: table 1 : it would interesting if the authors could give some insight on "fully anti-correlated" si and incubation period. under which circumstances, may this happen? i could imagine uncorrelated and fully correlated cases, probably, partially anti-correlated, but it was difficult for me to imagine the setting of fully anti-correlated cases. typos, etc: page 6, sixth line from above: probably, the authors could specify that it was the diamond princess cruise ship docked in japan. figure 3: i would recommend to make the lines grey and a bit transparent, so the reader would be figure 3 : i would recommend to make the lines grey and a bit transparent, so the reader would be able to distinguish between individual lines. github repo (very optional): the author could improve their readme.md file, so to make it as a graphical abstract of their paper. this may help to ease the access to the repo for the readers. is the study design appropriate and is the work technically sound? yes if applicable, is the statistical analysis and its interpretation appropriate? yes 1. variability/uncertainty through sensitivity analyses. figure 3 is difficult to read, can you use semitransparency to distinguish the density of simulated lines? do you have any estimate of how early people might become contagious before symptom onset? in figure 3 you show infectivity is persisting for up to a week after onset, but how many days before onset did infectiousness start? is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. reviewer expertise: infectious disease epidemiology i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. cmmid ncov working group: temporal variation in transmission during the covid-19 outbreak cmmid repository reference source 2. world health organization: novel coronavirus(2019-ncov), situation reference source 3. world health organisation: novel coronavirus (2019-ncov) situation report -48 reference source a well infant with coronavirus disease 2019 (covid-19) with high viral load radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study field briefing: diamond princess covid-19 cases, 20 feb update reference source epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore version 1 06 july 2020 reviewer report https://doi.org/10.21956/wellcomeopenres.17314.r39156 © 2020 lee e. this is an open access peer review report distributed under the terms of the creative commons attribution , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is license properly cited. department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa this report by liu is a clean and straightforward analysis that brings together early data on the et al. incubation period and serial interval of covid-19 to provide important insight into the contributions of pre-symptomatic individuals to onward disease transmission. the authors' analysis is strengthened by the inclusion of sensitivity analysis on assumptions about the incubation period and serial interval as well as scenarios that account for the impact of active case finding on the relative contribution of pre-symptomatic infections to onward transmission. i would like a more explicit description of how the pre-symptomatic transmission percentages were estimated. as is, i'm not sure why there are only point estimates presented in table 1 , unless only a single set of draws were performed for each category.can the authors comment on the sufficiency of follow up time for the studies that informed the serial interval estimates? are the authors reasonably confident that there is limited right censoring for individuals that were non-symptomatic and positive at identification (i.e., to see whether they may have instead been pre-symptomatic). this seems an important consideration that would bolster the validity of the authors' estimates.since this paper was originally posted, i am wondering whether there have been additional estimates of the serial interval under conditions of active case finding. presumably locations outside of china may have had time to prepare their surveillance systems so that serial interval estimates would directly reflect scenarios with active case finding (as opposed to making a cutoff assumption). are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. this is a nice simple analysis of the incubation period distribution and the serial interval distribution, showing that substantial pre-symptomatic transmission has likely been occurring.there is fairly good consistency in published estimates of the incubation period distribution, but relatively less consistency in published estimates of the serial interval distribution. authors deal with this variability/uncertainty through sensitivity analyses. key: cord-017016-twwa9djm authors: tomashefski, joseph f.; dail, david h. title: aspiration, bronchial obstruction, bronchiectasis, and related disorders date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_5 sha: doc_id: 17016 cord_uid: twwa9djm the conducting airways play a pivotal role in the spectrum of pulmonary pathology, not only as conduits for injurious agents to enter the lung, but also as an anatomic compartment that is affected by a diverse array of primary or secondary bronchocentric diseases. this chapter discusses aspiration and bronchial obstruction in detail, with emphasis on the aspiration of toxic, infective, or particulate matter. lung abscess, a frequent complication of obstruction or aspiration, is also reviewed. both aspiration and lung abscess are reconsidered within the context of pulmonary infectious disease mainly in chapter 8 on bacterial infections, and to some extent in the chapters on mycobacterial (chapter 9), fungal (chapter 10), and parasitic diseases (chapter 14). the conducting airways playa pivotal role in the spectrum of pulmonary pathology, not only as conduits for injurious agents to enter the lung, but also as an anatomic compartment that is affected by a diverse array of primary or secondary bronchocentric diseases. this chapter discusses aspiration and bronchial obstruction in detail, with emphasis on the aspiration of toxic, infective, or particulate matter. lung abscess, a frequent complication of obstruction or aspiration, is also reviewed. both aspiration and lung abscess are reconsidered within the context of pulmonary infectious disease mainly in chapter 8 on bacterial infections, and to some extent in the chapters on mycobacterial (chapter 9), fungal (chapter 10), and parasitic diseases (chapter 14) . bronchiectasis, which is frequently grouped with other forms of obstructive lung disease (see chapter 24) , is discussed in this chapter as a major pathway of airway remodeling that may be of inflammatory, postobstructive, or congenital etiology, and is, itself, an important sequela of aspiration. the topic of bronchiectasis cannot be considered without reference to inflammatory lesions of the small airways, which may follow or precede the development of bronchiectasis (see chapter 25) . the present chapter also discusses a variety of pulmonary disorders that may simulate, initiate, or complicate bronchiectasis. involvement of the large airways in systemic diseases such as amyloidosis (chapter 21), sarcoidosis (chapter 18), collagen vascular diseases, including relapsing polychondritis (chapter 20), connective tissue disorders such as marfan's syndrome (chapter 20), or as a complication of environmental dust exposures (chapter 26) is reviewed in each of their respective chapters. an excellent review of bronchiectasis in systemic diseases is that by cohen and sahn.l a discussion of asthma and its related conditions of mucoid impaction, bronchocentric granulomatosis, and allergic bronchopulmonary aspergillosis can be found in chapter 15. unique congenital lesions of the airways in 84 addition to the enigmatic intralobar sequestration are discussed respectively in chapters 6 and 7 on pediatric lung pathology. a variety of degenerative or so-called metabolic disorders that affect the large airways, such as tracheobronchopathia osteoplastica, are reviewed in chapter 21. finally, bronchial tumors are extensively covered throughout volume 2, which is devoted to pulmonary neoplasia. aspiration is the inhalation of liquid or solid materials into the lower respiratory tract, usually from the oral or nasal cavities, oropharynx, esophagus, or stomach. logically, the course of aspiration is determined by such laws of physics as inertia and gravity. larger, more solid materials, and finer more liquid materials, all follow the straightest and most dependent course after they enter the trachea. as explained in chapter 2, the right mainstem bronchus continues on a more direct course than the left (20 to 30 degrees compared to 40 to 60 degrees for the left mainstem bronchus); the wider angle of the left bronchus allows it to extend around the heart. larger, more solid objects that pass the larynx often lodge in the right mainstem bronchus, while smaller solid objects most frequently continue into the right lower lobe bronchus. 2 this has been well demonstrated in the aspiration of foreign objects by children, generally in the age group of 1 to 3 years. 3 ,4 during this age range, children examine almost everything by placing items into their mouths. in both children and adults, larger objects are sometimes stopped at the larynx and may be expelled by strong coughing. in adults, sudden death due to laryngeal obstruction by aspirated food (most frequently meat) has been termed the cafe coronary.s more than 80% of aspirated foreign bodies occur in children, and among all age groups only 5% are spontaneously expectorated. 6 ,7 sharper objects may perforate a bronchus and cause bleeding, or even penetrate the pleural cavity and cause pneumothorax. 8 a foreign body may migrate within the bronchi and cause wandering infiltra tes. 9 young children most frequently aspirate peanuts, beads, and other fragments of wooden or plastic toys. peanuts and sunflower seeds lead the list in western countries, whereas in arabic countries children most often aspirate melon seeds. 8 older children may inhale flowering grass fragments, which wedge themselves into more distal bronchi and resist expectoration. in any age group, teeth, fragments of bone ( fig. 5.1) , food, blood clots, tissue fragments, nasal pack components, lipids from oily nose drops or orally administered cathartics, bacterial fragments, and gastric content most commonly enter the lung. noguchi et a1. 10 reported a subacute reaction to mud aspiration in a victim of near-drowning, while aspiration of sand in children has been reported to cause a radiographic "sand bronchogram.,, 11 drowning, often thought of as occurring in fresh or salt water, has also occurred in large vats of beer, wine, liquid chocolate, and other interesting concoctions. a literary example of this is shakespeare's richard iii, in which the duke of clarence is finally dispatched in a large vat of wine (the "malmsey butt,,). 12 brock 13 .1 4 in 1942, beautifully illustrated the mechanics of aspiration with abscess formation, which most often followed the dependent course described. once within the lung, finer and more fluid ingredients flow into the first dependent orifices that are encountered. in the supine position, these are most often the posterior segment of the upper lobe and superior segment of the lower lobe ( fig. 5.2 ). in the more upright position, material flows preferentially into the basilar segments of the lower lobes. the basilar segments divide rather evenly, and localization within these segments is not as discrete as in other areas of the lung. when a person is in the lateral decubitus position, the axillary branches of the subsegments of apical and posterior upper lobe bronchi are favored. the more anterior portions of the lung are usually spared the effects of aspiration, unless aspiration occurs in the prone position, as in near-drowning. aspiration need not only be from external sources. rupture of large fluid-filled abscess cavities, tuberculous cavities, or other cysts might be followed by intrabronchial aspiration of infective or other types of material into dependent zones (see fig. 9 .7 in chapter 9). the most common conditions predisposing to aspiration include impaired consciousness, most frequently from alcohol, drugs, or anesthesia, followed by central nervous system disorders (e.g., epilepsy, stroke, dementia) or neuromuscular diseases. 4 next in frequency is aspiration secondary to obstructing masses or other functional defects of the esophagus or stomach. episodes of aspiration are eventually confirmed in about 85% of children, but in only about 30% of adults. 3 ,ls the difference in documentation is probably related to the altered level of consciousness in adults causing temporary amnesia. the common clinical manifestations of an aspirated foreign body constitute a triad of cough, wheezing or rhonchi, and decreased air entry. 3 the wheezing that is sometimes associated with aspirated foreign bodies in children may be ameliorated with theophylline, leading to diagnostic confusion with asthma. 16 early experimental animal studies showed that aspiration of material into the lungs regularly occurs when materials are placed in the nares or accessory sinuses during anesthesia. 17 ,18 myerson 19 found blood immediately postoperatively in the tracheobronchial tree in 79% to 100% of humans who underwent tonsillectomy, including those under general or local anesthesia. several experimental studies have also proven that normal adults aspirate with some regularity. quinn and meyer20 in 1929 introduced lipiodal (iodinated poppy seed oil) into the noses of sleeping subjects and found the material often entered the lungs. amberson 21 in 1937 reported placing barium in the mouths of normal subjects during sleep, with similar results. radiologists sometimes observe aspiration while performing upper gastrointestinal tract barium studies (see below). as reviewed by bartlett, 22 various markers placed in the stomach the night prior to surgery have been identified in lungs sampled during surgery the next day in 7% to 16% of patients. 23 • 24 huxley and associates 2s refined these techniques by placing indium-ll1 chloride in the posterior nasopharynx periodically during sleep. on lung scanning, this tracer was found in the lungs of 45% of normal individuals and in 70% of those with some alteration of the central nervous system. those in the normal group who did not aspirate were fitful sleepers who tended not to enter deep sleep. the implication is that most normal people who enter deep sleep, aspirate. nasogastric and oropharyngeal tubes, including endoscopes and tracheostomy tubes, increase the risk of aspiration. 26 normal individuals tend to clear such occult aspirations without difficulty or sequelae. an acute cough reflex is most important, but also valuable are intact mucociliary activity and alveolar macrophage response (see chapter 3) . the pathologic effects of aspiration are dependent on the character, volume, and frequency of the aspirated components. in this chapter, food and medicinals, gastric acid, lighter hydrocarbons, and heavier oils are separately considered. aspirated bacteria are covered in the section on abscess formation. retained squamous if. tomashefski, jr. , and d.h. dail cells from meconium in newborns are a sign of in utero distress. squamous cells in the lungs of adults are an indicator of oral, oropharyngeal, or esophageal aspiration ( fig. 5.3 ). in the absence of other evidence of aspiration, finding mixed bacteria in lung tissue is very suggestive of aspiration of oral content. lung injury following aspiration has been subdivided by marik 27 into aspiration pneumonitis and aspiration pneumonia. aspiration pneumonitis refers to acute chemical lung injury due to inhaled gastric acid with or without injury due to aspirated particulate matter, whereas aspiration pneumonia is an infectious process resulting from the inhalation of oropharyngeal secretions colonized by pathogenic bacteriay aspiration of gastric acid, a major cause of acute respiratory distress syndrome (ards), has been well studied in humans and in experimental animals. 22 in experimental models, it has been suggested that a ph of 2.4 or lower and a significant quantity of acid (estimated to be 20 to 25ml in an adult human or 1 to 4mllkg in an experimental animal) are necessary to induce a chemical pneumonitis.27 when gastric acid with methylene blue was put into anesthetized dog tracheas, dye was visible on the pleural surface 12 to 18 seconds later. 28 atelectasis oflung tissue was noted in 3 minutes. human studies date from the classic study of mendelson 29 in 1946, and acute gastric acid aspiration is sometimes called the mendelson syndrome. mendelson studied 61 cases of massive gastric aspiration in obstetric patients (0.15% incidence) under ether anesthesia. respiratory distress occurred soon after aspiration, with accompanying cyanosis, tachypnea, and tachycardia. bronchospasm occurred in most of his patients. chest radiographs initially showed rather widespread mottled densities, most of which cleared by 7 to 10 days. only eight of 61 patients became infected. this study was conducted before antibiotics were readily available, but other studies in humans with or without antibiotics or steroids have confirmed his findings. in general, later studies have found a lower incidence (about 30%) of bronchospasm, more frequent early temperature elevation, and increased mortality (in the range of 30% to 60%) despite therapy. hypotension and hypoxemia occurred more commonly than in mendelson's series. 22 the combination of acid and particulate aspiration exacerbates alveolar capillary injury. 30 bynum and pierce 3 ! studied 50 patients with welldocumented gastric acid aspiration. in their series, all these events followed altered consciousness, most often by a sedative drug overdose or a general anesthesia. as in mendelson's29 experience, respiratory symptoms developed rapidly and were very similar in all patients despite eventual outcome. three clinical outcomes were described: 12% died shortly after aspiration; 62% had rapid clinical and radiographic clearing on the average of 4.5 days; and 26% had rapid improvement followed by deterioration relating to bacterial infection. of this latter group 60% died, whereas 28% of the whole group died; death occurred between day 1 and 16, averaging 7.2 days. these authors found initial steroid or antibiotic therapy did not affect eventual outcome. in both humans and animals, edema, congestion, hemorrhage, and degeneration of bronchiolar lining cells and alveolar type i and ii cells follows early in the course (fig. 5.4) . after 4 hours alveoli are filled with polymorphonuclear neutrophils (pmns) and fibrin. hyaline membranes are formed by 48 hours, providing a histologic picture of diffuse alveolar damage (dad) (see chapter 4) . resolution begins at about 72 hours and may either lead to figure 5.4 . gastric acid aspiration. acute effects show hemorrhagic necrosis of lung parenchyma. complete restoration of alveoli or leave some residual scarring. of course repeated aspiration may lead to combined acute, subacute, and chronic appearances. in his review, bartlett 22 referred to this rapid and irreversible type of injury as comparable to a "flash burn," and noted that little can be done to prevent injury once acid has made contact with the lung (fig. 5.4 ). the reactions just described plus fluid extravasation help to dilute the acid, as do buffering components from serum and cell breakdown products, but these only occur after injury. more recent studies have indicated that the mechanism of lung injury following acid aspiration extends beyond the direct chemical effects of acid to involve various inflammatory mediators including tumor necrosis factora (tnf-a), interleukin-8 (il-8), adhesion molecules, and cyclooxygenase and lipoxygenase products. 32 -35 the role of reactive oxygen species, and the adverse effect of oxygen administration after an episode of aspiration were demonstrated experimentally by nader-djalal et al. 36 a primary role is currently placed on neutrophils and complement in mediating lung injury in this setting. 27 ,32,37.38 thus, aspiration pneumonitis represents a biphasic response composed of early-onset direct pulmonary injury due to acid, followed by delayed injury due to acute inflammation. 26 ,38 low-grade chronic aspiration of gastric content may escape easy detection. these occult aspirations may lead to interstitial fibrosis, and perhaps account for the 20% to 54 % incidence of associated and unexplained pulmonary fibrosis in patients with esophageal abnormalities, most commonly hiatal hernia or simple reflux,39,40 the role of reflux in asthma, chronic bronchitis, chronic cough, recurrent pneumonia, cystic fibrosis, and sudden infant death syndrome has been reviewed by allen et al. [see also section on cholesterol (endogenous lipid) pneumonia in this chapter].41 children, particularly those aged 1 to 5 years, are likely to ingest various lighter hydrocarbons, mostly lighter volatile petroleum distillates. these products include kerosene, turpentine, and other paint thinners; furniture or shoe polish; lighter fluid; gasoline; dry cleaning fluids; and some insecticides. the toxicity is greater with those products that disperse most easily, specifically those that cause the greatest decrease in surface tension, or have the least viscosity or the highest volatility. 42 although ingestion precedes aspiration, eade and associates 43 nicely reviewed the reasons that aspiration is the most important toxic pathway of injury. symptoms develop rapidly and radiographs often show localized pulmonary infiltrates, often in the aspiration zones mentioned earlier. experimentally, the lethal dose by figure 5.5. lentil bean. a. at top is thick outer coat; below, cotyledon compartments with starch cells. b. cellulose framework of legume cotyledon compartments is birefringent under ingestion alone is much higher than that usually ingested by persons who subsequently aspirate. sizable doses of distillates have been placed in the stomachs of experimental animals whose esophagi were ligated, and these animals did not suffer pulmonary toxicity. the pulmonary changes, almost identical to those of gastric acid aspiration, include diffuse congestion, hemorrhage, edema, hyaline membrane formation, and bronchopneumonia. atelectasis occurs early, apparently by direct toxic effect of these light hydrocarbons on s urfactan t. 44 about 75% of affected children have abnormal chest radiographs, but only 25% to 40% have pulmonary signs or symptoms. 43 ,45 in the past, death has been reported in about 2% to 10% of cases, but in two large series death occurred in 0.3% and 1 %, respectively.43a5 death usually occurs within 24 hours of exposure. most survivors experience few sequelae. various food particles, such as skeletal muscle, fat tissue, or fragments of bone, may be aspirated and identified histologically in lung tissue. cooking or digestion may result in poorly defined particles that appear foreign but defy further definition. as was well demonstrated by knoblich 46 and others,47-49 portions of legume seeds are one of the better markers of food aspiration. the legumes most commonly eaten are various peas, beans, and peanuts; because they are relatively inexpensive and nutritious, they occur in many products. (the word lentil is sometimes used in these references, but it is also j.f. tomashefski, jr., and d.h. dail polarized light. c. degenerated aspirated vegetable material retains compartmental structure and stains strongly with gomori methenamine silver. the name of a specific type of bean). the legume seed ( fig. 5 .5) consists of a thick cellulose outer coat, the cellulose walls of the inner food storage compartments, and the starch cells contained within these food compartments. cooking softens the outer shell and cell walls of the beans or peas and allows easy disruption of the content, with a resultant jelling effect of the starch particles (called "thickening" in cooking). the cellulose walls of the outer coat and starch compartments are more difficult to totally disrupt or digest, and therefore act as both a chronic irritant and a good marker of aspiration. aspiration of these fragments in both experimental animals and humans produces an acute exudative response within 24 hours (fig. 5.6 ), followed by a foreignbody giant cell reaction ( fig. 5.7) . these cell wall fragments are gomori methenamine silver (gms) positive, and usually birefringent under polarized light (fig. 5.5 ). the glycogen compartments, when intact, are vividly periodic acid-schiff (pas) positive ( fig. 5.7 a) . starch cells may be mistaken histologically for parasite larvae (fig. 5.6b ).47 at about 10 days (experimentally) an organized granulomatous reaction occurs around the aspirated particles, and eventually the starch cells disappear, leaving only the cellulose fragments. 48 the walls of carrots, onions, and most nonlegumes digest more readily and do not give rise to as much exuberant chronic reactions as seen with legumes. they do, however, undergo the same type of early changes if aspirated, and initiate acute and subacute pneumonia during digestion of the starch cells. some of the most offensive aspirated food fragments have undergone alterations in preparation, and some of the worst combinations are cooking oils and salts, as, for example, an aspirated potato chip ( fig. 5 .7b). eventually these areas become small fibrotic or fibrocalcific nodules, which may appear almost as degenerated parasites or as sclerosed blood vessels ( fig. 5.8 ). they may appear as small hyalinized granulomas or possibly as entrapped calcospherites, usually within a fibrous stroma. 46 at times the conditions for aspiration are chronic, and recurrent aspiration leads to the acute and chronic changes together or in close proximity in the same specimen. 50 respiratory bronchioles exhibit marked remodeling associated with proliferative bronchiolitis obliterans, foreign-body granulomas, and entrapped food particles. 51 the macroscopic appearance is that of scattered yellow miliary nodules that are reminiscent of miliary figure 5.8. chronic reaction from aspirated lentil beans. two hyalinized starch cells may be mistaken for fibrosed, obliterated blood vessels. 53 matsuse and colleagues 52 have designated this condition as diffuse aspiration bronchiolitis, which they observed in 31 of 4880 consecutive autopsies (0.64%). the mean age of patients with this condition was 81.2 years. affected individuals frequently were bed-ridden or had dysphagia due to underlying neurologic disorders. 52 high-resolution computed tomography (hrct) may show a striking pattern of centrilobular miliary opacities. 54 in chronically ill or hospitalized patients, aspirated medicinal products, such as intact or partially digested pharmaceutical tablets, may be associated with aspiration pneumonitis. inert tablet components such as microcrystalline cellulose, talc, and crospovidone may be identified histologically in conjunction with aspirated food, pneumonia, and foreign-body reaction (see chapter 26) 55 microcrystalline cellulose, like legume components, is brightly birefringent on polarization, and also positive with gms stain. 55 it can be distinguished from vegetable particles, however, by its fiber-like, or "matchstick-like" appearance. 55 aspirated tablet filler components, which reside within alveoli and bronchioles, usually can be discriminated from identical particles introduced by illicit intravenous injection of aqueous tablet suspensions, which localize within small pulmonary arteries or in a perivascular, interstitial distribution (see chapter 26) . aspirated sodium (or calcium) polystyrene sulfonate (kayexalate), a potassium-binding cation exchange resin, has a distinctive histologic appearance characterized by large dark eosinophilic or basophilic, angulated, "glassy" particles, that sometimes appear striated ( fig. 5.11 ). [56] [57] [58] [59] duct. b. angulated, "glassy" kayexalate particles have elicited a foreign-body giant cell reaction. kayexalate particles are weakly birefringent, and positive with pas, acid-fast, and gram stains, but negative with von kossa stain. 60 kayexalate has also been identified in tissue sections by infrared microspectrophotometry.6l in humans and experimental animals, kayexalate has been shown to produce a necrotizing or organizing pneumonia. 6l ,62 more frequently, kayexalate is a cause of mucosal ulcers of the gastrointestinal tract. 60 ,63 kayexalate histologically closely resembles the less frequently encountered material cholestyramine. 60 cholestyramine, however, is more opaque and pink rather than red on acid-fast staining. 60 the aspiration of an intact ferrous sulfate tablet may induce severe, potentially fatal, bronchial mucosal ulceration and hemoptysis. 64 -66 the endoscopic appearance of the ulcerated bronchus typically is of a golden-brown discoloration ( fig. 5.12a ).6465 histologically, ulceration, necrosis, foreign-body reaction, and brown or yellow pigment that stains blue with prussian blue stain may be seen ( fig. 5.12b ). the pathogenesis of this syndrome, colorfully termed "iron lung," is thought to be a chemical burn induced by oxidation of iron from the ferrous to the ferric form. 64 the radiographic contrast material barium sulfate (bas04) is also readily visualized by chest x-ray in patients who aspirate this material. barium is a fairly inert white powder that tends to produce minimal functional lung impairment. grossly, following barium aspiration, lung parenchyma is chalky, tan-gray, and slightly indurated ( fig. 5.13a ). histologically, fine, golden-tan, refractile, weakly birefringent particles of barium sulfate are present within the cytoplasm of alveolar macrophages. with chronicity, or upon repeated aspiration, barium-laden macrophages migrate into the interstitium (fig. 5.13b ). inhalation of barium in the industrial setting (barytosis) is discussed in chapter 26. activated charcoal is sometimes given therapeutically for oral drug overdose. however, if the airway is not protected, charcoal may be aspirated in copious amounts, causing "charcoal lung," in which coarse black carbon particles obstruct small airways. 55 oils that may be aspirated include mineral oils such as used in nose drops and cathartics, vegetable oils used in cooking, and animal oils such as cod liver oil or fat-soluble vitamin preparations. mineral oil, derived from petroleum products, is the most common agent of exogenous lipid pneumonia. 68 oil aspiration was first described by laughlen 69 in 1925 in a child who received oily oral and pharyngeal preparations for diphtheria; it was confirmed by him experimentally. the role of oil-based contrast media used for bronchoscopy was well reviewed by spencer. 70 animal oils cause a more severe inflammatory reaction than mineral or vegetable oils, and this difference appears related to the number of free fatty acids and increased viscosity of animal oi1. 42 ,71.72 mineral oils are fairly inert, as they have no fatty acids, and are rapidly emulsified and consumed by pulmonary macrophages. vegetable oil droplets may remain in alveoli for months without eliciting significant reaction, but eventually, due to low-grade chronic irritation, they cause scarring. animal and mineral oils, but only rarely vegetable oils, may be seen in regional lymph nodes. 71 aspiration of oils commonly occurs in older individuals, who may take oily nose drops or cathartics at bedtime. aspiration most frequently gravitates to the basilar segments of the lower lobes suggesting these patients usually sleep in a more upright position or experience aspiration before reclining.73 because of its weakly irritative nature, mineral oil can enter the tracheobronchial tree without stimulating glottic closure or cough reflex. 74 only two of 14 cases documenting exogenous lipid pneumonia at autopsy had reported significant clinical symptoms during life. 75 because these oils float in the stomach, it is possible they also are aspirated via reflux from the stomach,z2.75 in unselected autopsy series, oil aspiration has been documented in 2.5% to 14.6% of adults. 75 .76 other oily products that have been incriminated as being aspirated in the lung include fragments of lip balm, burning fats (an occupational exposure), a rapid drying agent in spray enamel paint, oils applied to tobacco products (blackfat tobacco), and possibly hair spray.77-81 children may aspirate oily medications if they are force-fed these while resisting and crying violently.82 atypical mycobacteria, particularly rapid growers such as mycobacterium jortuitum or m. chelonae, have been reported associated with oil aspiration pneumonia (see chapter 9) . [83] [84] [85] [86] [87] symptoms of lipid aspiration include fever (39% of patients), weight loss (34%), cough (64%), and dyspnea (50%), although in one large study lipid pneumonia was an incidental finding, without associated symptoms, in 41 % of patients. 68 lung function tests may indicate either obstructive or restrictive changes. computed tomography (ct) scans usually show alveolar consolidation or groundglass opacities in the lower lobes. subfissural clear zones may be interposed between densities, creating a "sandwich effect" on ct scan. 68 grossly, lungs affected by oil aspiration are often gray to yellow and rather solid (fig. 5.14a ). dense localized fibrotic lesions (paraffinomas) may grossly mimic cancer or complicated pneumoconiosis. 88 ,89 occasionally oily droplets exude from the cut surface. microscopically the lipid droplets are often dissolved by tissue processing. 71 one exception is cod liver oil, which remains as salmoncolored droplets on hematoxylin and eosin stain after tissue processing. fats may be seen with rapid watersoluble or oil red 0 stains on frozen section ( fig. 5 .15d); variably sized fat droplets and varying numbers of multinucleate giant cells are present ( fig. 5 .15a-c). when only a small amount of fat has been aspirated, the reaction may be contained in alveolar macrophages. this is most commonly seen in mild degrees of aspiration with a diluted fatty substance, as might be seen with milk aspiration. when larger doses of thicker and more toxic oils are aspirated or when oil aspiration is repeated, the areas become densely fibrotic with reduction of the background lung architecture ( fig. 5.14a ).89 occasionally, cor pulmonale results,90.91 transbronchial biopsies may provide enough tissue to make this diagnosis. precise identification of specific lipids can be determined by infrared spectrophotometry. 88 in the differential diagnosis is artifactual collapse of lung around remnant air bubbles (see fig. 1 .2c in chapter 1). exogenous lipid pneumonia can usually be distinguished histologically from endogenous lipid chronically ingested mineral oil as a laxative. note yellow oil layered on the surface of fluid. (cholesterol) pneumonia by subdivisions within fat droplets, coarse cytoplasmic vacuoles in macrophages, multinucleated foreign-body giant cell response, and, in more chronic cases, a greater degree of chronic inflammation and fibrosis with destruction of background lung parenchyma in exogenous lipid pneumonia ( fig. 5.15 ). at times some lipid is incorporated/entrapped in the interstitium ( fig. 5.15c ), resembling a similar appearance in diffuse pan bronchiolitis and xanthomatous bronchiolitis obliterans (see below and chapter 25). diffuse panbronchiolitis is centered more on terminalrespiratory bronchioles and is composed mostly of finely vacuolated fat. sputum cytology or cytologic aspiration specimens have been used to confirm lipid pneumonia. on bronchoalveolar lavage an oily layer is sometimes present on the surface of the collection tube ( fig. 5.14b ). in 1950, losner et al.,92 using oil stains, found lipid-rich macrophages in 19 of 20 suspected cases in contrast to two of 45 control patients. more recently, corwin and irwin 93 restudied this situation using bronchoalveolar lavage in various lung diseases including aspiration, hemoptysis, cancer in the lung (either primary or secondary), bronchiectasis, interstitial fibrosis, and sarcoidosis. when compared to normal lungs, samples from diseased lungs in general contained increased fat-filled macrophages. these authors warned that the simple presence of fatty macrophages in cytology preparations is not diagnostic of lipid pneumonia; however, the quantity of lipid was more abundant in aspirators than in these other groups. their figure 5.15. oil aspiration. a. subacute effects of oil aspiration show varyingly sized fat droplets, inflammation, lymphoid aggregates, and fibrosis. most of the lung architecture has been obliterated. b. higher power view shows foamy lipid-filled macrophages and multinucleate foreign-body cells. c. in the chronic state, oil droplets are still seen within the interstitium with "aspirator" group consisted mainly of patients with a history of upper gastrointestinal tract disease, including reflux in most. in young children the presence of numerous (>50) oil red o-positive lipid-laden macrophages on tracheal aspirate is highly specific for aspiration. 94 corwin and irwin emphasized that the size of the fat droplets in lavage fluid cannot be used to distinguish endogenous from exogenous lipid pneumonia. wherever it occurs, an abscess is a localized accumulation of inflammatory cells, initially having abundant neutrophils, that is usually accompanied by tissue destruction. in the lungs, "cross-country" necrosis occurs during the formation of an abscess (figs. 5. 16 bronchi, and arteries. in contrast, cavities that are more chronic and more slowly formed, such as tuberculous cavities, often leave remnants of fibrotic bronchopulmonary rays coursing through the cavity itself (see chapter 9) . some more slowly forming nontuberculous abscesses can do this, but most of the abscesses in the lung have an acute initial phase that destroys most of the tissue in the area ( fig. 5.17b ). bronchi frequently connect with abscess cavities, allowing drainage of the necrotic material, leaving an empty or partially empty cavity with or without an air/fluid level on chest radiograph (figs. 5.18 and 5.19). occasionally, inflammation seals off all such bronchial connections, resulting in a solid mass that may be suspected of being a tumor. adjacent organization in acute and subacute abscesses often accounts for an enlarged surrounding radiographic density (figs. 5.17 and 5.18). there are many etiologies for cavity formation in the lung, and abscess formation is but one of them. other pulmonary abscess formation, which were already well known by 1922, were summarized in this review. by 1936, a total of 2114 cases had been published. 97 aspiration is the most common cause of lung abscess. other instigators of this type of damage include b. gross specimen. note cavity is mostly drained, with freshappearing, thin lining without fibrous wall. note also persistence of some trabeculae, presumed bronchopulmonary rays, and variable but narrow surrounding inflammatory reaction. penetrating trauma, postoperative states, obstruction, hemorrhage or infarction, necrotizing pneumonia, infected emboli, infection of a preexistent cyst or bulla, or extension from nearby infected areas in the mediastinum, chest wall, diaphragm, or infradiaphragmatic locations. nonaspiration types of pulmonary abscesses of course do not follow the aspiration patterns of distribution, but occur as their coexistent factors dictate. for example, if there is an infarct or an obstructing tumor, infection would occur in the affected areas. septic emboli are hematogenously spread, and resultant abscesses are often multiple, small, and peripherally distributed (see fig. 28 .29 in chapter 28). as aspiration is the principal cause of pulmonary abscess formation, it is reasonable that conditions that favor abscess formation are identical to those favoring aspiration. 98 the locations are similar; men are more frequently affected than women; and the right lung is involved twice as often as the left. 98 -103 the posterior segments of the right upper lobe and superior segments of the right lower lobe are involved most frequently, followed by the corresponding segments on the left side. 103 the single most commonly associated event is an alteration of consciousness; the second most frequent association is poor dental hygiene; and the third is an immunodeficient status. poor dental hygiene was noted in cases without other apparent causes of pulmonary abscess in the early studies between 1927 and 1936.103-108 the spectrum of bacteria involved in abscess formation is almost identical to that of endogenous oral flora. 109 -114 moreover children and edentulous older people do not often develop lung abscesses.ll5 in children cases caused by aspiration must be separated from other cases of cavitary necrosis, such as pneumatoceles in primary staphylococcal pneumonia. 116 -1l8 anaerobic bacteria are the only organisms cultured in about one half to two thirds of lung abscesses; in the remaining cases either aerobic or facultative aerobic bacteria are isolated, or no bacteria are culturedys the anaerobic bacteria most frequently found are peptostreptococci (i.e., gram-positive anaerobic cocci), pigmented gram-negative bacilli (including bacteroides), and the fusobacteria.98.1l9.120 spirochetes were described morphologically in earlier studies and seemed to be significant, as they were present in the growing rims of necrosis; however, they have not been mentioned much recently, perhaps because they are difficult to culture.los. 121 about half of cultures with anaerobes also contained aerobic bacteria capable of necrosis, specifically staphylococcus, streptococcus, haemophilus, pseudomonas, klebsiella, and escherichia spp.122 patients with predominantly anaerobic pulmonary abscesses often present with indolent symptoms, in contrast to those with necrotizing aerobic abscesses. the latter may be more common in nosocomial-acquired lung abscess. 9s -98 immunocompromised patients often acquire gram-negative necrotizing pneumonias and vasoinvasive necrotizing fungal pneumonias, both of which may lead to cavitation. one clue to aspirated bacteria in lung abscesses is finding mixed-type organisms on smear gram stain, tissue gram stain, or culture. the oral cavity abounds with mixed bacteria and is estimated to contain some 200 different types of organisms. 121 this is one reason sputum cultures are notoriously difficult to interpret, and why even oral contamination of a bronchoscope interferes with most lung cultures. 122 transtracheal and transthoracic needle aspirations, however, correlate well with blood culture results. i 19. 123 the third most frequent factor in abscess formation is host response. factors that compromise normal host defenses include alcohol ingestion, diabetes mellitus, renal failure, malnutrition, malignancy, and other debilitations, along with treatment with immunosuppressive agents for any reason. patients with these factors do more poorly with pulmonary abscesses (and most other insults) than those without. [124] [125] [126] pathologically, acute cavities have only a thin transition zone into the reactive adjacent lung parenchyma (see numbers of neutrophils and macrophages, along with tissue necrosis. the nearby lung parenchyma has variable findings depending on the rapidity of spread. rapidly growing cavities necrose nearby lung parenchyma, destroying any early attempts at organization. there may be adjacent hemorrhage, exudate, and fibrin extravasation. in those that are slightly more stable, beginning organization occurs in the surrounding lung parenchyma, sometimes chronic cavities heal with a thin fibrous border and may retain a coagulum of necrotic debris in their lumen. this apparently indicates that the nearby bronchi have been sealed off. chronic cavities may resolve by collapse and fibrosis, or may remain open. in the open variety they may become reepithelialized, first with a squamous lining and then with ciliated respiratory lining. 127 in the latter case, the distinction from bronchiectasis or bronchocele may be somewhat confusing, but multiple bronchial connections in a chronic abscess cavity distinguish these entities. spontaneous healing may occur, but healing is greatly aided by appropriate antibiotic dosage. weiss l28 noted in appropriately treated and monitored cavities that 13% of cavities disappeared by 2 weeks of therapy, 44% by 4 weeks, 59% by 6 weeks, and 70% by 3 months. surgery is sometimes indicated for nonhealing cavities or when complications develop, such as hemoptysis, persistent sepsis, bronchopleural fistulas, or empyema. the incidence of abscess formation has greatly decreased during the past 50 years, partly because antibiotics are available and frequently used early in pulmonary infections, and partly because factors leading to of fibrin (2) resting on cellular granulation tissue (3). b. wall of chronic abscess cavity composed of dense, fibrotic, mature granulation tissue. abscess formation are better understood; for example, surgery is avoided with the patient in the upright position or on patients with food in the stomach. in the pre antibiotic era approximately 34% of the patients treated either conservatively or by surgery died, and another one third had chronic residual lung disease. 97 more recently the prognosis is much better, but the mortality associated with established abscess formation remains in the range of25% ? although the term gangrene of the lung has been applied to necrotizing, sometimes putrid, pneumonia in any location, it has more recently been used more specifically to indicate massive necrosis and sloughing of lung associated with severe infection. this entity almost always involves the upper lobes, usually on the right side, and radiographically evolves through typical changes of diffuse infiltrate into multiple cystic spaces that become confluent, leaving a crescent of lung density compressed medially, or occasionally laterally, with a final walled-off area of pus and necrotic lung in an otherwise empty structureless space. curry and colleagues l29 and penner et a1. 130 attribute the first description of pulmonary gangrene to laennec in the 1820s. phillips and rao,l3l who reported four cases, refer to sir william osler's132 description of diseased lung "converted into a horribly offensive greenish, black mass, torn and ragged in the centre" (fig. 5.21) . the patients may cough up large pieces of necrotic lung, which in one case was therapeutic. 130 these fragments histologically show ghosts of lung parenchyma and thrombosed vessels. vasculitis has been described in some reports. 129 ,130 assorted microorganisms cultured from these cases include klebsiella pneumoniae, pseudomonas aeruginosa, haemophilus injluenzae, staphylococcus aureus, streptococcus pneumoniae, and mucor species, 131 anaerobic bacteria probably also playa significant role. 130 in their reviews, phillips and rao l3l and penner and colleagues130 note that similar predisposing factors as those with community-acquired pneumonia, such as aspiration and abscess formation, pertain to this entity, but the location helps distinguish it from the other typical sites of aspiration, when in the upper lobes, it appears to progress through necrotizing pneumonia with thrombosis of arteries (pulmonary and bronchial) and veins, [129] [130] [131] although not strictly abiding by the foregoing definition (of localization in upper lobe), in one case total unilateral lung gangrene was attributed to hilar vessel involvement following treatment of a massive hilar recurrence of hodgkin's disease. 133 pulmonary gangrene is life threatening, and surgical removal of necrotic lung tissue if. tomashefski, jr. , and d.h. dail figu re 5.21. pulmonary gangrene, lung parenchyma is greenish-black, necrotic, and cavitated in this specimen from a patient with central bronchogenic carcinoma, bronchial obstruction, pulmonary artery invasion, and thrombotic occlusion. note hyperemic rim between necrotic lung and apical lung parenchyma. proteus, e coli, and enterococcus cultured, is often indicated since only a few patients survive with antibiotic therapy alone,134 an entire lung, or even one lobe, may rotate around the hilar structures and cause congestion, hemorrhage, or infarction,135 cases of torsion involving only single lobes most commonly occur postoperatively when a portion of ipsilateral lung has been removed,135,136 torsion is an infrequent event. a review of the literature in 1994 by schamaun 137 documented 17 cases of postoperative torsion, five posttraumatic occurrences, and four spontaneous events. in a poll of british thoracic surgeons, however, 35 of 117 responders (30 % ) had encountered at least one instance of torsion,137 torsion has also been documented to occur in transplanted lungs or as a result of pneumothorax or mass lesions. 138 ,139 due to the simultaneous compromise of pulmonary and bronchial arteries and the pulmonary vein, fatal gangrene may occur (see above), the entity of obstructive, golden, or endogenous lipid pneumonia is most commonly seen secondary to tumor obstruction of large airways, but any of the causes of obstruction can lead to obstructive pneumonia. 140 the obstructive effect accounts for a much larger infiltrate on the usual chest radiograph than is caused by tumor alone. the involved area is primarily supplied by the affected bronchus, and the larger the obstructed bronchus, the greater the area involved. however, even when obstruction occurs in the smallest bronchioles, there may be secondary effects in the centriacinar regions. cholesterol pneumonia may also spread into the adjacent nonobstructed segment, and occasionally throughout the lobe. 141 the latter pattern of disseminated spread is often associated with more poorly differentiated or cavitated carcinomas. 141 the involved lung is reduced in size, but not to the extent expected in simple atelectasis. the difference is due to the infiltration by abundant inflammatory cells. the microscopic hallmark of obstructive pneumonia is flooding of air spaces initially by edema followed by fat-filled, finely vacuolated, so-called foamy alveolar macrophages (fig. 5.22a golden-yellow color, hence the term golden pneumonia. 142 obstructed secretions, increased cell breakdown products, and possibly leakage from vessels and interstitium, may give rise to the fat seen in this characteristic reaction. as these products are derived from the lung, this is called endogenous lipid pneumonia. early in its course the alveolar outlines are well defined though distended with foamy macrophages. if the pneumonia is rapidly reversed, lung function may return. gradually, permanent damage ensues, including fibrosis and vascular sclerosis, and it is then difficult to restore lung function even though the obstruction may eventually be reversed. some degree of intraalveolar organization may also be present in approximately 57 % of cases. l44 features of superimposed infection, such as acute inflammation, necrosis, or abscesses are seen in a minority of cases. 142 in contrast to exogenous (aspiration) lipid pneumonia, endogenous lipid pneumonia is characterized by finely vacuolated fat, absence of a foreign-body response, and minimal inflammation and fibrosis of the underlying lung architecture (figs. 5.15 and 5.22b,c). sometimes there are changes that suggest pulmonary alveolar lipoproteinosis (see below). 145.146 at times parenchymal changes very similar to those just described may be present, although no obstruction of bronchus can be identified, so-called idiopathic cholesterol pneumonia. 147 ,148 in 1949, robbins and sniffen 148 described 11 cases of chronic nonobstructive cholesterol pneumonia, 10 of which (91 %) occurred in men aged 32 to 67 years, the only female being a 12-year-old girl. in extent, five of their cases involved most of the lobe and six cases included a portion of one or more segments, often with pleural adhesions; some had small abscess cavities. the involved areas were wedge-shaped with their bases on the pleura and were described as bright yellow; they were accounted for histologically by abundant, finely vacuolated foamy macrophages, but otherwise these areas presented as mass effects. mucoid or mucopurulent exudate filled some bronchioles and bronchi, but no other cause of obstruction was present. bronchiectasis was absent, although focal necrotizing bronchitis was noted. the lobar distribution was not documented. these authors argued against aspiration as they noted the usual aspiration to be more diffuse, often multifocal, and more often seen in lower lobes. lawler able foreign material in 11 (37%) and strongly suspected aspiration in another six for a total of 57% in this series. all except two cases (82 %) of confirmed aspiration were solitary lesions. it seems reasonable that at least some of both chronic organizing pneumonia and idiopathic cholesterol pneumonia, even when not so confirmed, may be the result of aspiration, usually involving ingredients other than exogenous lipid. secondary chronic organizing pneumonia, involving large portions of a lobe or presenting as a mass lesion, is not to be confused with the distinctive form of interstitial lung disease termed cryptogenic organizing pneumonia (bronchiolitis obliteransorganizing pneumonia), which is further discussed in chapter 4. fisher et al. 153 have described a series of patients (six children [<13 years of age] and two adults) with progressive diffuse interstitial lung disease having a combination of histologic features including endogenous lipid pneumonia, interstitial cholesterol granulomas, and patchy alveolar proteinosis (fig. 5.23 ). three patients in this study had severe combined immunodeficiency, two had pulmonary hypertension, and one each had cystic fibrosis (cf), trisomy 10q, and ventricular septal defect (vsd), or lysin uric protein intolerance. all patients exhibited delayed growth, five had digital clubbing, six had depressed appetite or anorexia, five were anemic, and three experienced hemoptysis. six of eight patients also had evidence of gastroesophageal reflux, which the authors suggest is important in the pathogenesis of this condition. 153 pulmonary function tests in four patients showed either restrictive or mixed restrictive and obstructive physiology. chest x-rays predominantly showed nodular pulmonary opacities, while bronchiectasis and perihilar or mild hazy parenchymal infiltrates occurred in one patient each. 153 the mechanism whereby gastroesophageal reflux might elicit this interesting triad of histologic findings is through recurrent micro aspiration of gastric content with associated bronchospasm (see discussion of alveolar proteinosis in chapter 21). 153 the differential diagnosis of cholesterol pneumonia also includes drug reactions, notably reactions to amiodarone. the clinical history and documentation of amiodarone therapy should facilitate the correct diagnosis (see chapter 22 and fig. 22.8) . while the foamy macrophages in both of these disorders resemble each other histologically, electron microscopy demonstrates more abundant osmiophilic dense bodies and giant lamellar bodies in many different cell types in amiodarone toxicity compared to similar but smaller inclusions within macrophages in endogenous lipid pneumonia.146.153~156 compared to endogenous lipid pneumonia, amiodarone toxicity also encompasses a prominent inflammatory response that may include fibrosis, organizing pneumonia and diffuse alveolar damage (see chapter 22) . 156 other drugs that have been implicated as a cause of endogenous lipid cholesterol granulomas not only are associated with endogenous lipid pneumonia and pulmonary alveolar proteinosis, but also have been attributed to pulmonary hypertension, organizing hemorrhage, and a unique case of excessive consumption of apples.160-162 kay and colleagues 163 suggest that cholesterol granulomas in patients with pulmonary hypertension are more likely due to other concomitant processes characterized by type ii pneumocyte hyperplasia and degeneration. atelectasis is the collapse of aerated lung. most often it is caused by internal bronchial obstruction of the air flow (absorption atelectasis) (see fig. 15 .2 in chapter 15), but it may result from external compression of the lung, such as by empyema, mesothelioma, constrictive pleural fibrosis, or tumors, or by internal compression, secondary to a bulla, tumor, or other space-occupying lesions (compressive atelectasis). pneumothorax is an important cause of atelectasis in the ipsilateral lung. atelectasis may also be caused by a change in metabolism or surface-wetting balance such as with hyaline membrane disease, ards, infection, or gastric acid or other aspiration with loss of pulmonary surfactant. it may also have vascular causes as with embolism, postoperative splinting, obesity (e.g., pickwickian syndrome), or secondary to nerve or muscle dysfunction of diaphragm or chest wall. following complete airway obstruction, atelectasis occurs when alveolar oxygen and nitrogen are absorbed. atelectasis is also commonly seen around chronic inflammatory reactions such as bronchiectasis, and may be part of the sequence of events leading to bronchiectasis (see below). pathologists must be cautious, however, because most often, when observed histologically, atelectasis is artifactual. upon the release of negative pressure, air escapes from the lung when the thorax is opened or when lung tissue is excised, and this collapse may cause confusion with preexisting atelectasis (see fig. 1 when bronchial obstruction is partial, it may easily lead to air trapping, as discussed in chapter 15 (see asthma). obstruction of segmental bronchi usually does not cause atelectasis because of preserved collateral ventilation. a collapsed lung may be an isolated cause of fever, but is also a frequent site for superinfections, such as in postoperative patients. 164 chronic atelectasis may lead to irreversible scarring. the special situation of rounded atelectasis is discussed in chapter 27 on asbestos-related pathology. bronchiectasis simply defined refers to dilatation of bronchi. included in this broad definition are conditions such as traction bronchiectasis secondary to parenchymal scarring; airway dilatation accompanying parenchymal loss as in emphysema; or reversible dilatation, which may be seen radiographically in atelectasis or pneumonia. 165 -172 a more selective definition of bronchiectasis, and the type usually understood by pathologists, is irreversible fixed airway dilatation associated with inflammation and destruction of bronchial matrix components. 173 • 174 bronchiectasis can be further categorized as localized or diffuse/multifocal. causes of localized bronchiectasis, the most important of which is airway obstruction, are listed in table 5 .1. localized bronchiectasis may also have an infectious etiology, most notably pulmonary tuberculosis (see fig. 9 .13 in chapter 9). obstructive bronchiectasis is most commonly seen beyond endobronchial tumors (fig. 5.24 ), but foreign bodies, concretions such as broncholiths, secretions such as inspissated mucus in mucoid impaction and allergic bronchopulmonary aspergillosis (see chapter 15), strictures, or compression as by tumor or enlarged nodes may play a role. rarely, lack of cartilaginous support with airway collapse, bronchial atresia, or mucosal webs may be associated with bronchiectasis. obstructive bronchiectasis occurs anywhere obstruction occurs, but there are some localizing factors in a few of these conditions: the upper lobe in allergic aspergillosis or with primary epithelial tumors, which are more common in this site; the middle lobe with its tendency toward airway compression (middle lobe syndrome); and localized bronchiectasis governed by the usual routes of aspiration (covered earlier in this chapter). there are many exceptions, and bronchoscopy is usually indicated in both children and adults to diagnose the type of obstruction. localized bronchiectasis is often successfully treated by surgical resection or elimination of the cause of bronchial obstruction. diffuse or multi focal bronchiectasis is usually of the nonobstructive type, the major causes of which are listed in table 5 .2. it is this type that is more frequently a cause of significant chronic obstructive pulmonary disease (copd) and respiratory failure. nonobstructive bronchiectasis occurs most frequently in the basal segments of the lower lobes, often sparing the superior segment and the anterior basal segment. it is found more than twice as frequently in the left lower lobe as in the right. 17s -j77 next in frequency are the right middle lobe and its counterpart, the lingula. these areas of the lung may have the poorest drainage. the upper lobes may be involved but are usually not solely involved by nonobstructive bronchiectasis. tuberculosis more selectively causes bronchiectasis in the upper lobes, and cystic fibrosis should be considered in cases of upper lobe involvement without a definitive etiology. about one third of the cases of nonobstructive bronchiectasis have bilateral involvement. 176.178,179 macroscopically bronchiectasis typically involves the second to the eighth order of segmental bronchi, sparing the larger, more proximal airways, which are protected by a firmly supporting cartilaginous network. 179 -181 more distal airways are often obliterated or effaced as the number of bronchial divisions is reduced (fig. 5.25 ).173,182 within a bronchopulmonary segment there may be patchy involvement (see fig. 1.9 in chapter 1). there is also apparent loss of more distal lung parenchyma as the dilated airways approach the visceral pleura ( fig. 5.25) . 173, 182 bronchiectasis has been divided into many different patterns grossly and radiographically. the most widely applied classification is that suggested by reid l82 : (1) saccular (cystic), (2) cylindrical (fusiform or tubular), and (3) varicose. in the saccular form, the distal extensions of the bronchi are more dilated than proximal portions, described by reid as "globular ballooning." usually, the second-to fourth-order bronchi are involved. cylindrical bronchiectasis consists of evenly enlarged tubular dilatation of bronchi, usually involving the sixth-to eighthorder bronchi. is i on bronchograms dilated bronchi come to an abrupt, square-ended termination thought to be caused by impacted endobronchial secretion.173 the varicose type describes focal dilatation separated by more narrow areas (see fig. 1.9 in chapter 1). these various gross patterns were previously best visualized by bronchography, and are not specific for any given etiology, although certain clinical-pathologic correlations have been made (see below). whitwell 175 integrated histologic features into his classification of follicular, saccular, and atelectatic bronchiectasis. follicular bronchiectasis most frequently begins in childhood as the sequela of viral infections, most notably adenovirus. 183 saccular bronchiectasis, in whitwell's series, was often found to be postinfective or idiopathic. the pathogenesis of atelectatic bronchiectasis was linked to lobar bronchial obstruction, often by enlarged lymph nodes. congenital bronchiectasis, purportedly due to cartilage deficiency in the bronchial walls, is a controversial entity discussed elsewhere in this chapter under the williams-campbell syndrome. 180 ,1s1,184 not to be confused with congenital bronchiectasis are those hereditary conditions, such as cystic fibrosis or primary ciliary dyskinesia, that predispose to the subsequent development of progressi ve bronchiectasis (see below). 174, 180 patients with bronchiectasis typically present clinically with forceful cough, purulent sputum production, wheezing, and recurrent pneumonia in the bronchiectatic zones. wet bronchiectasis refers to abundant inflammation and mucus hypersecretion, whereas dry bronchiectasis refers to minimal sputum production. dry bronchiectasis is most common in the upper lobes, is often of the cylindrical type, and probably relates to better drainage in this zone. hemoptysis commonly presents as blood-streaked sputum, but may be massive and life-threatening. frequently purulent sinusitis accompanies bronchiectasis and may contribute to its development. the chest radiograph is usually abnormal in bronchiectasis, including specific features of ring-like shadows due to dilated airways seen on end, or of tram lines when the airways are visualized longitudinally. iss high-resolution ct scan is the best current modality for diagnosing bronchiectasis, revealing airways that are dilated relative to the adjacent blood vessels, lack of airway tapering, constrictions along the path of the airway, and terminal balloon-like cysts. is5 ,is6 pulmonary function tests show obstructive changes with reduced forced expiratory volume in 1 second (fev1)/ forced vital capacity (fvc) and frequently airway hyperresponsiveness. 185 various theories have been proposed to explain nonobstructive bronchiectasis. inflammation seems to best account for the changes that are observed, including the fact that the involved zones of lung are those most difficult to drain. it was known even in the 1930s and confirmed in subsequent decades that respiratory infection often preceded bronchiectasis. 175 ,176,187-192 usually older children and young adults have the well-developed disease pattern, but also have a history of infections before the age of 2 or 3 years, with recurrent respiratory problems dating from this time. 187 many patients may appear stable and do well for some time, and then develop a progressive course of recurrent infections and systemic toxicity. 174, [177] [178] [179] [180] [181] [182] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] in approximately 50% of cases of bronchiectasis, however, a specific inciting factor is not identified-so-called idiopathic bronchiectasis . 195 in these patients, childhood respiratory infections especially those likely to have produced bronchiolitis obliterans, are presumed to have initiated the process of bronchiectasis. 173 ,195,196 viral infections may be important in many cases. 167 ,194,196-203 glauser and associates 204 noted in an extensive review that measles and pertussis immunizations probably have played a significant role in decreasing the incidence of bronchiectasis. bacteria also playa significant role, both in primary infections (see following) and in superinfections or reinfections in areas of previous injury. aggressive treatment of pediatric pulmonary infections with antibiotics has helped to make bronchiectasis a disappearing disease. 204 historically, the impact of immunizations and antibiotics on the declining incidence of bronchiectasis occurred at about the same time, and it is difficult to differentiate their effects; nonetheless, this association supports the role of early infection in initiating bronchiectasis. excluding cases of kartagener's syndrome, the concurrence of sinusitis and bronchiectasis is greater than expected. as early as 1929, quinn and meyer20 noted a 58% incidence of chronic sinusitis in cases of bronchiectasis. aspiration of infective material from the sinuses may playa role. however, another study noted a 15% incidence of sinusitis in cases with less than 5 years of symptoms of bronchiectasis compared to the 44% incidence in all cases of bronchiectasis. 179 h. infiuenzae, a common pathogen of the upper respiratory tract, is also found with some regularity in lung cultures from patients with bronchiectasis. anaerobic bacteria, reflecting endogenous oral flora , may also be cultured from bronchial secretions. long-term antimicrobial treatment may be required for complete eradication of these organisms. the role of recurrent infection in perpetuating and aggravating bronchiectasis cannot be overemphasized. this has been documented in children and adults. the dilatations of the bronchial contours, their irregularities, their relative stenosis at the proximal end, altered secretions and exudate, surface mucosal ulcerations, and metaplasia all playa role in hampering adequate drainage. a vicious cycle ensues as the injured area perpetuates further injury, leading to increased damage and progressive bronchiectasis. necrotizing inflammation involves bronchial walls and adjacent parenchyma (fig. 5.26 ) . some scarring probably takes place in healing, with retraction of surrounding tissue. retraction occurs circumferentially, and bronchial dilatation results. as noted, the more distal bronchi and bronchioles are often destroyed. there is also general lung contracture due to atelectasis of involved zones, while nonaffected lobes may undergo compensatory hyperinflation (fig. 5.26) . the basic principles of fibrosis and contraction also apply to traction bronchiectasis seen in interstitial fibrosis and honeycombing.172 traction bronchiectasis is usually not as marked as primary bronchiectasis, and is localized and most severe in the peripheral subpleural zones where fibrosis is often most prominent (see chapter 19) . 172 grossly, the involved lung tissue is usually atelectatic, gray-blue, shrunken, and rubbery. there may be zones of golden or obstructive pneumonia, and sometimes these zones form layers around the dilated bronchial tubes. it may be difficult or impossible to adequately inflate such a chronically contracted specimen. the involved bronchi are dilated instead of following their smoothly contoured courses as they extend peripherally. these dilated bronchi almost reach the pleural surface and run in a somewhat parallel or radial fashion without interbronchial connections (figs. 5.25 and 5.26). partially or totally circumferential thin folds in the mucosa extend internally from the wall and are seen as transverse infolded pleats on the bronchial cast (see fig. 1.9 in chapter 1). these give the appearance of webs or bands of mucosa. there are variably sized outpouchings, larger ones between the remnant bronchial cartilages, and dilated smaller pits that appear to be dilated submucosal glands. grossly, elastic fibers can be seen still running through the wall, but these are more widely separated than is normal because of the stretched diameter of the bronchus. in wet bronchiectasis there is thickening of the wall, and mucinous, granular, semisolid material accumulates within the lumen (fig. 5.27 ). occasionally this material hardens and even calcifies (see broncholithiasis, below). in dry bronchiectasis the wall is thin, almost translucent, and gray-pink without mural thickening. microscopically the respiratory mucosa may be intact, show squamous metaplasia, or be ulcerated or inflamed (fig. 5.28 ). the bronchial walls are usually chronically inflamed. submucosal glands and surface goblet cells are not prominent and may decrease, although they may occasionally increase. elastic tissue is preserved except in areas of necrosis (fig. 5.28 ). smooth muscle is usually present and often shows some degree of hypertrophy; occasionally this is atrophic. cartilage seems less obvious and occasionally is eroded, but most often appears normal histologically. in advanced saccular bronchiectasis cartilage is markedly reduced or absent. 173 ,205,206 neutrophils, macrophages, and desquamated and mucinous debris are present in the bronchial lumen in wet bronchiectasis. acute inflammatory cells may infiltrate the bronchial wall or the adjacent lung parenchyma depending on the status of inflammation and active infection at the time of lung removal. as these patients are subject to recurrent infections, acute pneumonia may also be present. lymphocytes and plasma cells usually predominate in bronchial wall and surrounding lung tissue. in follicular bronchiectasis, hyperplastic lymphoid follicles may appear to constrict the bronchial lumens (fig. 5.29 ).175 there may be a degree of obstructive pneumonia correlating with the gross yellow color. small granulomas are present in a few cases, apparently as a reaction to inspissated material within the bronchi. if granulomas are extensive or present in the adjacent lung parenchyma, in more normally contoured segmental and subsegmental bronchi, or in lymph nodes, one must consider fungal or mycobacterial infections. if granulomas are confined to the injured areas, one must also consider aspiration. bronchioles are often constricted or obliterated beyond the dilated bronchi (fig. 5.30 ). other small airways may be dilated and sometimes mucus-filled probably because of their obstruction at the junction with the larger bronchi. foci of carcinoid atypical proliferation (tumorlets) occur with some frequency in bronchiectasis (see chapter 36) . bronchial arteries respond to sustained inflammation, and may exceed 1 mm in diameter. 207 ulceration of these systemic arteries accounts for the bright-red appearance of hemoptysis. the right middle lobe and occasionally its left-sided counterpart the lingula, have lobar bronchi that branch from their parent supply at a more acute angle than most other dividing bronchi (see chapter 2) . the middle lobe bronchus is relatively narrow, and there are frequently moderately prominent nodes in the angle of bifurcation that may compress and further constrict the bronchus. the subcarinal node may even approach this angle. several authors have also suggested there is less effective collateral ventilation in the middle, compared to the adjacent upper lobe. 208 .209 because of these anatomic characteristics there is a greater tendency toward middle lobe and lingular atelectasis, inflammation, nonspecific scarring, broncholith formation, and bronchiectasis-collectively termed middle lobe syndrome (mls) .2io in addition to peribronchial lymphadenopathy, mls can result from numerous disorders including asthma, tuberculosis, foreign bodies, cf, broncholiths, endobronchial silicosis, cardiovascular and of bronchiolar wall. white arrow indicates cross section of separate, obliterated airway (patient with cf) (elastic van gieson stain). bronchopulmonary malformations, and allergic bronchopulmonary aspergillosis. 2 11-213 the pathologic findings in resected lung specimens of 21 patients with middle lobe syndrome have been comprehensively described most recently by kwon and colleagues,212 and are delineated in table 5 .3. although the histologic findings are nonspecific, a combination of bronchiectasis, bronchiolitis, and atelectasis is typical. 212 in this series, a mechanical obstruction (broncholith) was identified in only one patient. 212 as early as 1966, culiner 208 also recognized bronchial patency in most cases of middle lobe syndrome. the current understanding suggests that mls is due to recurrent infection related to poor lung drainage, possibly associated with intermittent obstruction of the precariously situated bronchi in the setting of reduced collateral ventilation of the middle lobe.208.212 broncholiths represent calcified material in the airways.214-220 they most commonly are calcified lymph nodes that compress bronchi and either partially or completely erode through the bronchial walls (fig. 5.31 ).217.221 they then may be expectorated (lithopytsis) (fig. 5.32) calculus (scale equals 1 cm). c. rare yeast-like organisms, consistent with histoplasma, were identified in the necrotic center of the broncholith (gomori methenamine silver stain). or aspirated and cause hemorrhage or obstructive changes, including cough, atelectasis, pneumonia, abscess formation, bronchiectasis, or air trapping. broncholiths form less often from chronic reaction to retained aspirated material or eroded fragments of calcified or ossified bronchial cartilage. 18o they may also occur with retained mucus as in bronchiectasis. 180 historically, "spitting stones" dates back to descriptions by aretaeus, galen, and aristotle. 218 although usually less than 1 cm in diameter, a record-sized calculus of 139g c/3ib) occurred in a patient who also had produced multiple sand-like or melon-seed-sized calcified particles. 214 the pathognomonic finding of lithoptysis is fairly rare and was seen in only two of 43 (5%) cases by faber et al. 218 and six of 41 (15%) cases by schmidt et au 16 the regional nodes usually calcify from old granulomatous disease, and tuberculosis is the most common etiology worldwide while histoplasmosis is the most common etiology in the united states. 219 other infectious agents include coccidioides, cryptococcus, actinomyces, or no cardia. 219 ,22o,222,223 the latter two organisms probably represent superinfections of necrotic debris.224 silicotic lymph nodes may also cause a similar reaction. 225 ,226 men and women are about equally affected, and although calcified nodes may occur at any junction of the bronchial tree, they are 2 to 6.5 times as common on the right side, and favor the anterior superior segment of the upper lobe and the bronchus intermedius, along with the right middle lobe bronchus, where they may produce the middle lobe syndrome. 217 ,227,228 the superior segment of the lower lobe is also a site of occurrence. occasionally, erosive calcified nodes may cause bronchopleural fistulas and are the most common cause of bronchoesophageal fistulas, [228] [229] [230] retraction diverticula of the esophagus may also occur secondary to peribronchial fibrosis and calcified mediastinallymph nodes associated with broncholiths. 218 ,231 calcified nodes have also been studied with ct scans.232 in the retrospective series by conces et al.,227 of 15 patients with ct-proven broncholiths, 11 (73 % ) had juxtabronchial calcified nodes identified on chest radiographs. calcified intraparenchymal nodules were seen radiographically in only four (27%) patients. bronchoscopy is less accurate in detecting calcifications, ranging from 28% to 56% of cases. rarely, calcifying tumors such as an ossifying bronchial carcinoid or endobronchial hamartoma can cause confusion (see chapters 36 and 40) . 232, 233 histologically broncholiths appear similar to calcified fibrocaseous lymph node lesions. the outer surface of the often sharp-edged calculus may be coated with inflammatory exudate or, in cases of actinomyces superinfection, eosinophilic rays (splendore-hoeppli phenomenon). 221, 223 the gms stain may disclose histoplasma yeast forms in the centrally necrotic area of the calculus (fig. 5.32b) . 224, 234, 235 the airway in which the calculus is lodged is typically stenotic, with mural fibrosis and chronic inflammation. within the chest, fistulas may be bronchopleural, bronchocutaneous, bronchomediastinal, or bronchoesophageal in their connections. an aortobronchial fistula is a rare (and often fatal) complication of previous aortic or cardiac surgery,236 bronchopleural fistulas are the most common form and often are secondary to surgery, such as from a leaking postoperative bronchial stump. other causes include necrotizing pneumonia or abscess, penetrating wounds, eroding granulomatous disease, penetrating broncholiths, or malignancies. see chapter 6 for congenital causes. extrathoracic bronchial fistulas include connections with bile ducts, pancreas, and other assorted sites. bronchocele means one or more dilated bronchi filled with fluid, which may be mucinous (bronchomucele) or purulent (bronchopyocele ).237 this condition is caused by stenosis or occlusion of the proximal end of dilated sac(s), and therefore differs from bronchiectasis and mucoid impaction, in which proximal ends are generally still patent. it may be either congenital, or early or late acquired, usually of postinflammatory nature but sometimes of malignant nature.238 localized emphysema, which occurs around the bronchocele, may be caused either by inflammation early in lung growth with continued traction-type effects on nearby lung, or by sustained air-trapping due to airway obstruction. 237 ,239.24o many cases are reported as bronchial atresia.241-245 bronchocele/ atresia may present in adults or children and typically affects the left upper lobe. a characteristic ct appearance is that of a branching mass surrounded by hyperlucency. 246 an irregularly cylindrical (sometimes branched) thin-walled cyst (fig. 5,33a) grossly and histologically resembles a bronchocele lined by respiratory or squamous epithelium. 238 ,24o,242 occasionally a scar or intrabronchial web proximal to the lesion represents the remnant atretic or occluded bronchus. the adjacent bronchial arteries may appear hypertrophic, especially if there have been recurrent infections (fig. 5.33b) , bronchocele may be a relative of saccular bronchiectasis and may be the etiology for some so-called intraparenchymal bronchogenic cysts (see below and chapter 6).247 mucoid impaction may also be related to an allergic effect, often to noninvasive aspergillus (see chapter 15), usually does not have proximal bronchial stenosisocclusion, and has more eosinophils and cellular debris in the mucus, in addition to intraluminal hyphae. bronchocele/atresia is distinguished from intralobar seques bronchogenic cysts are closed sacs lined by respiratory mucosa, usually with bronchial glands, smooth muscle, and cartilage in their walls. they often represent congenital fragments that drop off or are remnants of the original budding of the lungs from the primitive endodermal canal. they are most common in the middle mediastinum where they account for 10% to 15% of all primary mediastinal masses but can be seen as isolated masse es) in the lung. within the lung, some may form as bronchoceles as discussed previously. a series of 86 cases of bronchogenic cyst, with 66 (77%) in the mediastinum and 20 (23%) in the lung, was presented by st. georges et al. 248 from montreal. a similar distribution was recorded in adult patients by patel and colleagues. 249 of interest, 75% to 90% of those in the lung were symptomatic at the time of operation, most often because of infection or bronchial obstruction,z48,249 although suspected, a preoperative diagnosis was not correctly made in any case in the large montreal series. 248 the presence of bronchial epithelial cells on trans bronchial fine-needle aspiration (fna) was found not to be specific for the diagnosis of bronchogenic cyst. 249 most occur in the lower lobes, but all lobes may be affected.248-z50the ct appearance is that of a well-defined hypertrophic bronchial arteries (ba). lumen of cyst is at top (movat stain). ovoid lesion, with surrounding mosaic and band-like linear attenuation consistent with emphysema and bronchiolar metaplasia/fibrosis. 250 a bronchioloalveolar cell carcinoma arising in a bronchogenic cyst in a 37-year-old woman has been reported as a rare association.251 bronchogenic cysts are uncommon in adults and are further discussed in children in chapter 6. bronchorrhea is arbitrarily defined as production of more than 100ml of sputum per day.252 although it is a clinical symptom, pathologists may ponder the differential diagnosis if faced with this history on a specimen request card. bronchorrhea may be idiopathic, or secondary to chronic bronchitis, bronchiectasis, scleroderma, asthma, mucinous bronchioloalveolar carcinoma, metastatic mucinous adenocarcinoma, tuberculosis, or relapsing polychondritis.252-258 cytology exams, cultures, or trans bronchial biopsies may help evaluate at least some of these possibilities. cystic fibrosis is a prototypic example of bronchocentric inflammation and bronchiectasis and the most common lethal genetic disease among caucasians, having a frequency of approximately 1 in 2500 live births.259-261 the molecular defect of this autosomal recessive disorder was discovered in 1989 to involve mutations in a 1480 amino acid polypeptide, the cystic fibrosis transmembrane conductance regulator (cftr), encoded by a gene on the long arm of chromosome 7. 262 ,263 over 1000 different mutations of the cftr gene have so far been identified, but the most frequent mutation worldwide and the most severe genetic lesion, is the deletion of phenylalanine at position 508 of cftr (af508), accounting for over 70% of affected patients. 26o , 264 cystic fibrosis transmembrane conductance regulator functions as a cell membrane-associated, cyclic adenosine monophosphate (camp)-regulated chloride channel, which also has regulatory activity on the absorption of sodium through a separate epithelial channel (enac).264-266 the structure of cftr is schematically depicted in figure 5 .34. mutations in cftr have been grouped into six major types, each of which may present phenotypically as cf: (1) lack of synthesis of cftr; (2) defective processing of cftr such that it does not reach the cell membrane; (3) aberrant regulation of ion transport due to dysfunctional cftr; (4) abnormal conductance of chloride ions; (5) partly defective production and processing; or (6) accelerated turnover at the cell surface ( fig. 5.35) . 259, 261, 267, 268 the af508 mutation is a type 2 defect in which abnormal cftr is sequestered within cellular organelles leading to reduced insertion into the cell membrane, markedly limiting the ability of chloride to cross the membrane. 269 in epithelial cells of bronchi, biliary tract, and intestine, impaired transport of intracellular chloride and its accompanying water molecules leads to dehydration of ductal and lumen secretions. 26o in bronchial epithelium there is also enhanced intracellular absorption of sodium ions, which further dehydrates secretions within the airway lumen. 26o ,264,266 in contrast, the uptake of extracellular chloride is inhibited in sweat ducts, causing an elevation of sweat chloride concentration, a key diagnostic indicator of cf. 260 ,270 the manifestations of cf are protean, involving nearly every organ system either directly or secondarily. the correlation between genotype and phenotypic expression is best exhibited for pancreatic function and is relatively poor for pulmonary manifestations. 267 ,271,272 however, certain mutations such as a455e or the ivs8 5t allele are associated with relatively mild lung disease that may initially present in adults.m-276 a unifying feature of the pathophysiology of cf is impaction of viscid secretions in exocrine gland ducts leading to cardinal manifestations such as intestinal obstruction (e.g., meconium ileus); pancreatic acinar atrophy and fibrosis with consequent metabolic insufficiency (due to intestinal malabsorption); organ maldevelopment (e.g., congenital bilateral absence of the vas deferens); hepatic fibrosis (focal biliary cirrhosis); and infection associated with mucus stasis (e.g., infective bronchitis).26o,277 pulmonary involvement is usually the lungs in cf are structurally normal at birth. dilatation of mucous gland ducts followed by intrabronchial mucus stasis are the earliest pulmonary lesions seen in infants. 261 ,279,28o it has long been recognized that patients with cf are predisposed to lung infection. 28 1.282 current hypotheses suggest that susceptibility to infection may be related not only to entrapment of bacteria in thick bronchial secretions, but also possibly to abnormal binding and reduced uptake of bacteria by epithelial cells, or impaired epithelial antimicrobial protection provided by defensins (natural antibiotics of the innate immunity system).259.283-289 even in infants without apparent infection, however, bronchoalveolar lavage studies document ongoing bronchial inflammation associated with increased levels of endobronchial il-8, a potent cytokine that recruits neutrophils into the inflammatory response, and relatively decreased levels of il-lo, an inhibitor of proinflammatory cytokines. 29 g-294 it is as yet undetermined whether or not intrinsically exaggerated inflammatory responses are the direct result of mutations in cftr. 295 infection and inflammation stimulate bronchial mucus secretion leading to a vicious cycle of worsening airway infection and obstruction, progressing to chronic bronchitis, bronchiolitis obliterans, and bronchiectasis. 286 the chronic pulmonary complications of cf evolve from the airway disease.277 hyperinflation or collapse is the direct result of bronchial obstruction. air trapping and postinflammatory cystic lesions underlie an increased susceptibility to recurrent pneumothorax. pulmonary hypertension and cor pulmonale derive from sustained hypoxia, while hemoptysis is a direct effect of bronchiectasis and bronchial artery hypertrophy. 277 endobronchial infection tends to occur in sequential fashion, initiated by s. aureus, followed by h. injluenzae, and finally by p. aeruginosa (mucoid strains).260.2 8 submucosal glands are enlarged and chronically inflamed behind ducts that are obstructed by dense, inspissated, eosinophilic secretion (a characteristic, but not pathognomonic feature of cf) (fig. 5.36 ).300-302 although bronchial smooth muscle in individual patients may appear hypertrophic, its mean volume density is within the normal range. 300 in patients with cf-associated lung disease, saccular bronchiectasis is usually present beyond 4 months of age.27r although all bronchopulmonary segments may be affected, bronchiectasis tends to be more severe in the upper lobes (fig. 5.37 ).301.303.304 blind-ended ectatic airways, devoid of cartilage, are surrounded by atelectatic, chronically inflamed, and fibrotic parenchyma (see fig. 5 .27).206 bronchial mucosa is frequently denuded or ulcerated leaving the bronchial surface lined by highly vascular granulation tissue that is rich in histiocytes (see fig. 5 .28). in severe disease, bronchi terminate in large, juxtapleural, thin-walled cavities that present radiographically as contiguous, bubble-like cysts. intrapleural blebs or emphysematous bullae are less common forms of cystic lesions, which contribute to an increased incidence of pneumothorax.305.306 extensive acute and chronic bronchiolitis and bronchiolar mucoid impaction impart a finely nodular texture to the parenchymal surface and account for a micronodular radiographic appearance ( fig. 5.38 ).307 bronchiolitis obliterans, predominantly of the constrictive type, contributes importantly to airway obstruction, and likely precedes the development of bronchiectasis (see fig. 5 .30).301.308 occasionally, occlusion of respiratory bronchioles by polypoidal protrusions of fibroblastic tissue accompanies interstitial and organizing pneumonia?09 small airway density decreases with age and is most significantly reduced in patients with hypercapnia. 310.311 the lung parenchyma is grossly indurated by multifocal, bronchocentric chronic pneumonia and fibrosis, with features of both organizing pneumonia and endogenous lipid (cholesterol) pneumonia. a variable degree of acute bronchopneumonia may also be seen at autopsy. some patients who are colonized by burkholderia cepacia undergo an accelerated decline due to acute necrotizing pneumonia (see figs. 8.61 and 8.62 in chapter 8). other patients colonized by burkholderia follow a more protracted course, similar to those colonized by p aeruginosa. [312] [313] [314] fungi and nontuberculous mycobacteria may also colonize cf airways and contribute to lung destruction. 288 ,315,316 bhargava and colleagues 317 identified fungal organisms histologically in 21 % of cf patients retrospectively studied at autopsy. the dilated, obstructed airways of cf patients are predisposed to fungal colonization, accounting for an increased prevalence of allergic bronchopulmonary aspergillosis (abpa) of approximately 2.0%.318 infrequently the pathologic features of abpa, including bronchocentric granulomatosis, are superimposed on chronic cf-associated airways disease (see fig. 15 .19 in chapter 15).277 in cf patients with non tuberculous mycobacterial infections (often due to mycobacterium avium or rapidly growing strains like m. chelonae or m. abscessus), necrotizing fibrocaseous granulomas may be present. 315 ,316,319,320 pulmonary lesions are most likely to be found in patients with repeatedly positive sputum cultures for mycobacteria (see chapter 9) . 320 the pulmonary vascular changes of cf-associated lung disease are usually pronounced, chronic hypoxia and inflammatory changes contribute to pulmonary artery medial hypertrophy and intimal fibrosis of muscular pulmonary arteries and medial myxoid degeneration of elastic arteries.321-326 postmortem arteriograms often show abnormally tapered arteries with a reduced background haze (see fig. 1 .8 in chapter 1).277 morphometric studies provide evidence of a decreased density of arteries, which correlates inversely with the degree of right ventricular cardiac hypertrophy,321 the dropout of arteries may be related to impaired postnatal growth or to vascular destruction secondary to chronic hypoxia or sustained inflammation. 321 right ventricular cardiac hypertrophy, seen at autopsy in approximately 84% of cf patients older than 3 years of age, is a direct consequence of pulmonary artery remodeling and associated pulmonary hypertension. 321 bronchial arteries also undergo significant hypertrophy as a response to sustained bronchial inflammation, bronchiectasis, and bronchocentric abscesses (fig. 5.39 ).327-329 the source of hemoptysis in cf patients is most frequently the delicate capillaries within airway granulation tissue (see fig. 5 .28),330,331 occasionally, mucosal ulcers erode into hypertrophied bronchial vessels leading to life-threatening massive hemoptysis (fig. 5.39 ). interventional bronchial artery embolization of metal coils, polyvinyl alcohol (ivalon), or gelfoam particles is undertaken to induce thrombosis and control bronchial artery bleeding. 331 ,332 degenerated remnants of embolized polyvinyl alcohol may surround stenotic or occluded bronchial arteries in patients who have undergone this procedure. 333 bronchopulmonary arterial anastomoses may further allow the paradoxical entry of small embolized particles into the pulmonary arterial circuit (see fig. 28 .42 in chapter 28). 334 other less frequently reported complications of cystic fibrosis include systemic amyloidosis, intralobar sequestration, and anaerobic lung abscess. 335 -338 emphysema is usually a minor feature, localized to bronchiolocentric scars or as paraseptal emphysema in the upper lung zonesys,30s,329 cystic fibrosis patients with indwelling venous access devices may surreptitiously inject aqueous suspensions of psychoactive pharmaceutical tablets leading to pulmonary artery obstruction due to embolized tablet filler materials (see chapter 26).339 primary ciliary dyskinesia (pcd) is an autosomal recessive disorder, occurring in approximately 1 of 15,000 to 30,000 persons, characterized by the absence or dysregulation of ciliary movement mainly due to ultrastructural defects in the ciliary axoneme. (see chapter 2)?40-344 cilia on the respiratory epithelial surface play an important role in propelling mucus, bacteria, and inhaled particulate debris out of the lung (see chapter 3). as a result of impaired clearance due to ciliary malfunction, patients with pcd are predisposed to chronic sinusitis, serous otitis, and recurrent bronchopulmonary infections beginning in early childhood. 345 ,346 primary ciliary dyskinesia has also been implicated as a cause of neonatal respiratory distress syndrome. 347 ,348 male patients are usually infertile due to poor flagellar motility of sperm. 345 approximately 50% of patients with pcd also have situs inversus secondary to abnormal rotation of embryonic epithelia consequent to the lack of ciliary movement. 343 the syndromic triad of situs inversus, sinusitis and bronchiectasis was first proposed by kartagener in 1933, and is now designated kartagener's syndrome (fig. 5.40 ).349.350 while pcd is an important cause of bronchiectasis, the prognosis is generally more favorable than that of cf. 340 in 1976 afzelius 340 and pedersen and mygind 341 were among the first to recognize that ultrastructural abnormalities of ciliary dyne in arms were associated with kartagener's syndrome. originally termed immotile cilia syndrome by afzelius, it is now recognized that there are numerous structural variations that may contribute to if. tomashefski, jr., and d.h. dail pcd, and that cilia are not always immotile?51 when compared to normal (fig. 5.40a) , the most commonly observed ultrastructural defects are the complete absence of dyne in arms or the selective absence of either inner or outer arms (fig. 5.40b ). other derangements of the axoneme contributing to pcd include defective or absent radial spokes (fig. 5.41d ), transposition of microtubules (fig. 5.41c ) (well seen in longitudinal sections of cilia), central microtubular agenesis, absence of nexin links, agenesis of cilia, or rarely, bizarre cystic dilatation of ciliary shafts. 352 -356 nonspecific findings such as ciliary blebs, megacilia, compound cilia, and displaced microtubules may accompany the more specific defects, but are also frequently present in inflammatory airway disease of diverse causes including infectious bronchitis, cf, or air pollution. 35 6-361 some patients with structurally normal cilia and a normal ciliary beat frequency may develop the clinical manifestations of pcd due to ciliary disorientation, resulting in uncoordinated ciliary motion (fig. 5.40e ).362,363 ciliary disorientation has also been described in individuals with infectious bronchitis (including cf), but the degree of disorientation is usually not as great as in those in whom the defect is primary, and the disorientation secondary to infection typically resolves after effective antibiotic treatment. 299 the diagnosis of pcd is established by ultrastructural analysis of respiratory epithelium in conjunction with typical clinical manifestations, exclusion of other causes of chronic airway inflammation, and documentation of abnormal ciliary motion by phase contrast microscopy.344.364,365 mucosal samples obtained by endoscopic biopsy or brushing are examined by transmission electron microscopy. in patients with pcd, nasal mucosal samples are reflective of bronchial changes when most cilia are abnormal. when only few cilia are structurally abnormal in a patient in whom the diagnosis of pcd is highly suspected, a bronchial sample is required. 366 abnormalities in sperm flagella may differ in type and quantity from those of respiratory cilia within the same patient, suggesting separate genetic control of axone mal structures at differing loci? the lung pathology in pcd is postinfective in appearance. both saccular and cylindrical bronchiectasis may be present with the predominant histologic pattern of follicular bronchiectasis. 175 ,350,370 neither bronchial mucus stasis nor squamous metaplasia is prominent chronic interstitial pneumonitis, peribronchial fibrosis, and atelectasis accompany the bronchiectatic changes. 349 ,370,371 studies to date suggest that pcd is a genetically heterogeneous disorder. the molecular basis of pcd has been localized in a few instances to mutations in the human dyne in axonemal heavy chain (dnah5) located on chromosome 5, or in the intermediate dynein chain gene 1 (dnail) on chromosome 9,372-374 a mutation in the dyne in axonemal heavy chain type 11 (dnahll) has been associated with pcd and situs inversus, without evident ultrastructural ciliary changes, 372 ongoing studies on genetically engineered knockout mice may uncover other genetic defects associated with pcn,372,375 in 1970 donald young, a urologist, reported a series of patients with obstructive azoospermia, 54 % of whom had associated respiratory conditions including bronchitis and bronchiectasis,376 this condition was initially designated as berry-perkins-young syndrome and later shortened to young's syndrome. 377 patients, nearly all had chronic cough, sputum production, and recurrent pulmonary infections. bronchiectasis and chronic sinusitis were each present in about two thirds of patients. in one study it was estimated that young's syndrome accounted for approximately 3% of all patients who presented with bronchiectasis of unknown etiology (equivalent to the prevalence of cf and slightly greater than that of pcd). 195 azoospermia in young's syndrome is the result of retention of semen in an enlarged epididymal head. motility studies have demonstrated impaired upper airway mucociliary transport; however, ciliary beat frequency and ultrastructure are normal. 377 ,378,382,383 in patients with young's syndrome, sweat chloride concentration and the electrical potential difference across the nasal epithelium are normal. 384 the respiratory symptoms in young's syndrome have been suggested to be the result of altered viscoelastic properties of airway secretions, but the basic molecular defect is unknown. 385 an association with mercury toxicity has been hypothesized. 386 friedman and colleagues 380 evaluated mutations of cftr in a cohort of patients with young's syndrome and found that the prevalence of mutations did not differ significantly from the expected carrier frequency in the general population. pulmonary involvement is generally less severe than in cf. 379 bronchiectasis tends to occur at an early age and predominantly involves the lower lobes. pulmonary function tests indicate mild obstruction with decreased fev1 and increased residual volume, although a number of patients have undergone lung resections for bronchiectasis, the pathologic features of bronchiectasis in young's syndrome have not been well described, and it is uncertain if there are any distinctive histopathologic changes. from a diagnostic standpoint it is important to exclude cf and pcd, each of which may be clinically misclassified as young's syndrome?87,388 the distinguishing characteristics among these three conditions are presented in table sa . williams-campbell syndrome is a rare disorder in which extensive loss of bronchial cartilage is associated with diffuse cystic bronchiectasis without other recognized predisposing factors. 184 ,402 the clinical presentation that commences in infancy may include cough, dyspnea on exertion, cyanosis, and clubbing.403 on chest radiograph large thin-walled cysts reside in hyperinflated lungs. highresolution ct scan characteristically shows central, cystic, thin-walled airways that collapse upon expiration. 404.405 the clinical course is one of recurrent pulmonary infections leading to respiratory failure. patients may survive into adulthood and require lung transplantation. 406 as described in the original report by williams and campbell and substantiated in subsequent morphologic studies, the lungs grossly exhibit extreme saccular and cystic bronchiectasis (fig. 5.43a ).406-409 microscopically, dilated airways have very thin walls with minimal inflammation (fig. 5.43b ). cartilage is absent or markedly deficient from the fourth to the eighth divisions of subsegmental bronchi. first-and second-order bronchi usually have a normal cartilage investment. pan acinar emphysema or emphysema localized to the peribronchial zone is usually also present.406.407 bronchiolitis obliterans has also been reported.403.407 the williams-campbell syndrome has been considered to be the result of a congenital absence of cartilage in the subsegmental airways. morphologic studies documenting absent cartilage and insignificant inflammation, and rare reports of familial occurrence have been used to support this view.402.409.410 however, given the propensity for cartilage loss in acquired saccular bronchiectasis of diverse etiologies, the williams-campbell syndrome remains a controversial entity, and its congenital origins have yet to be proven beyond question. 175.205.206.407 the williams-campbell syndrome has not been associated, nor is it to be confused, with congenital lobar emphysema, in which cartilage is focally deficient, usually in upper lobe bronchi, leading to bronchial collapse and air trapping (see also chapter 6).411 tracheobronchomegaly (tbm) is a condition of marked dilatation of the trachea and major bronchi, often associated with recurrent respiratory infections. tbm can be congenital, or at least evident in early life, in which it is termed mounier-kuhn syndrome (see chapter 6) . it has occurred in several cases of ehlers-danlos syndrome, suggesting it may be related to poor elastic support, or perhaps to loss of other matrix components as in chondromalacia. 412 tracheobronchomegaly occurs in adults, mostly in men in their fourth and fifth decades, and can be an acquired condition secondary to sustained inflammation affecting the trachea, such as in chronic tracheobronchitis secondary to tobacco abuse, cystic fibrosis, trauma, emphysema, or pulmonary fibrosis. 412, 413 a comprehensive review and an intriguing study of various pulmonary fibrotic reactions associated with this entity was reported by woodring et al. 412 these investigators evaluated the tracheal diameter on plain chest radiography in a series of 34 cases of fibrotic lung reactions, and found enlargement of the trachea in 10 (29%). the associated lung diseases were idiopathic pulmonary fibrosis and sarcoidosis in four patients each, and progressive histoplasmosis in two patients. in seven of these patients as well as in nine of 24 patients (38%) who did not meet initial radiographic criteria for tracheal dilatation, tracheomegaly developed or progressed over time, tracheobronchomegaly was usually associated with moderate-to-severe restrictive pulmonary defects, and it was proposed that shrinkage of the lung tissue retracts all adjacent spaces including the trachea in a manner similar to traction bronchiectasis. 412 regardless of the cause oftbm, airway dilatation may extend distally, bronchomegaly simulates bronchiectasis, probably impairs lung clearance, and promotes recurrent bronchopulmonary infection, which paradoxically may induce secondary bronchiectasis. 412 roditi and weir 414 identified tracheobronchomegaly in 17% of patients with evidence of bronchiectasis on ct scan, thereby emphasizing the frequent association and possible causal connections between these two conditions. 414 tracheobronchomegaly is predominantly a radiologic diagnosis, and its pathologic features have not been well characterized. associated radiographic features include marked tracheal wall thinning, scalloping due to mural infolding, bronchial diverticula, and collapse on expiration. 415 . 416 the diagnostic criteria of tbm by ct scan are a tracheal diameter of greater than 3cm (measured 2cm above the aortic arch) and diameters of 2.4 and 2.3 cm for the right and left main bronchi, respectively.415al? tracheobronchomegaly must be distinguished from saber-sheath trachea, seen in some patients with emphysema, in which there is a decrease in tracheal coronal diameter and increased sagittal diameter. 415 .4 1 8 patients with immune deficiency, especially hypogammaglobulinemia due to x-linked agammaglobulinemia or common variable immunodeficiency (cvid), are predisposed to develop bronchiectasis secondary to recurrent pulmonary infections. 419 -421 chronic pulmonary disease is the most common long-term complication in patients with hypogammaglobulinemia. 422 common variable immunodeficiency, a heterogeneous immunodeficiency syndrome characterized by depressed levels of serum immunoglobulin g (igg) and defective antibody response to antigen challenge, is associated with sinusitis, recurrent pneumonia, and chronic sputum production in up to 90% of patients.423 patients with cvid also have an increased incidence of autoimmune diseases, and as with other primary immunodeficiency syndromes, a tendency toward lymphoproliferative disorders (see chapter 32) . 422, 424 in this population there is a spectrum of lung abnormalities including interstitial fibrosis (>80% of patients), pneumonia, lymphoid interstitial pneumonia (lip), sarcoidosis-like granulomatous disease (10%), lung abscess, and bronchiectasis. 422 ,425 bronchiectasis is the most common radiologic finding and may be identified in over 30% of patients by chest x-ray, and in up to 80% of patients by hrct. 424 ,426 by hrct bronchiectasis may be either focal or multilobar, and is of the cylindrical or rarely cystic type. 420 ,426 the lower and middle lobes tend to be predominantly involved.420.424 although mucociliary clearance is impaired in these patients, ciliary ultrastructure is normal. 427 there is little information on the histopathology of bronchiectasis in cvid. 42 8. 429 hill and colleagues 429 noted severe bronchiectasis, emphysema, fibrosis, and granulomas in the lung explant of a 37-year-old man with cvid. no unique features of bronchiectasis were described. patients with cvid are treated with immunoglobulin replacement therapy, which reduces the severity and frequency of respiratory infections. symptomatic bronchiectasis, identified by hrct scan, has also been reported in patients with hiv disease in whom it is associated with rapidly progressive airways obstruction. 43o , 431 king and colleagues 432 correlated airway dilatation on ct scan with increased neutrophils on bronchoalveolar lavage. a single report of a transbronchial biopsy showed only nonspecific lymphocytic peribronchiolitis. 431 the pathogenesis of bronchiectasis in hiv patients is likely consequent to bronchial damage from recurrent pneumonia and bacterial bronchitis in this immunosuppressed population. 430 .433 frequently cultured microorganisms include h. injluenzae, p. aeruginosa, and s. pneumoniae. 430 ,433 bronchiectasis also occurs as a complication of lung transplant-associated immunosuppression and bronchiolitis obliterans and is further discussed in chapter 23. 434.435 rheumatoid arthritis symptomatic bronchiectasis is estimated to occur in 1 % to 3% of patients with rheumatoid arthritis (ra), although with hrct scan, up to 30% of patients with ra can be shown to have cylindrical bronchiectasis. 185 .4 3 6,437 early autopsy studies of patients with ra provided a prevalence of bronchiectasis of 0% to 12%. [438] [439] [440] [441] in some studies bronchiectasis typically preceded the development of arthritis, leading to the interesting hypothesis that chronic suppurative airway disease is involved in the pathogenesis of ra.442-444 shadick and colleagues,444 however, reported 23 patients with bronchiectasis and ra, of whom 18 developed bronchiectasis as a late complication of severe ra. bronchiectasis may also be more frequent in patients with ra-associated sjogren's syndrome.442a45 bronchiectasis associated with ra cannot be adequately ascribed to either traction bronchiectasis or therapeutic immunosuppression. 436 ,444 the morphologic features of ra-associated bronchiectasis are not well documented. by ct scan, cylindrical bronchiectasis primarily involves the middle and lower lung zones. 437a44 , 445 bronchiectasis is part of the spectrum of lung involvement in patients with inflammatory bowel disease (ibd), ulcerative colitis more so than crohn's disease (see also chapter 20) .446,447 suppurative bronchiectasis may also develop after proctocolectomy for either of these conditions. 447 ,448 in the 1993 literature review of camus et al.,446 bronchiectasis was identified in six patients with ulcerative colitis out of 33 patients (18%) with ibd-associated lung disease 446 histologically a dense cuff of lymphocytes typically occupies the submucosa, and squamous metaplasia replaces the overlying epithelium (fig. 5.44 ). the chronic inflammatory infiltrate involves bronchial glands and ducts; however, lymphoid germinal centers are usually absent, distinguishing ulcerative colitis-associated bronchiectasis from the usual pattern of follicular bronchiectasis (fig. 5 .44b).175,446 neutrophils infiltrating the mucosa and spilling into the dilated bronchial lumen impart a suppurative appearance in some cases. 44 6--449 direct immunofluorescence staining of bronchial biopsies in three patients with ulcerative colitis showed deposits of immunoglobulin and complement in bronchial structures. 448 lung biopsy in patients with crohn's disease and bronchiectasis may show features of either granulomatous bronchiolitis or suppurative-appearing acute bronchiolitis (see fig. 20 .27 in chapter 20).450 inhaled steroids were of durable benefit in patients with ibd-associated chronic bronchitis, but less so in patients with bronchiectasis. 446 speculations on the pathogenesis of bronchiectasis in ibd are presented in a provocative editorial by stockley.451 bronchiectasis is reported as a late sequela of heroinassociated pulmonary edema. 452 the bronchographic features include diffuse or localized cylindrical and varicose bronchiectasis. 453 itis are etiologic factors in some patients, and bronchial ulceration and foreign-body giant cells have been observed at autopsy.454 other cases of diffuse bronchiectasis in heroin users appear to be unrelated to aspiration. 453 see chapter 31 for other pathologic features of heroin toxicity. in adults, severe direct chemical injury 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inflammatory bowel disease the impact of substance abuse on the respiratory system bronchiectasis: a cause of pulmonary symptoms in heroin addicts pulmonary complication of heroin intoxication. aspiration pneumonia and diffuse bronchiectasis bronchiectasis following ammonia burns of the respiratory tract: a review of two cases fatal anhydrous ammonia inhalation bronchiectasis following pulmonary ammonia burn said sl bronchiectasis and progressive respiratory failure following smoke inhalation acknowledgments. the authors are deeply appreciative to diane gillihan for expert secretarial assistance, vince messina for photography, and the staff of the brittingham memorial library for bibliographic support. key: cord-281603-3308f8hm authors: souza, william marciel de; buss, lewis fletcher; da silva candido, darlan; carrera, jean paul; li, sabrina; zarebski, alexander; vincenti-gonzalez, maria; messina, janey; sales, flavia cristina da silva; andrade, pamela dos santos; prete, carlos a; nascimento, vitor heloiz; ghilardi, fabio; pereira, rafael henrique moraes; santos, andreza aruska de souza; abade, leandro; gutierrez, bernardo; kraemer, moritz u. g.; aguiar, renato santana; alexander, neal; mayaud, philippe; brady, oliver j; souza, izabel oliva marcilio de; gouveia, nelson; li, guangdi; tami, adriana; oliveira, silvano barbosa; porto, victor bertollo gomes; ganem, fabiana; almeida, walquiria ferreira; fantinato, francieli fontana sutile tardetti; macario, eduardo marques; oliveira, wanderson kleber; pybus, oliver; wu, chieh-hsi; croda, julio; sabino, ester cerdeira; faria, nuno r. title: epidemiological and clinical characteristics of the early phase of the covid-19 epidemic in brazil date: 2020-04-29 journal: nan doi: 10.1101/2020.04.25.20077396 sha: doc_id: 281603 cord_uid: 3308f8hm background: the first case of covid-19 was detected in brazil on february 25, 2020. we report the epidemiological, demographic, and clinical findings for confirmed covid-19 cases during the first month of the epidemic in brazil. methods: individual-level and aggregated covid-19 data were analysed to investigate demographic profiles, socioeconomic drivers and age-sex structure of covid-19 tested cases. basic reproduction numbers (r0) were investigated for sao paulo and rio de janeiro. multivariate logistic regression analyses were used to identify symptoms associated with confirmed cases and risk factors associated with hospitalization. laboratory diagnosis for eight respiratory viruses were obtained for 2,429 cases. findings: by march 25, 1,468 confirmed cases were notified in brazil, of whom 10% (147 of 1,468) were hospitalised. of the cases acquired locally (77.8%), two thirds (66.9% of 5,746) were confirmed in private laboratories. overall, positive association between higher per capita income and covid-19 diagnosis was identified. the median age of detected cases was 39 years (iqr 30-53). the median r0 was 2.9 for sao paulo and rio de janeiro. cardiovascular disease/hypertension were associated with hospitalization. co-circulation of six respiratory viruses, including influenza a and b and human rhinovirus was detected in low levels. interpretation: socioeconomic disparity determines access to sars-cov-2 testing in brazil. the lower median age of infection and hospitalization compared to other countries is expected due to a younger population structure. enhanced surveillance of respiratory pathogens across socioeconomic statuses is essential to better understand and halt sars-cov-2 transmission. the first case of covid-19 was detected in brazil on february 25, 2020. we report the epidemiological, demographic, and clinical findings for confirmed covid-19 cases during the first month of the epidemic in brazil. individual-level and aggregated covid-19 data were analysed to investigate demographic profiles, socioeconomic drivers and age-sex structure of covid-19 tested cases. basic reproduction numbers (r 0 ) were investigated for são paulo and rio de janeiro. multivariate logistic regression analyses were used to identify symptoms associated with confirmed cases and risk factors associated with hospitalization. laboratory diagnosis for eight respiratory viruses were obtained for 2,429 cases. by march 25, 1,468 confirmed cases were notified in brazil, of whom 10% (147 of 1,468) were hospitalised. of the cases acquired locally (77·8%), two thirds (66·9% of 5,746) were confirmed in private laboratories. overall, positive association between higher per capita income and covid-19 diagnosis was identified. the median age of detected cases was 39 years (iqr 30-53). the median r 0 was 2·9 for são paulo and rio de janeiro. cardiovascular disease/hypertension were associated with hospitalization. co-circulation of six respiratory viruses, including influenza a and b and human rhinovirus was detected in low levels. socioeconomic disparity determines access to sars-cov-2 testing in brazil. the lower median age of infection and hospitalization compared to other countries is expected due to a younger population structure. enhanced surveillance of respiratory pathogens across socioeconomic statuses is essential to better understand and halt sars-cov-2 transmission. to investigate individual-level diagnostic, demographic, self-reported travel history, place of residence and likely place of infection, differential diagnosis for other respiratory pathogens, as well as clinical details, including comorbidities, we collected case data notified to the redcap database 8 from february 25 to march 25, 2020. data was contributed by public health and private laboratories. diagnosis and case definitions (see appendix, pp.1) were based on world health organization (who) interim guidance. to explore the time-lag between the number of imported cases and of local cases we used the granger causality test 9 . geospatial analysis of covid-19 cases, demographic and socio-economic data based on data from the first covid-19 reports in brazil 10 , we hypothesized that rates of incidence and testing for covid-19 are higher in areas of higher per capita income. for the greater metropolitan region of são paulo (gmrsp), per capita income at the gmrsp neighbourhood level (517 zones) were retrieved from the 2017 pesquisa origem e destino survey (www.metro.sp.gov.br/pesquisa-od/). 13,913 notified cases (covid-19 confirmed, ruled out, and without final diagnosis) resident in the gmrsp were geocoded based on self-reported address using the galileo algorithm and verified using google api. per capita income for each zone was linked to each notified case based on residential address. we compare per capita income for all notified cases between those tested (positive and negative) and untested, and for confirmed cases by rt-pcr. full details on the statistical analysis can be found in the appendix, pp.1. to quantify transmission potential of covid-19 in brazil, an exponential model was used to represent the incidence of covid-19 at the national level and in são paulo and rio de janeiro states. time series of confirmed cases were modelled as samples from a negative binomial distribution with a mean equal to a fixed portion of the incidence. the analysis was carried out in a bayesian framework with uninformative priors on all parameters apart from the removal rate, which was given an informative prior. the informative prior ensured that the average duration for which an . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. to investigate which factors are associated with a confirmed covid-19 result and with hospitalization summary statistics were calculated for continuous variables and for categorical variables and summarized as medians (range and interquartile range, iqr), as appropriate. missing data were removed (assumed missing at random) (see appendix table s1 and fig. s3 ). uni-and multivariate analysis included only cases with complete information for the relevant variables. these analyses compared demographics, symptoms, clinical signs and comorbidities between confirmed covid-19 cases (rt-pcr positive) and ruled-out covid-19 cases (rt-pcr negative). additionally, separate multivariate logistic regression models were built to predict hospitalisation (binary variable: hospitalised vs. not hospitalised) based on symptoms, clinical signals and comorbidities, and to predict testing status (positive or negative for rt-pcr sars-cov-2). the associations between the outcome and independent variables were reported as adjusted odds ratios (aor) with 95% confidence intervals and likelihood ratio test (lrt) using the univariate and multivariate logistic regression models. model diagnostics were performed to check for model specification errors, multicollinearity and influential observations. a 0·05 significance level was applied. the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint by march 25, 2020, four weeks after the first report of covid-19 in brazil, 67,344 covid-19 cases had been notified as covid-19 suspected infections from 172 cities across all five administrative regions of brazil. of these, 1,468 cases were confirmed (2·18% of all notified cases) and notified through the redcap system (fig. 1a) (fig. 1b) . the epidemic curves of locally-acquired cases followed the curves from imported cases with a lag of two days (granger causality test) (fig. 1a) . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint these locations used a bayesian approach to fit an exponential growth model to covid-19 aggregated incidence data. consistent with previous studies in china and overseas 12 , we find that epidemic spread in são paulo and rio de janeiro states is characterized by similar r 0 values of 2·9 (95% ci 2·1-4·4) and 2·9 (95% ci 2·2-4·5). the r 0 for brazil was slightly higher with median of 3·2 (95% ci 2·4-5·4) (fig. 2) . analysis of the age-sex structure of confirmed and notified cases compared to the brazilian demographic structure revealed a disproportionately lower proportion of confirmed covid-19 infections reported in younger categories (0-9, 10-19 years of age) and a slightly higher proportion in middle-age categories (20-29 and 30-39 years of age) (fig. 3) . specifically, compared to the proportion of the total brazilian population per age category, the proportion of confirmed covid-19 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint infections in the 0-9 and 10-19 years of age categories are 16·4-and 5·3-fold lower compared to brazilian demographic structure (fig. 3a) . we found that most confirmed cases were in males (776 [54·7%] of 1,420 -46 confirmed cases had missing information for sex and/or age) (fig. 3a) . the median age of cases was 39 years (iqr, 30-53, range: newborn-93 years). nearly half (695 [48·9%] of 1,420) of the confirmed cases were in the age range of 20 to 39 years of age (fig. 3a) . similarly, 51·6% (2,288 of 4,438) of cases tested for sars-cov-2 belonged to this age-group (fig. 3b) , which is substantially higher than the corresponding fraction of the brazilian population (68,451,093 [32%] of 211,755,692). 9·5% (133) of cases were health care workers. overall, only four newborns, three infants (6 to 8 month-old), ten children (1 to 12 years old), and twelve adolescents (12 to 17 years old) were diagnosed with covid-19. in addition, nine patients were pregnant, one in the first trimester, one in the second trimester, four in the third trimester and 3 had missing information). six cases were hiv-positive. proportion (%) of the country's population in each age-sex class is shown as faded bars. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. (fig. 4a) . to test whether notified tested cases were associated with socioeconomic status, we evaluated the association between covid-19 diagnosis and socioeconomic status in the subset of cases in the greater metropolitan region of são paulo (gmrsp) region with geocoded residential information using an ordinal probit model. we found that the proportion of tested cases in gmrsp increased as income per capita increases (z-score = 0.19, likelihood ratio test p-value <0.01) (fig. 4c, table s2 ). moreover, the increase in the proportion of tested cases for a unit-increase in income is higher in weeks 2, 3 and 4 compared to week 1. for the range of income per capita observed, given the same amount of income per capita, the proportions of tested cases were lower in weeks 2, 3 and 4 than week 1. overall, there was a noticeable upwards trend in the association between testing rate and per capita income uncovering a widening socioeconomic disparity in testing practice as the number of cases expands. the income distribution of the untested fraction increasingly approximates the average for gmsp, whereas the tested and confirmed cases (both laboratory and clinical epidemiological) are consistently higher over the study period. we also analysed the results for other respiratory pathogens tested in brazil as part of the differential diagnosis by central public health laboratories and national influenza centres (brazilian , fig. s4 ). . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. in a univariate analysis of4,387 cases with a final classification as confirmed (n=1,101) or discarded (covid-19 ruled out) (n= 3,286), we found that increasing age, symptoms (cough, difficulty breathing, dyspnoea/tachypnea, sputum production, nasal congestion, nasal flaring, nausea/vomiting, headache, irritability/confusion, difficulty swallowing, intercostal retraction and alteration on chest auscultation) and clinical signs (fever and conjunctival congestion) were higher associated with a negative sars-cov-2 results (see appendix table s4 ). overall, a total of 12·5% (184/1,468) of confirmed covid-19 cases had at least one comorbidity. most common comorbidities were heart disease, hypertension, diabetes, and chronic respiratory disease. (figure 5b) . interestingly, age was not significantly associated with hospitalization after accounting for co-morbidities. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. of the four fatal cases, one had cardiovascular disease/hypertension, one had both cardiovascular disease/hypertension and renal disease, and two fatal cases had no reported comorbidities. only one case had reported close contact with a confirmed covid-19 case reinforcing that local transmission was already well established in brazil by march 25, 2020. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint these findings provide evidence that sars-cov-2 transmission in brazil shifted rapidly from a scenario of imported to local transmission. we found that the proportion of tested cases is higher in zones with higher per capita income. we showed that during the first month of covid-19 in brazil, only 33·1% of the reported confirmed cases were conducted in public health laboratories. our results support similar transmission potential (r 0 ) of sars-cov-2 in brazil to other geographic regions. overall, our clinical findings demonstrate that chest x-ray abnormalities and o 2 saturation <95% are strongly associated with hospitalization. the combination of universal access to diagnostic and the success of interventions will dictate the fate of covid-19 in brazil. overall, these findings 14 . secondly, our retrospective study has focused predominantly on symptomatic patients (92%) that presented themselves to health services for testing. therefore, we cannot describe the full spectrum of disease. population-based serologic surveys are urgently needed to properly determine the asymptomatic and oligosymptomatic fraction. finally, many patients remained hospitalized when the dataset was extracted, and, we were unable to estimate clinical outcomes given the long duration of infection. together with changes in surveillance guidelines, socioeconomic bias in testing suggests that the number of confirmed case counts may substantially underestimate the true number of cases in the population. additional reasons for underreporting include (i) a significant proportion of asymptomatic infections 15 , (ii) people with mild and even moderate disease are unlikely to present to health services for testing, (iii) limited testing capacity in public health service in brazil in face of the large number of cases due to delays in importing reagents and kits used in molecular testing. close monitoring of state-and municipality-level data will further help to inform mitigation strategies. our results suggest that approximately 50% of the covid-19 cases in brazil were skewed towards age groups between 20 to 39 years with substantially fewer cases in younger age groups. this pattern could be explained by (i) a higher risk of exposure of this group due to more frequent international travel (travel bans were only implemented on march 23, 2020), and (ii) younger age . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint groups being less likely to acquire an infection and/or less likely to acquire significant symptoms upon being infected 16 . covid-19 infections were reported in paediatric and pregnant patients [17] [18] [19] . paediatric infection appears to typically be of mild or moderate severity; we observed a similar proportion of asymptomatic infections compared to reports in 36 children in china (24% vs. 28%) 19 . also, the onset symptoms of pregnant women were similar to those reported in non-pregnant adults with covid-19 infection. on the other hand, proportion of hospitalisation of paediatric patients in brazil was lower than those observed for children in china (3.3% vs. 38.9%) 19 . also, in contrast to china, none of the pregnant women that tested positive for covid-19 in brazil had pneumonia or were hospitalized 17, 18 . however, the absence/lower number of hospitalisations could be explained by resource availability and local clinical practice guidelines. despite the small sample size, our findings in pregnant and paediatric patients in the early-phase covid-19 pandemic in brazil require further understanding of sars-cov-2 infection in these groups. although clinical features in brazil are similar to those recently reported in other countries 1,4,5 , we observed that 8% of confirmed cases reported no symptoms. this should not be considered as an estimate of the asymptomatic fraction. firstly, it is not possible to distinguish true asymptomatic infections from cases in the pre-symptomatic phase. secondly, routinely collected data tends to be incomplete. thirdly, these cases were tested because they were in contact with a known confirmed case. lastly, there is an ascertainment bias towards symptomatic infections due to the case definition used for notification (appendix). other estimates of the asymptomatic fraction have varied widely, including 18% on the diamond princess ship 15 , 50-75% in the italian village of vo'euganeo 20 and 31% based on repatriation flight screening 21 . overall, 10% of covid-19 cases in brazil were hospitalized compared to 19% in the usa 22 . as mentioned above, these differences may reflect factors other than disease severity, for example, resource availability, local clinical practice guidelines and testing availability. on the other hand, they may also reflect right censoring, whereby cases that were notified towards the end of the period studied had not yet been hospitalized. this would be expected given the median lag of four days between symptom onset and hospitalization observed in brazil. although age was not a risk factor for hospitalization after controlling for comorbidities, is should be noted that the age distribution among patients who were hospitalized differed from that reported in china, with a higher proportion of younger (<39 years: brazil, 24.5% vs. china, 10%) and older patients (>70 years: brazil, 21.8% vs. china, 15%) 18 . however, such comparisons need to be taken cautiously due to different testing and notification practises in the two countries. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. we showed that patients with pre-existing cardiovascular diseases/hypertension were at increased risk of hospitalization. the prevalence of at least one comorbid condition among infected individuals in brazil was similar to that reported in china (12.5% vs. 10.5%) 23 . previous studies suggest that persons with underlying health conditions, such as cardiovascular, diabetes and chronic lung diseases, appear to be at higher risk for severe covid-19 infection than persons without these conditions 22,24 . pre-existing cardiovascular disease appears to be particularly important, potentially due to the involvement of the renin angiotensin system signalling pathway 25 . this study provides new information on co-circulation and co-detection of other respiratory pathogens in the early phase of the covid-19 epidemic in brazil. particularly, we found cocirculation of eight other respiratory viruses, the most common respiratory infections were influenza a and b, and human rhinovirus (hrv). co-detection of sars-cov-2 with influenza a and human metapneumovirus (hmpv) have also been reported in china 26,27 . here we found co-detection of sars-cov-2 with influenza a and hmpv, and we expanded the description of the other multiple codetection scenarios of sars-cov-2 with other respiratory viruses, including hrv, influenza b, human respiratory syncytial virus, and other coronaviruses (i.e. coronavirus 229e/nl63, hcov oc43/hku1). although, viral co-infection has been reported with many other respiratory viruses, no difference in clinical disease severity between viral co-infection and single infection has been reported 28 . in conclusion, we provide the first description of covid-19 in brazil. our study provides crucial information for diagnostic screening and health-care planning, and for future studies investigating . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04.25.20077396 doi: medrxiv preprint clinical features of patients infected with 2019 novel coronavirus in wuhan, china coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of coronavirus disease 2019 in china first case of 2019 novel coronavirus in the united states coronavirus disease 2019 (covid-19) in italy world-health-organization. coronavirus disease (covid-2019) situation reports research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support investigating causal relations by econometric models and cross-spectral virological assessment of hospitalized patients with covid-2019 the reproductive number of covid-19 is higher compared to sars coronavirus percepção do estado de saúde, estilos de vida e doenças crônicas the lancet infectious estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study. the lancet infectious clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study. the lancet infectious covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village international journal of infectious diseases : ijid : official publication of the international society for infectious diseases united states characteristics of and important lessons from the coronavirus the authors thank the clinicians and epidemiologist for technical support. this work was supported key: cord-288770-hquc2v2c authors: gupta, rajan; pal, saibal kumar; pandey, gaurav title: a comprehensive analysis of covid-19 outbreak situation in india date: 2020-04-11 journal: nan doi: 10.1101/2020.04.08.20058347 sha: doc_id: 288770 cord_uid: hquc2v2c the outbreak of covid-19 in different parts of the world is a major concern for all the administrative units of respective countries. india is also facing this very tough task for controlling the virus outbreak and has managed its growth rate through some strict measures. this study presents the current situation of coronavirus spread in india along with the impact of various measures taken for it. with the help of data sources (till 7th-8th april 2020) from various state units of india and ministry of health and family welfare, government of india, this study presents various trends and patterns. this study answers six different research ques-tions in a comprehensive manner. it has been reported that growth rate of infected cases has been controlled with the help of national lockdown, however some uncontrolled mass level events had negatively impacted the infected cases. with the help of exponential and polyno-mial regression modelling, the predictions of up to 75000 cases have been done by the end of april 2020. it has also been seen that there are some prominent clusters and patient nodes in the network of patients which are the major influencers for covid-19 spread. also, death rate case predictions have been done through multi-class classification models with an accuracy of 75%. at the end, strategies for continuation for lockdown has been discussed and presented. it appears that only essential services should be open for the citizens of india and the national lockdown should be carried on for next 2-4 weeks. this study will be useful for the government of india and various states of india, administrative units of india, frontline health workforce of india, researchers and scientists. this study will also be favorable for the administrative units of other countries to consider various aspects related to the control of covid-19 outspread in their respective regions. united kingdom. with respect to the recovered patients list, china is at the top of the list followed by spain, germany, italy, iran and the united states of america. india was placed comfortably out the list of infected nations by huge margins, but recent events led to its rise to 27 th position which is a point of concern. the mortality rate is controlled at less than 3% right now, which is better than the ~5.5% mortality rate of world, but the model of spread is slowly moving towards an exponential trend which can lead to massive loss of lives and infrastructure. india is being looked upon by various nations now as a world leader and even who acknowledged that world is looking towards indian strategies to contain the outbreak of this epidemic [3] . india accounts for almost one-fifth of the world's population and is second leading country in terms of population in the world. india contributes heavily to the world's gdp and is amongst the most prominent developing country in the world with fairly strong economic growth percentages [4] . india's good camaraderie with majority of the nations in the world and its helpful nature makes it a perfect ally for other countries. therefore, the analysis of covid-19 outbreak in indian region is closely watched and monitored by the world and there is a need of comprehensive analytical studies based on different strategies taken by indian administrators from time to time. india has been following a nationwide lockdown since 22-march-2020, which was a one-day lockdown, followed by a 21-day lockdown after two days. every activity in india since then has been happening with permission from various administration units and almost all the domestic and international travels have been either banned or monitored closely. india is yet to get into the third phase of covid-19 outbreak i.e. the community outbreak as seen by various countries around the world, but the cases have been rising continuously. india's lockdown period has been impacted by two major events in the recent days which were related to the mass exodus of laborers and workers from one state to other states (especially from delhi to neighboring states) and conduction of a religious event in delhi which led to spike in the number of cases in various states of india. during this time, the indian prime minister has been trying to connect with indian citizens through innovative strategies and coming up with various engagement activities which are impacting the whole nation. with so much happening in india right now, it becomes imperative that we study the current situation and impact of various such events in india through data analysis methods and come up with different plans for future which can be helpful for the indian administrators and medical professionals. the current study explores various aspects associated with the covid-19 outbreak in india and the various regions situated in india. the specific research questions (rq) explored in this study are as follows.  rq1: how has the situation changed in post-lockdown period in india i.e. what is the outbreak situation after 22-march-2020 in india as compared to pre-lockdown period?  rq2: what are the short terms predictions for the number of infected cases in india for the next 3-4 weeks based on current situation?  rq3: has the lockdown been followed by the indian citizens after 22-march-2020? has the social distancing worked for indian citizens? what are the mobility changes in the various regions of india?  rq4: whether the community outbreak spread started in india with the conduct of a religious event in delhi? how is the outbreak different for citizens related to event and for citizens not related to the event?  rq5: which are the prominent clusters which were formed in the last few weeks with respect to covid-19 outbreak in india? does network analysis provide influential points in the infected patient network? 3  rq6: whether the national lockdown should be opened after 14 th april 2020 in india or should it continue? are there any partial regions for which lockdown can be removed? which all essential services should be opened in india after one week under restricted lockdown? the current study is divided into five sections. first section has laid the context of the study. the second section discusses various literature review and analytical techniques followed by the different researchers across the world. the third section presents the methodology and research variables of the study. the fourth section presents the results and findings of the study along with the discussion of the achievement of the various research questions explored in this study. and finally section five concludes this study and present limitations and future directions for this research work. as per different papers available in literature, there are a few studies that focus on the trend analysis and forecasting for indian region. the studies [5] [6] on indian region presents long term and short term trend, respectively. these studies use time series data from john hopkins university database and present forecasting using arima model, exponential smoothing methods, seir model and regression model. however network modelling and pattern mining are not attempted in these versions of the studies and that too at the regional level, hence the current study attempts to do that. also, the studies in indian region from the past are more focused on presenting time series analysis based on the overall data for indian region rather than covering other sources of information apart from just considering the number of infected patients, so the need to analyze the patients background and information is required for the authorities to get better insight about the situation. similarly, there are other mathematical models that were developed for analyzing the trends of covid-19 outbreak in india. a model [7] for studying the impact of social distancing on age and gender of the patients in india was presented. it compared the country demographics amongst india, italy and china and suggested the most vulnerable age categories and gender groups amongst all the nations. the study also predicted the rise of infected cases in india with different lockdown periods. similarly, a network structure approach was used by one of the study [8] to see whether any specific node clusters were getting formed. but only travel data nodes were considered by the authors to check which the prominent regions are affecting indian travelers coming back to the india. also, the study presented the sir model to see the rate of spread of the corona virus amongst patients in india. analysis on the testing labs and infrastructure was also presented by earlier authors. work of medical doctors and frontline health workers was also presented by some studies [9] . it was found that in india, the role of health workers was less stressed as the spread stage of corona virus was still in phase two or the phase of local transmission rather than the community transmission as compared to other nations like italy, spain and usa. however, it was also claimed that indian healthcare infrastructure is not very strong as per the who guidelines and in case of community spread, the indian government may find it difficult to manage the spread. some detailed discussion on the nature of the corona virus was also presented by some studies [10] [11] . apart from india, a few models are also available for other countries primarily for china, italy and usa as the number of infected patients was high. studied like [12] [13] [14] [15] [16] worked on various mathematical models to determine the spread of the disease, predict the number of infected patients, commenting on the preparedness for each country in tackling covid-19 4 spread and finding the patterns of flattening curve in different conditions. a lot of researches are still in preprint stage for the world level and are yet to be peer reviewed. with respect to the research activities conducted in the indian region, the studies are yet to work on the impact of different policies working towards containment of the corona virus. even in the preprint databases, there are fewer evidences available which worked in the indian region with more granularities and came up with analysis that can support the decision making of the various administrators in india to curb the lockdown and work on future strategies. therefore, this study attempts to work on a comprehensive level to analyze the covid-19 spread in india and impact of various strategies imposed by the government at both state level and central level. to answer the different research questions, specific methodologies have been used which covers up different data set, data sources, modeling techniques and outcome variables. the overall variables covered up in the study are shown in figure 1 . for the first research question, the time series data from the time period of 30 th january 2020 till 7 th april 2020 has been covered from the indian database of covid-19 [17] . this dataset was divided into three parts of 30 th january 2020 to 4 th march 2020, 5 th march to 22 nd march 2020 and 23 rd march 2020 to 7 th april 2020. trend analysis and average number of infected cases were compared at both the national level. on the same dataset, the second research question has been answered using the exponential modelling to predict the short term trends for the next three weeks. the exponential modelling can be done using equation 1. where, n 0 and n are the total cases at time t 0 and t, respectively, and γ is the growth rate. by definition [18] , n = 2n 0 needs the doubling time (α) that is expressed as α = ln(2)/γ through ln(n/n 0 ) = ln(2) = γt. if the growth rate evolves with time, depending on the effective prevention strategies, then the doubling time changes with time. thus, the time-dependent growth rate γ(t) causes the time-dependent doubling time α(t), defined as α(t) = t ln (2) for the third research question, the mobility data set [19] from google has been considered after 22 nd march 2020 to see the patterns in mobility of indian citizens at prominent places in . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint india. also, the number of operational places and events conducted during lockdown data has been analyzed through news reports and channels. based on the news reports, the major events were traced and people getting infected due to such events have been covered from the indian data repository created using crowdsourcing channels [20] . the patients were categorized as infected from the event as compared to other patients who were not related to the event. the trends and patterns were analyzed after conduction of this event i.e. in the first week of april 2020. the data was also confirmed from various news reports in india. to understand the patterns existing in the patients of covid-19 in india, their demographic details were studied in detail since 30 th january 2020 from the crowdsourced database in india [20] and rules were generated for the patients with potential probabilities. network analysis [21] was also conducted on the patients and node centralities [22] were calculated the patients. based on the patient database and number of infected cases along with the various secondary reports from the news media and consulting firms, the success of engagement activities and lockdown strategies have been analyzed, which answers the research question 7. the findings of the different types of data analysis conducted on various types of datasets are presented in the current section as follows. the national lockdown for one day was announced on 22 nd march 2020 by the central government of india, before which majority of the schools, colleges, markets, cinema halls, etc. were already shut down by respective state governments. merely, two days after this one day curfew, a 21-day lockdown was announced by the central government banning all the movement and restricting the indians to stay at home. the citizens were allowed to step out only in emergency situations and that too with prior permissions from the local administration. all these instructions were given in the hope of flattening the curve of infected cases and to restrict the exponential growth of the patients in india. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 6 there are almost 5000 confirmed cases reported in india as on the morning of 7 th april 2020 with more than 90% cases being active. the death rate has been keeping under 3% at all the stages of the covid-19 spread. looking at the graph in figure 2 , it is clearly evident that a spike has been reported in india after 22 nd march 2020 i.e. the time when lockdown was announced. it clearly shows that indian authorities were quick enough to sense the spread rate in indian region and taking necessary steps of maintaining social distancing by announcing a rigid step of lockdown. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. looking at the growth rate of infected cases on daily basis (blue line) and the trend line of expected growth (red line) in figure 3 , it appears that in early days of infection the growth rate was quite high due to low number of cases. the growth rate has been calculated as the difference in number of cases between two consecutive days divided by the count of infected cases on the previous day of the two days under consideration multiplied by 100. since, in the early days the count was in single digits, so the growth rate was pretty high. hence ignoring that period, considering the second phase of time period from 5 th march to 22 nd march 2020 i.e. exactly before the lockdown, the growth rate has been hovering around 20% with trend line forecasting it to be maximum around 28%. however, for the time period after lockdown i.e. from 22 nd march 2020 onwards, the growth rate slightly increased, but it remained around a similar mark of 20%. and the trend line also predicted the growth rate on per day basis to be around maximum of 28%. therefore, it can be said that the national lockdown has been able to contain the growth of the number of cases of covid-19 patients. without lockdown, the growth might not have been contained in india and may have gone into the exponential zone too quickly. this gives all the state level and national level administrators and health workers to get prepared for the rising number of cases. exponential modelling has been used to predict short term predictions at national level. first, the growth for doubling days was calculated, i.e. the number of days to double the number of infected cases have been calculated. as seen from figure 4 , the first image shows that prior to the lockdown period average number of days to double the cases was majorly above four, while the average period drop down near to three after lockdown. as per the predictions . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 8 (trend line in red) the average number of days is constantly decreasing when the number of cases is rising in india. based on the exponential model, the predictions for the next 3 weeks were made for the infected cases in india. considering that doubling rate is going as per the historical evidences, the number of predicted cases in india is shown in table 1 . based on the exponential modelling based growth of the number of covid-19 cases in india, polynomial regression line was plotted with different degree values. a total of 5 degrees were checked between 2 to 6 and root mean error (rme) was checked for all cases. the lowest rme reported was 237.58 for . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 9 the model with degree 4. thereafter, the prediction model of polynomial regression of degree 4 was built and used for the prediction values. for training set, data for 31 days was considered starting from 3-march-2020 to 3-april-2020. this was due to the fact that prior to 3 rd march, only 5 cases were reported in indi within 40 days which was impacting the prediction model. for evaluation, data from a short time period of 4-april-2020 to 7-april-2020 was used. other train and test sets were also considered and predictions were similar. with these prediction values, it is estimated that the values for infected cases may rise near to 75,000 in india which may not be a really good situation in india. the concept of social distancing was suggested by a lot of health experts around the world as it was important that the chain of physical interaction between humans must be broken. the major step in this direction was to announce lockdown at national level. indian government took this suggestion seriously and started with the one-day implementation of lockdown on 22 nd march 2020 and then announcing a 21-day lockdown till 14 th april 2020. it was welcomed by a lot of citizens in india. the same was suggested by google through its mobility report as shown in figure 5 . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint as per the google's method of calculation, every day changes for the number of visits and length of stay at different places were compared the baseline values. the baseline value was considered as the median value for the corresponding day of the week. there were six different categories which were considered important for social distancing and there was a significant drop down seen in five of these categories. graph shows a 77% decline as compared to the baseline in the visits at retail and recreation places like restaurants, cafes, shopping centers, theme parks, movie theatres, etc.; 65% decline as compared to the baseline in the visits . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 11 to the grocery & pharmacy places like markets, food shops, farmer places, drug stores, pharmacies, etc.; 57% decline as compared to the baseline in the visits to the parks like public beaches, dog parks, marinas, national gardens, public gardens, etc.; 71% decline as compared to the baseline in the transit stations like bus stations, public transports, metro stations, train stations, etc.; and 47% decline as compared to the baseline at the workplaces like private and government offices. it was seen that a 22% increase as compared to the baseline was observed at the residential places which implies that more indians are now staying at their homes. all these numbers cannot be 100% as a lot of frontline health workers, administrators, pharmacists, grocery store and people involved in other essential services were on duty during the lockdown as well. however, the basic purpose of lockdown to maintain social distancing has been achieved to good potential. citizens are spending more time at their homes as compared to crowded places. so it can be said that indian citizens have been able to follow the lockdown effectively. during the lockdown period, there were two mass events reported in india. one was related to the exodus of the laborers to their respective states in india [23] and the other was a religious event which happened in new delhi at a mass level [24] . there are around 25% citizens living below poverty line and have to depend on the daily wages to feed their families. once the lockdown was announced, the fate of these 1.3 billion people was under scanner and that is why even the government of india came up with a package of more than 22 billion usd to help these workers and laborers. all the respective state governments also came up with the different infrastructural setup for providing food and money to the needy citizens. some agencies reported that in providing food security, india and the neighboring nations depending on india may fall short of food [25] while the others reported loss of millions of job [26] during the lockdown due to such mass exodus. however, the numbers of infected cases were not impacted much by this mass movement as majority of workers were not carrying any infection with them during their movements from workplaces to their native places. as seen from figure 6 , the average number of days to double the infected cases from corona virus without any cluster event was estimated to be 7.1 as per the health ministry, while it is 4.1 after delhi's religious event took place [27] . this event resulted in formation of clusters in the whole country as people who attended this event went to different parts of the country without following any rules of getting quarantine. some of them even came from out of india 12 to attend this event and no rules were followed by them regarding covid-19 protection. this resulted in a sharp spike in the number of infected cases after 31 st march 2020. as per the figure 7 , at least 36% of the total infected cases reported on 6 th april 2020 were linked to the religious event in delhi [28] . out of over 4000 cases reported, a minimum of 1445 cases were suspected to be due to the religious event in delhi. this event took the graph sky high in a week's time with growth slowly moving towards exponential path and india entering into the third phase of community transmission for covid-19 virus. many regions of india which were totally isolated with the infection of corona virus, also reported their first case in the first 3-4 days of the conduction of the event. therefore, it can be said that religious event that happened in delhi has really pushed the bars higher for infected cases from coronavirus in india. it has far more serious consequences than it seems right now and authorities needs to be really alert as people linked to this event have gone back to different parts of the nation. based on the data available from crowdsourced database at covid-19-india dot org [17] , network of the patients and their demographic details was created. the patient id, the countries of travel, and any mass events were considered as the nodes and connection between each patient and his travelling history or event attending history was considered as edge in the network. presenting the visualization of the network consisting of 551 nodes was out of scope for this paper; however degree centrality of important nodes was calculated and is presented in table 2 . degree centrality of the nodes is calculated as the number of connections with that particular node divided by the total number of edges present in the network. the top 7 nodes based on their nodes centralities were religious event in delhi, italy, gulf countries, united kingdom, mumbai, saudi arabia and the first patient. it was found that these were the major hotspots responsible for the rapid spread of coronavirus in india. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint based on the patient database, classification model was developed to check that on the basis of the demographic features, can a patient have the probability to be deceased. other patterns were mined for the rules for covid-19 patients; however rules were not significant enough for the positive cases as data for negative cases was not available. based on the age group of covid-19 infected patients, it was found that majority cases are in the age group of 31-40 years in india, as shown in figure 8 . the deceased patients are majorly above 60 years of age group and majority people who are infected either travelled back from italy or attended the religious event in delhi. [17] there was a classification model developed around patients who lost their lives after getting infected from the coronavirus. decision tree classification model [29] was used to classify that out of the given covid-19 infected patients, whether a patient will be deceased or not. three features were found to be significant which were the age of the patient, gender of the patient, and the state/region of the patient. although a total of 5000 patients have been infected from covid-19 virus so far, but demographic details of all the patients is not known. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 14 there were 912 living patients and 129 dead patients modelled on the dataset. out of 129, only 24 patients had complete demographic details available for the modelling, however for rest of the patients, relevant data imputation techniques [30] were used and missing values were filled up scientifically. accuracy and precision values were computed based on the confusion matrix generated from decision tree classifier model. the model generated a 60% accuracy as the data points were too less for the model to be trained and tested. moreover, the three-variable model was not sufficient enough to make an significant relevance out of the model. however, the deaths have found to be more prevalent in the age group of 60 or more years and males were more prominent to getting deceased. also, infants were least impacted by coronavirus in terms of life and death. the state of maharashtra and madhya pradesh were found to be the significant regions for the dead cases. not major inferences can be drawn from the classification model as numbers of cases were low, but as per the records of deceased patients, a hint can be taken by the medical and administrative authorities as in which type of patients need extra critical care. considering the numbers in previous sections, there are five major reasons due to which the national lockdown will be difficult to be abolished. firstly, the growth rate of infected cases is continuously increasing. secondly, the doubling time for number of infected cases in india is declining very fast making it a dynamic spread. thirdly, majority of the indians are following the social distancing fairly well with mobility rates going down for public places in india. this is due to the combined efforts of the administrative authorities, engagement activities by the office of prime minister and citizen's will to stay away from social events. fourthly, the sudden mass events are ruining the efforts of the indian authorities to contain the spread of coronavirus. and lastly, the penetration of the people infected from religious event has been really deep in india currently. there is a lot of tension created in india due to spread of infected people and the numbers are rising with each day. thus, it is very difficult for india to completely ban the national lockdown after 14 th april. in extreme conditions, the state wise rate of infection spread needs to be considered and then decision over lifting the lockdown may be taken. table 3 shows the current number of infected cases in different states of india and the associated growth rates for the last 7 days for each state, respectively [17] . it can be seen that majority of the states have witnessed more than 200% of growth rate in last one week, which is worrisome situation for all the state authorities. in fact, the national growth has also been towards 180% mark which is very in the last seven days. the administrators for the regions with less than 50 infected cases can consider the uplifting of ban on lockdown in their respective regions, however uplifting national lockdown looks difficult. the social distancing must be followed for next few weeks so that curve flattening process can continue for some more time. with respect to transportation services, only intra-state travel may be allowed in states where population density is less and reported cases are less than 50 right now. no inter-state domestic travel should be allowed for the time being as it may result in transferring the covid-19 carriers from one region to another region. international travel flights must not be activated as the number of cases in majority of the regions of the world is very high. european and us region are already facing most of the turmoil, while other parts of the world are seeing rapid rise in number of infected cases. starting inbound international flights to india may disturb the covid-19 action plan in india. only essential services should be open for the citizens of india and lockdown should be carried on for next 2-4 weeks. this study presented a comprehensive analysis of the covid-19 outbreak situation in india. the cases are rising very fast and they need aggressive control strategies from the administrative unit of india. there are six different aspects covered up in this study and six research questions have been answered comprehensively. they are related to presenting the growth trends of infected cases in india, predictions for the number of infected cases for next few days, impact of social distancing on the citizens of india, impact of mass events on the number of infected cases in india, network analysis and mining of patterns on the patients suffering from coronavirus, and analyzing the strategies for uplifting lockdown in india. the current study implemented various techniques to present the analysis and the results are in sync with few limited studies available in the literature. this study will be useful for the govern-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20058347 doi: medrxiv preprint 16 ment of india and various states of india, administrative units of india, frontline health workforce of india, researchers and scientists. this study will also be favorable for the administrative units of other countries to consider various aspects related to the control of covid-19 outspread in their respective regions. coronavirus disease (covid-19) pandemic, who novel coronavirus (covid-19) cases, provided by jhu csse india's swiftness in dealing with covid-19 will decide the world's future, says who, quartz india india is now the world's 5th largest economy, world economic forum trend analysis and forecasting of covid-19 outbreak in india seir and regression model based covid-19 outbreak predictions in india. medrxiv age-structured impact of social distancing on the covid-19 epidemic in india network structure of covid-19 spread and the lacuna in india's testing strategy. available at ssrn 3558548 covid-19: how doctors and healthcare systems are tackling coronavirus worldwide a review of coronavirus disease-2019 (covid-19). the indian journal of pediatrics world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) early dynamics of transmission and control of covid-19: a mathematical modelling study. the lancet infectious diseases the effect of travel restrictions on the spread of the 2019 novel coronavirus real-time forecasts of the covid-19 epidemic in china from critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response risk assessment of novel coronavirus covid-19 outbreaks outside india covid-19 tracker 2020 doubling time tells how effective covid-19 prevention works see how your community is moving around differently due to covid-19 india covid-19 tracker 2020 centrality and network flow visual analysis of network centralities india: coronavirus lockdown sees exodus from cities, aljazzera news channel coronavirus: search for hundreds of people after delhi prayer meeting, bbc news will coronavirus lockdown cause food shortages in india? bbc news india lost more jobs due to coronavirus lockdown than us did during depression, the print 4.1 days doubling time for coronavirus worrisome, deccan chronicle coronavirus: 1,445 cases liked to tablighi jamaat event, total crosses 4,000, india today news a new decision-tree classification algorithm for machine learning a comparison of imputation techniques for handling missing data key: cord-283294-fvhq8yud authors: skalet, alison h.; allen, richard c.; shields, carol l.; wilson, matthew w.; mruthyunjaya, prithvi; gombos, dan s. title: considerations for the management and triage of ocular oncology cases during the covid-19 pandemic date: 2020-04-21 journal: ocul oncol pathol doi: 10.1159/000507734 sha: doc_id: 283294 cord_uid: fvhq8yud nan dear editor, sars-cov-2, the virus that causes covid-19, is a novel pathogen. it is spreading rapidly in the usa, and our understanding of its behavior, transmissibility, and best practices to reduce risk for spread remain limited. rationing of resources is already occurring in response to the covid-19 pandemic. the purpose of this document is to lay a basic framework for ocular oncology care during the current pandemic. however, it is important to recognize the complexity of ocular oncology care and the need for flexibility within any guidelines. exceptions will occur. patients will need to be evaluated on a case-by-case basis. treatment of ocular and adnexal malignancies is not considered elective. to the extent possible, ocular oncology surgical cases for malignant tumors and some visionthreatening benign tumors should proceed during the current covid-19 pandemic. it is important to recognize that there is risk for transmission of sars-cov-2 during patient care, even by asymptomatic individuals. therefore, risk to patients, physicians and staff, and the community at large, must be balanced with the necessity for urgent care. in some cases, the balance may shift to delayed care being more appropriate for safety and conservation of limited resources. clinicians faced with making these decisions should consider the severity of a negative outcome (death, loss of an eye, loss of vision) together with the probability of such an outcome. in ocular oncology, particularly with regard to retinoblastoma screening, events may be quite rare but consequences of delayed care devastating. the balance during a pandemic should favor the value of maximizing benefits to the population [1] . the availability of local resources and perceived risk for covid-19 exposure asso-mruthyunjaya/gombos ocul oncol pathol 2 doi: 10.1159/000507734 ciated with care given a region's burden of disease may also factor into decision-making. these decisions will not be simple, and in some cases, consultation with medical ethics may help with medical decision-making. in addition to the need to reduce the risk for virus transmission and preserve personal protective equipment (ppe), for ocular oncologists there may be competing obligations between patient survival, globe salvage, and vision. at this time, there is no recommendation to alter treatment algorithms to favor enucleation over globe salvage; however, patient survival must be prioritized. informed consent to include covid-19 risk is recommended. as the pandemic worsens, it is likely that prioritization of only the most urgent and emergent procedures may be necessary. there is precedent for such prioritization of surgical oncology cases by the american college of surgeons [2] . in these situations, malignant tumors are prioritized over benign tumors, and the highergrade malignancies expected to more immediately lead to death and/or permanent disability are prioritized over less aggressive malignancies. lower risk and benign case may be amenable to deferred care via telemedicine. multidisciplinary discussions for urgent and semiurgent cases may facilitate institutional case allocation by or committees. in some cases, ophthalmic pathology resources may also be limited. telepathology may be an option in these circumstances. in march 2020, the authors communicated with numerous experts within the usa as well as internationally, in both pandemic hotspots and in regions less impacted, to assist in creating this document. separately, multiple formal surveys were initiated to gauge current covidassociated practice patterns that reflect variations in practice focus and location. preliminary results from one such study, assessing practice patterns among north american ocular oncologist members of the collaborative ocular oncology group, have recently been made available online [3] . strong pre-existing research networks and subspecialty societies have aided ocular oncologists in the rapid conduct of research, an often overlooked but critically important component of the public health response. the results of these studies will assist in the development of any future guidelines for ocular oncology care during a similar public health emergency. four levels of urgency in ocular oncology have been established: emergent, urgent, semiurgent, and nonurgent. − emergent cases should be performed within 24 h or as soon as possible to preserve life and/or sight − urgent cases should be performed within the week, considering the availability of resources − semiurgent cases should be performed within 1-2 months, considering the availability of resources. it is important to note that while retinoblastoma care does not fall into an urgent category due to optimal timing for serial interventions, the continuation of retinoblastoma care including examinations under anesthesia is a critical need and should be prioritized − nonurgent cases should be deferred for at least 2-3 months or until improved availability of local and national operating room resources some surgical cases are believed to carry a higher risk for transmission of sars-cov-2 [4, 5] . in ocular oncology, these higher-risk procedures are primarily in the oculoplastics domain and are indicated with an asterisk below. special precautions, including presurgical covid-19 testing and use of full ppe, should be considered when performing these surgeries in which aerosolization of virus may be more likely to occur. for these procedures, if covid testing is positive or unavailable and the case cannot be deferred, full ppe including powered air-purifying respirator is strongly recommended. the following procedures are considered emergent: − orbital biopsy for malignancy in a child (suspected rhabdomyosarcoma) − enucleation for intractable glaucoma/globe perforation from intraocular tumor (retinoblastoma, uveal melanoma) the following procedures are considered urgent: − examination under anesthesia for newly suspected retinoblastoma − enucleation for retinoblastoma − orbital biopsy for processes causing optic neuropathy and vision loss − * orbital decompression for impending visual loss (optic neuropathy or corneal perforation) secondary to orbital tumor the following procedures are considered semiurgent: real chemotherapy, plaque radiotherapy, cryotherapy, transpupillary thermotherapy, laser photocoagulation) must continue on necessary schedule to control disease, typically every 3-4 weeks − examination under anesthesia for retinoblastoma evaluation for patients with stable disease, who have received treatment within the past 6 months − examination under anesthesia for children at high risk for retinoblastoma due to family history or known rb1 mutation − intraocular injection of chemotherapy agents for highgrade neoplasia − biopsy of suspected intraocular malignancy (fine-needle aspiration biopsy or other) − excision/drainage of iris cyst with pain or glaucoma orbital tumor − biopsy of suspected orbital malignancy (case by caserapidly growing tumor may need urgent biopsy; slowly growing suspected lymphoma is semiurgent) − biopsy of suspected orbital lymphoma (extended delay may be appropriate under certain circumstances) − * exenteration (case by case: rapidly growing tumor may need urgent biopsy; a slowly growing one could be semiurgent) the following procedures are considered nonurgent: fair allocation of scarce medical resources in the time of covid-19 american college of surgeons. covid-19 guidelines for triage of cancer surgery patients covid-19 pandemic: ocular tumor triage and careeyenet precautions for endoscopic transnasal skull base surgery during the covid-19 pandemic academy supports cms, offers specific nasal policy the authors wish to thank their colleagues from throughout the usa and around the world for their personal communications and contributions. they thank dr. robert macauley for discussions pertinent to the medical ethics considerations. the authors have no proprietary or commercial interest in the materials discussed in this article. dr. alison skalet is a consultant for castle biosciences inc. and immuncore inc. this work was supported by grant p30 ey010572 from the national institutes of health (bethesda, md, usa) and by unrestricted departmental funding from research to prevent blindness (new york, ny, usa). a.h.s. and d.s.g. conceived the presented idea. a.h.s. and r.c.a. wrote the manuscript with input from all authors. a.h.s., r.c.a., c.l.s., m.w.w., p.m., and d.s.g. contributed substantively to the final paper. key: cord-290206-fmy4zrim authors: lim, jue tao; dickens, borame l; cook, alex r; khoo, ai leng; dan, yock young; fisher, dale andrew; tambyah, paul anantharajah; chai, louis yi ann title: the costs of an expanded screening criteria for covid-19: a modelling study date: 2020-08-12 journal: int j infect dis doi: 10.1016/j.ijid.2020.08.025 sha: doc_id: 290206 cord_uid: fmy4zrim objectives: nosocomial infection is an ongoing concern in the covid-19 outbreak. the effective screening of suspected cases in the healthcare setting is therefore necessary, enabling the early identification and prompt isolation of cases for epidemic containment. we aimed to assess the cost and health outcomes of an extended screening strategy, implemented in singapore on 07 february 2020, which maximizes case identification in the public healthcare system. methods: we explored the effects of the expanded screening criteria which allows clinicians to isolate and investigate patients presenting with undifferentiated fever or respiratory symptoms or chest x-ray abnormalities. we formulated a cost appraisal framework which evaluated the treatment costs averted from the prevention of secondary transmission in the hospital setting, as determined by a branching process infection model, and compared these to the costs of the additional testing required to meet the criteria. results: in the base case analysis, an [formula: see text] of 2.5 and incubation period of 4 days, an estimated 239 (95% ci: 201-287) cases could be averted over 150 days within the hospital setting through esc. a corresponding $2.36 (2 – 2.85) million usd costs could be averted with net cost savings of $124 000 (95% ci: -334 000–516 000). in the sensitivity analyses, when positive identification rates (pir) were above 7%, regardless of [formula: see text] and incubation period, all scenarios were cost-saving. conclusion: the expanded screening criteria can help to identify and promptly isolate positive covid cases in a cost-saving manner or within acceptable cost margins where the costs incurred from the testing of negative patients could be negated by the averted costs. outbreak control must be sustainable and effective; the proposed screening criteria should be considered to mitigate nosocomial transmission risk within healthcare facilities. since its emergence in wuhan, hubei province, china, the coronavirus disease 2019 has continued to spread with an escalating numbers of cases and countries affected. 1 to prevent ongoing transmission, effective screening of suspected cases is necessary which enables the early identification and prompt isolation of cases for epidemic containment. these screening measures for testing have been largely guided by the world health organization's (who) case definitions, 2 which serves as a valuable epidemiologic tool for public health officials to track the disease and is regularly updated with the changing epidemiology and demographics of the disease. this definition includes the presentation triad of fever, chest symptoms as well as a positive contact with an active infection or travel to an area with confirmed local transmission. covid-19 shares many clinical similarities to other respiratory and febrile illnesses, creating significant uncertainty at the point of testing as to whether cases should be treated as a potential positive infection. although symptom case reports have emerged over the ongoing outbreak, [3] [4] [5] the case definition is dynamic, being continually updated using clinician feedback through research or policymakers who collate and publish. both pathways however have an inevitable time delay in information dissemination. this affects clinicians and healthcare workers (hcws) at the front line who rely on the case definition to screen and triage patients, where this epidemiologic case definition lag can lead to problems of case misclassification. the screening process should therefore maximise sensitivity to minimise the number of missed cases and risk of nosocomial transmission, which occurred in singapore during another coronavirus outbreak of severe acute respiratory syndrome (sars) in 2003. 6 during this outbreak, frequent inadequately protected patient-to-hcw interactions among untested and unidentified cases 6, 7 led to 97 hcws becoming infected. 8 due to the presence of nosocomial transmission, clinicians at the national university hospital, singapore (nuhs) quickly responded with a revised testing protocol. they screened patients with a substantial expansion of the admission and isolation criteria to quickly identify and isolate infected individuals whose symptom profiles were too mild to be considered for testing 9, 10 or for patients who sought medical attention with atypical disease presentations, especially in the pediatric or geriatric population. 11 the implementation of this expanded screening criteria (esc) placed all patients with undifferentiated fever or respiratory symptoms or chest x-ray abnormalities in isolation until all symptoms and fever were resolved or an alternative diagnosis was proven, regardless of travel history or confirmed previous exposure. using esc, nuh clinicians were able to identify and isolate 13 patients who did not fit the who sars criteria but were eventually confirmed to have sars, who would have otherwise been potential spreaders (the number of positive and negative cases for sars are presented in supplementary table 1.) 12 clinicians responded to the covid-19 crisis similarly, implementing esc on 7 th february 2020, which was approximately three weeks after the first imported case. with widespread concerns raised regarding the low detection rate of infected travellers where most present j o u r n a l p r e -p r o o f mild symptoms (12.1% with no fever, 67.7% with cough) 13 and high asymptomatic rates 14 an estimated 1.8% ascertainment rate in wuhan. 15 a wider screening policy for covid-19 and future novel respiratory viruses requires assessment in terms of its efficacy and costs. the clinical management and costs of a large proportion of negative outcomes comes at the expense of resources which can be invested in other parts of the healthcare system. the efficacy of the esc in long term should thus be assessed in terms of its prevention of nosocomial transmission which may not only negate these costs but also save resources for the healthcare system. this paper therefore assesses the practicality of esc through a cost-effectiveness analysis by estimating the number of cases that can be averted through esc, and calculating the associated costs from the testing of negatives and cost savings from potentially averted cases. the first case definition used by the ministry of health, singapore for the testing of suspect covid-19 cases was based on who and other international bodies recommendations. 16 this centered on the presence of fever, respiratory illness or pneumonia, and recent travel to wuhan, china (namely, regions with reported infections) or close contact with a case of covid-19 patient 17 (table 1 ). recognizing the limitations of these case definitions and extending from our experience from sars, 7,12 we proposed an expanded screening criteria (esc) simplified for execution at the hospital triage, which was then implemented on 7 february 2020 with immediate effect. any patients with undifferentiated fever or respiratory symptoms or chest x-ray abnormalities are placed into isolation on admission with respiratory precautions, investigated until symptoms and fever resolve or an alternative diagnosis was determined (table 1 ) we used our institution in singapore, a 1,000-bed tertiary hospital, as a study example. 18 as of 8 february 2020 to 21 february 2020, 14 cases have been identified through esc which would have otherwise been missed. we determined the total number of infected cases that could occur from each non-identified admitted covid-19 cases in a hospital setting using a branching process infection model (figure 1 ), which estimates the number of new infections at each generation. each day, a set of imported cases is designed to be able to start multiple generations of transmission. the model characterizes this influx of cases into the hospital and subsequent infections that may occur. the baseline generation represents the first covid-19 positive case which is not deemed to be a suspected case under the non-esc criteria (who criteria until 31 january 2020 or ministry of health criteria until 25 january 2020) but would otherwise have been isolated under esc. under non-esc screening, the daily influx of symptomatic and positive cases is parameterised by a binomial distribution with a probability determined by the positive identification rate (pir), otherwise referred to as the proportion of confirmed symptomatic cases observed out of the total number of observed respiratory illness cases . all symptomatic and asymptomatic cases are assumed to be able to transmit the infection due to the closed environment within the hospital setting so are not distinguished. the number of importations in the hospital facility is therefore, the number of infections incurred from these daily incoming non-isolated cases is determined by a poisson distribution with a rate of infection λ, and the incubation period length which determines the infectiousness period . therefore the total number of infections at time , to time and incubation period , is defined as, where , denotes the size of the generation descending from ancestor , and determined by, after each newly infected person's incubation period, the individual is assumed to be in the process of being identified and have a decreasing rate of infectiousness of 10% until their end point at 20 days. after the period + 1, an individual's infectiousness is consequently greatly reduced, where a person is expected to have follow up with the development of new symptoms or unexpected ongoing symptoms from the new infection of covid-19 and is consequently isolated, or if asymptomatic, is discharged. therefore, is updated to, for the main analysis, we ran 50 simulations with a set of parameters taken from literature; a rate of infection ( ) of 2.5 and incubation period (ip) of 4 days, 19 and pir rate 8.0% which is based on the upper bound for case identification in the sars epidemic in 2003. [20] [21] [22] [23] [24] [25] the full analysis was run for different parameter distributions (table 2) . for each independent combination, 50 simulations were run where the incubation period was varied between 4 to 8 days, the rate of infection λ from 1.5-3.5 increments, and the rate of influx from 1.0% to 30.0% in 1.0% increments based on a range of positive identification rates (pirs) from literature on covid-19, cov-sars and cov-middle east respiratory syndrome. [20] [21] [22] [23] [24] [25] all simulations were run across 150 days (approximately 5 months) and performed in r version 3.6.3. after determining the number of new cases occurring within healthcare facilities, we assessed the healthcare system costs and outcomes of implementing the proposed esc and followed the consolidated health economic evaluation reporting standards (cheers). the cost framework examined the impact of the esc on hospitalization and testing costs while considering the transmission dynamics and importation rates of covid-19 cases in a hospital setting. a constant daily influx of 84 patients was assumed across 150 days based on 1182 general ward patients being admitted across 14 days (8 february 2020 to 21 february 2020, supplementary table 2 ). the total number of patients being admitted to the isolation wards for undifferentiated fever or respiratory infection or an abnormal chest x-ray according to the esc was taken to be 14.7% (174 isolated out of 1182 admitted patients, supplementary table 2) . a proportion of these, according to the pir, will be covid-19 positive. all costs presented are in us dollars with no discounting for the year 2020. the net cost of esc is calculated as the total hospitalization costs for the isolation of negative patients subtracted from the total hospitalization costs averted from the isolation of positive cases which would have otherwise been missed by a non esc criteria. for the former, the total hospitalisation cost for the isolation of negative cases was the total number of negative diagnoses at the cost of $1201 each, as all patients receive 2 coronavirus polymerase chain reaction (pcr) tests and a respiratory isolation room cost (supplementary table 3 ). this represents the costs to the healthcare system which should be minimised as they require no further isolation measures. for the latter, the total costs averted from the isolation of positive cases under the esc was determined by the branching process model with an associated testing and treatment cost each. these covid-19 positive patients inherit the same initial costs as negative patients at the testing phase and have subsequent treatment costs depending on whether they require intensive care unit (icu) stay with a probability of 26.6% based on patient outcome profiles from literature. 4 table 4 ). patients who required icu received an additional cost for 7 days of intensive care management, followed by the standard respiratory isolation procedures for a non-icu patient at a total of $12 658 (supplementary table 5 ). the sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. at an of 2.5 and incubation period (ip) of 4 days, approximate to estimates by wu and colleagues 29 for wuhan, an estimated 240 (95% ci: 200-290) cases would be prevented over 150 days through esc by isolating patients presenting fever or respiratory symptoms such as cough with their travel and exposure profile not considered (table 2) . with an observation of 84 patients being admitted per day into hospital and a corresponding total of 12 600 across the 150-day period (supplementary table 2 ), 1900 patients would be expected to enter respiratory isolation of which 200 (95% ci: 170-230) to 300 (95% ci: 250-370) would be covid-19 positive, depending on the ( table 2) . a total cost between $2.0 million (95% ci: 1.7-2.3 million) to $3.0 million (95% ci: 2.5-3.7 million) could be potentially averted from the isolation of these patients. a corresponding cost saving at an of 2.5 or greater can be achieved with $120 000 (95% ci: -330 000-520 000) to $740 000 (95% ci: 120 000 -1 400 000) saved. of note, if the proportion of positive cases is taken to be 8.0%, which represents the upper bound of sars patient identification during the testing period for the 2003 epidemic, 20-25 the total cost of testing for negative patients is $1.9 million at this pir. figure 3a) . a higher causes esc to yield larger cost savings with this difference in cost savings being more pronounced at higher pir values. at a pir of 1.0%, having an of 1.5 versus 3.5 increases the cost savings by $153 000 with 14 more infections averted ( figure 3b ) but at a pir of 30.0%, this difference widens to $5.0 million ( figure 3a ) with 440 more infections averted ( figure 3b ). however, provided that the pir is above 8.0%, regardless of , cost savings can be achieved through esc implementation. notably, less than a 3.0% difference was recorded in net cost savings across the of 1−3.5 at a pir of 1.0%, with the largest change being a -$49 000 decrease in net cost savings at the of 3.5. across all and pir values, changing the ip from 4 to 8 days decreased the net cost savings of esc by an average of -$52 000. j o u r n a l p r e -p r o o f discussion among the lessons learnt during the sars epidemic of 2003-2004 was the importance of adapting official case definitions to provide effective triage and screening, 7 especially as the definitions are expected to rapidly evolve for a new disease. for the current covid-19 outbreak, all early diagnosed patients had travelled from china but by the 4 th feb 2020, suspected local transmission was evident therefore negating the usefulness of this travel history 30 . furthermore, as symptom profiles were identified as being very heterogeneous with the two dominant symptoms fever and cough at 88.7% and 67.8% prevalence among 1099 patients in mainland china, 5 the expanded screening criteria (esc) was designed to capture all of these patients to reduce the risk of nosocomial transmission. as of 7 february 2020, esc was implemented as an emergency response to operate alongside intense contract tracing. 31 the results demonstrate that provided the positive identification rate (pir) is sufficiently high at 7% or greater, costs-savings can be created across the values 1.0−3.5, making the screening strategy sustainable for the healthcare system in the long term. at lower pir vales and low values, the strategy should still be considered by policymakers during the initial part of the epidemic to ensure the majority if not all cases are captured at healthcare facilities. nosocomial transmission should also be prevented through the isolation of these cases at the point of admission during testing with the costs accounted for. the prevention of 200-300 covid-19 infections in a hospital at an of 2.5 and incubation period (ip) of 4 days can also prevent transmission within the community and relieve the need for ppe resources which are already under strain. 32 the branching process shows the rapidity of infection spread and highlights the urgency of enhancing hospital surveillance to detect any potential transmission chains when a case is identified, which is already carried out for all atypical pneumonias, some upper respiratory illnesses and hospitalized acute respiratory illnesses. 31 our findings are relevant to cities and highly urbanized regions with well-developed healthcare infrastructure and resources to undertake considerable epidemiological investigation for suspected cases. the costs for areas with fewer resources or different health systems however are likely to be considerably lower but potentially at the cost of a lower pir. a total of 40 000 cases have been confirmed in singapore to date (25th march 2020; a full case listing is provided by gov.sg 33 ) with limited local transmission currently suspected. elsewhere, ~8 million cases and over 435 000 deaths have been confirmed across 6 who regions. 34 the analysis performed here can be utilised for all countries regardless of outbreak stage as it can prevent nosocomial transmission at hospitals with incoming cases of local transmission and prevent imported cases from establishing transmission chains altogether. it also supports the general body of literature highlighting the importance of a highly sensitive standardised criteria for case identification in an epidemic involving a new infectious pathogen. for covid-19, this has been particularly challenging as it shares symptoms with many respiratory illnesses, making it difficult for practitioners to distinguish positive cases. similar issues already exist for influenza between national and regional surveillance programmes where most use measured or reported fever with cough and/or sore throat. 35 during the initial wave of the 2009 influenza a h1n1 pandemic in singapore, serological surveillance on 727 patients from june to october 2009 demonstrated that the revised who definition had the highest reported positive predictive value (ppv) in comparison to three others. 36 this variety in case definition across institutions demonstrates the ongoing challenges in place for both diagnosis and surveillance, which can be difficult for practitioners to interpret and implement. for covid-19, complete case finding is being prioritised where possible in singapore as ppv will substantially change with increasing disease prevalence. provided the case definition is highly sensitive, it can continue to be rapidly updated with greater specificity over time as symptom profile data continues to be shared and assessments carried out on missed cases, therefore sensitivity at the initial phases should be greatly prioritised. the model can be used by policy makers to estimate the costs saved using esc within the hospital setting for the ongoing covid-19 pandemic. esc is cost-effective in the long term, especially at higher 0 and pir values, preventing undiagnosed cases from infecting other individuals, which is especially important during the initial outbreak phase or where the epidemic is suppressed from lockdown measures. it should be noted however that there are several limitations in our analysis, primarily due to the ongoing uncertainties regarding the parameters of covid-19 infection profile and differing operational procedures in hospitals. firstly, the pir is currently unknown and the is likely to vary considerably in space and time across singapore. infectiousness will also vary between hcws and patients which may not be fully explained by the branching process. second, the asymptomatic rate has yet to be determined as part of the continually evolving symptomatic profile. 37, 38 third, we only considered the hospital costs for the institution and did not account for the productivity and economic losses from unnecessary isolation or those saved from averting of nosocomial cases. this also includes the excess manpower costs from hcws who are currently working long hours to ensure patient wellbeing. fourth, revisions may be required to make these findings applicable to other countries or cities through adaption of the isolation and testing procedures. lastly, the potential for superspreading events, as witnessed in sars, 39 has not been accounted for as the movement patterns of hcw were not modelled. esc should be implemented in the early phase of an epidemic where local community transmission is suspected or where imported cases are being repeatedly recorded. cost-savings or relatively minor costs are expected to occur whilst reducing the risk of uncontrolled outbreaks occurring in nosocomial settings. this is critical considering the ongoing anxiety and strain on healthcare systems worldwide, which are reflected within the public and impossible to fully quantify 40, 41 . measures such as esc, which can aid national control efforts, should be considered in order to relieve this pressure through the exhaustive testing and isolation of individuals which have good potential to be positive. 18 contributors jtl, bld, lyac designed the experiments, jtl, bld, arc created the models, bld, jtl, lyac, arc, alk, yyd, daf, pat interpreted the results, bld, jtl, lyac, arc wrote the manuscript, alk and lyac performed data collection for the models. singapore population health improvement centre (nmrc/cg/c026/2017_nuhs) to bld and arc. clinician scientist award (csa) funded by the national medical research council to lyac. ethical approval was not required incubation period 4-8 days lauer 26 linton 27 [20] [21] [22] [23] [24] [25] all costs are rounded to the nearest ten is greater than 10, nearest hundred if greater than 100, thousand if greater than 10 000, and hundred thousand if greater than a million. costs are also in usd. an interactive web-based dashboard to track covid-19 in real time surveillance case definitions for human infection with novel coronavirus clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of coronavirus disease 2019 in china sars 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infections transmission characteristics of mers and sars in the healthcare setting: a comparative study a comparative study of clinical features and outcomes in young and older adults with severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia comparative study of patients with and without sars who fulfilled the who sars case definition the incubation period of covid-19 from publicly reported confirmed cases | annals of internal medicine | american college of physicians incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study -the lancet nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study highlights: confirmed cases of local transmission of novel coronavirus infection in singapore investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures rational use of personal protective equipment (ppe) for coronavirus disease (covid-19) world health organisation revision of clinical case definitions: influenzalike illness and severe acute respiratory infection performance of case definitions for influenza surveillance eurosurveillance | pattern of early human-to-human transmission of wuhan 2019 novel coronavirus (2019-ncov) the rate of underascertainment of novel coronavirus (2019-ncov) infection: estimation using japanese passengers data on evacuation flights indirect health care costs. in: weintraub ws, ed. cardiovascular health care economics indirect costs and cost-effectiveness analysis we declare no competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.j o u r n a l p r e -p r o o f key: cord-279330-vy0ohgia authors: adamik, b.; bawiec, m.; bezborodov, v.; biecek, p.; bock, w.; bodych, m.; burgard, j. p.; krueger, t.; migalska, a.; ozanski, t.; pabjan, b.; rosinska, m.; sadkowska-todys, m.; sobczyk, p.; szczurek, e. title: estimation of the severeness rate, death rate, household attack rate and the total number of covid-19 cases based on 16 115 polish surveillance records date: 2020-11-03 journal: nan doi: 10.1101/2020.10.29.20222513 sha: doc_id: 279330 cord_uid: vy0ohgia background: estimating the actual number of covid-19 infections is crucial for steering through the covid-19 pandemic crisis. it is, however, notoriously difficult, as many cases have no or only mild symptoms. surveillance data for in-household secondary infections offers unbiased samples for covid-19 prevalence estimation. methods: we analyse 16115 polish surveillance records to obtain key figures of the covid-19 pandemic. we propose conservative upper and lower bound estimators for the number of sars-cov-2 infections. further, we estimate age-dependent bounds on the severe case rate, death rate, and the in-household attack rate. results: by maximum likelihood estimates, the total number of covid-19 cases in poland as of july 22nd, 2020, is at most around 13 times larger and at least 1.6 times larger than the recorded number. the lower bound on the severeness rate ranges between 0.2% for the 0-39 year-old to 5.7% for older than 80, while the upper bound is between 2.6% and 34.1%. the lower bound on the death rate is between 0.04% for the age group 40-59 to 1.34% for the oldest. overall, the severeness and death rates grow exponentially with age. the in-household attack ratio is 8.18% for the youngest group and 16.88% for the oldest. conclusions: the proposed approach derives highly relevant figures on the covid-19 pandemic from routine surveillance data, under assumption that household members of detected infected are tested and all severe cases are diagnosed. the covid-19 pandemic has led to dramatic changes in the everyday life worldwide. the most appropriate countermeasures are sought against a further spread. to this aim, key disease characteristics need to be described, such as the severeness and attack rates. mounting evidence indicates that many infected with sars-cov-2 show no or only mild symptoms and remain undetected. 1,2,3 the lack of accurate knowledge of the actual number of infected highly complicates epidemic control. 4 computational approaches to estimating both severity of covid-19 disease and the unknown total number of infected range from statistical and machine learning to model-based. 5,6,7,8,9,10,11 with more detailed data, these approaches gain more statistical power and need to make less educated guesses. the report by bi et al. proved contact tracing and surveillance data to be useful in characterizing epidemiology and transmission of sars-cov-2 in china. 5 to our knowledge, such detailed case data has not been reported in europe, and has not been used for the estimation of the total number of cases. here, we rely on detailed covid-19 surveillance data in poland, recording the source of transmission, household contacts and hospitalization status for 16 115 cases. these data allow to differentiate from the cases that were first detected in each household from the secondary cases that were infected in the household. while the former cases are often diagnosed based on their symptoms and are biased towards worse disease progression, the latter can be considered unbiased samples of the susceptible population. using this unbiased sample, we derive upper and lower bounds on the age dependent rates of severe progression, death rates and in-household attack rates. we show that the severeness rates grow exponentially with age. unlike easily obtainable case fatality rates, our bounds on death rates give insight into the actual lethality of covid-19. based on these estimates, we further provide bounds on the total number of sars-cov-2 cases. our results pave the way for utilizing surveillance data in the covid-19 spread for unbiased estimation of its key characteristics and the unknown total number of infections. in the following, we analyze covid-19 surveillance data, with each case assigned to a household. for each household, the first detected case in this household is referred to as an index case. the index cases may be subject to some bias, since they may be diagnosed due to the severity of their disease, different kinds of ex-ante social activity or other exposure factors. all remaining household members (all apart from the index cases) are referred to as the susceptibles. the number of susceptibles is equivalent to the sum of the household sizes reduced by the number of the index cases, and is denoted n * . in contrast to the index cases, it is plausible that the infections among the susceptibles are unbiased with regard to clinical progression, severeness rate and attack rate. those remaining infections in the household are called secondary household infections. let i * denote the number of the known (recorded in the data) secondary household infections. denote by t * the true total prevalence of secondary household infections (including undetected cases). let i * ,sev be the number of severe cases among these infections, and assume that all severe cases among the susceptibles are observed. there is a close relation between the rate of severe disease progression, household attack rate and the undiagnosed fraction of covid-19 infections, as they can all be estimated using the above-mentioned quantities. below, we first derive estimators for the bounds on covid-19 severeness and attack rates, as well as the total number of infected, without taking account for such factors as age or sex. next, we extend the derivation to consider these factors. we considered two alternative definitions of severe cases. first, we assumed severe cases are those hospitalized for 10 days and more or dead, and second we assumed they were hospitalized for 14 days and more or dead. finally, we describe how the susceptible population size can be estimated from external census data in the absence of records of household sizes for the index cases. given the true total number of secondary household infections t * and the number of severe secondary infections i * ,sev , the severeness rate is easily estimated as σ = i * ,sev t * . (1) i * ,sev can easily be obtained from the existing data records, since severe cases are likely found in a functioning healthcare system. contrarily, e.g. due to asymptomatic cases, t * may be unknown. we hence derive upper and lower bounds on the severeness rate, which are derivable from the observed 3 quantities. first, denote the observed severe case rate α by α := i * ,sev i * . (2) if all infected persons were diagnosed, i * = t * and α would be the true severe case rate among the infected. in common case, when only some of the infected are diagnosed, i * is the minimum of secondary infected in the observed households. hence, α defines an upper bound on the severe case rate. this rate is lower bounded by as n * is the maximum number of possible infected in the observed households. in section 2.3 we compute different bounds on severeness rates, separately for different values of other factors, such as age. finally, smooth estimates on the functional dependence of the severeness bounds on the age are obtained using restricted cubic splines. further details are depicted in section a of the supplementary online material (som). the exact same equations as for the bounds on the severeness rates can be used to estimate the death rates, but inserting for i sev the number of deaths instead of the number of severe cases. let t sev be the total number of severe cases in the entire population and t be the unknown true total number of sars-cov-2 cases. assuming that the severeness rate among secondary infected individuals, σ, is the same as it is in the entire infected population, we have from which we obtain the estimate of the total prevalence t . since σ is often not directly obtainable from the data, we can use it's lower bound β and upper bound α to derive the conservative maximum likelihood upper and lower bounds on t , respectively aŝ andt if the secondary infections in the households are diagnosed precisely, this lower bound (6) will be near the expected number of infected. a gap between officially recorded infections and the estimated lower bound could stem from a poor testing of secondary infections. if this can be ruled out, the gap indicates the expected minimum of additional undiagnosed infections. conservative one-sided 1% lower confidence bound on β (denoted β q ), and 99% upper confidence bound on α (denoted α q ), are derived using and extension of the clopper-pearson interval method 12 (som b). based on this, the one-sided 99% upper confidence interval bound for the upper bound of infected is given by while he one-sided 1% lower confidence interval bound for the lower bound estimator of infected is given by for a rapidly growing number of infections, which was not the case of the polish epidemic as of july 22nd, 2020, one would have to adjust for a time delay from infection to the recording of severe cases. in som c we describe how to correct for this time lag. 2.3 accounting for population strata to estimate of the total number of infections above we assumed that the rate of severe disease progression among infected individuals is the same. it is known, however, that this rate depends on factors like age, sex, and the comorbidity status. 9,13,14,15 when stratifying the population according to these factors, the between-class variance is removed from the total variance. hence, the estimate becomes more efficient. we thus adapt the approximation to account for these strata. again, we make use of the fact that the secondary infected in the households constitute a severeness-rate-unbiased sample. instead of calculating the rates α and β over all units in the sample, they are calculated in the classes known to affect the severity of infections. let us number the classes of all combination of age, sex, comorbidity values consecutively with l = 1, . . . , l . note that the resulting classes, as in anovas, have to be big enough such that some severe cases s * ,sev l in the l-th class are observed. the severe case rates in class l yield 5 . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint therefore, for obtaining an upper bound estimator of the upper bound of infected we sum up this figure over all classes. accordingly, the estimators t q post , t 1 2 post , and t 1 2 post can be obtained. in poland and other countries, neither the negative tests nor the household sizes are recorded. in that case it is necessary and possible to estimate n * based on external data. here, we obtain the household size distribution conditioned on demographic characteristics of the index cases from census data. via this information a distribution of n * is derived by bootstrapping, as described in som d. the bootstrap draw of a household size was conditioned on the age of an index case, minimal household size information and residing voyvodship. adjustment for the estimation of the confidence bound for the lower bound β on the severeness rate is described in som b. there are two natural ways to define the household attack rate. the first is a probability λ * of an infected household member to infect a non-infected household member. we refer to this quantity as the attack probability. λ * is estimated based on the secondary case data, taking into account the fact that the infections proceed consecutively within a household (som e). the second is the ratio of the expected total number of infections t * to the expected number of susceptibles n * , given λ * and the distribution of the household size (som e). we refer to this quantity as the attack ratio and denote it g(λ * ). arguably, in contrast to the attack probability, the attack ratio is not a medically-relevant intrinsic characteristic of the disease. it is, in particular, dependent on the household size distribution. thus, for example, for the same attack probability, the attack ratio would be different for countries with different typical household size. an estimator of the attack ratio is then given by: since the true number of secondary infections t * is unknown, as we instead compute the lower bound on 6 . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; the attack ratio as g(λ * ) = i * /n * . g(λ * ) depends on the probability λ * , which in turn constitutes a lower bound on λ * . since the upper and lower bounds on the severeness rates depend directly on the i * and n * , respectively, one can associate attack probability λ * directly with the upper bound α on the severeness rate, and an attack probability 1 with the lower bound β. furthermore the relation α · g (λ * ) = β between attack rate, rate of severe progression and the g -function holds. analog computations can be performed for age-dependent attack probability and ratio (som e). in these computations, we assume that the probability and ratio depend on the age of the susceptible (the person acquiring the disease). the analyzed data was collected as part of routine covid-19 surveillance in poland, which was implemented based on a data collection system functioning for other notifiable infections. the mandatory reporting was ordered both for clinical diagnoses of covid-19 and positive laboratory tests of sars-cov-2. the notifications were sent to the local public health departments, which were responsible for conducting epidemiological investigation, contact tracing and, if necessary, -ordering quarantine. according to the protocol, all quarantined cases were tested in case of symptoms. testing of all individuals in the quarantine was optionally applied. the results of the epidemiological investigations were documented in the epidemiological reports registration system (srwe). the data was to be updated once the case outcome was known. however, given the strain on the public health system, this information could be missing or delayed. the srwe database includes basic demographic and clinical information, exposure category, hospitalization history and use of mechanical ventilation and moreover detailed information on established links between cases. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.29.20222513 doi: medrxiv preprint the total number t sev of the severe cases the polish population was computed as the number of all such defined severe cases in the analyzed households (including the index cases), multiplied by a factor d/c , where d was defined as the total number of officially diagnosed cases in poland (as of july 22nd, 2020), and c the total number of all diagnosed in the analyzed database ( 41 162 16 115 = 2.554). the factor is intended to scale up the database level estimates to a national level of the entire polish population. we characterized a total of 16 123 covid-19 surveillance records (table 1) , out of which 11 895 (73.8 %) were the index cases and 4228 (26.2 %) were the secondary cases. the patients were divided into four age groups, including a group of 0-39 years old (39.8 % of all records). this wide age group was formed to reliably estimate per-group severe case rate, as there were no or only a few severe cases among children. the proportion of females was slightly larger (51.6%) than of males, and similar in both index cases (51.1%) and secondary cases (52.9%). the index cases can be regarded as detected based on their symptoms and the secondary cases as an severeness-rate-unbiased sample of the population. the index cases were more often hospitalized (with hospitalization rate 30.3 %) than the secondary cases (18.2 %). in addition a larger fraction of hospitalization for longer than 14 days is observed (12.7 % for index cases vs 7.2 % for secondary cases) . on 22/07/2020, the final outcomes were known for 6 360 out of 16 123 cases, with 488 deceased and 5872 recovered. again, for the index cases, the death fraction was larger than for the secondary cases. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; to estimate upper and lower bounds for the covid-19 severeness rate in poland, we focused on the secondary case data ( table 2 ). the expected number of susceptibles n * , i.e., the estimate for the number of all inhabitants of the analyzed households, except for the index cases, was computed using the polish census data (som d) as 39 102. first, we obtained the bounds on the severeness rate for the different age groups using the maximum likelihood estimators, in equation 3 for the lower bound, and in equation 2 for the upper bound, respectively. for the definition of severity, we used two alternative thresholds of hospitalization, 10 and 14 days respectively. for the 14 days threshold, the young population (0-39 year old) had the smallest lower bound on severe case rate (0.2%). the bound increases for older age groups, reaching 5.7% for the group older than 80. across the age groups, the upper bound on the severeness rate α is roughly an order of magnitude larger than the lower bound β and also increases for older age groups. next, we obtained smooth estimates of the severity rates and death rates among secondary cases as a function of the age together with the 98% bootstrap intervals (figure 1 ). the death rate is presented only for people over 60 years, because number of deceased among secondary cases among younger people was too small to get credible intervals. to get estimates at this resolution we estimated mortality with logistic regression model with age transformed with tail linear restricted cubic splines (som a). overall, severity and mortality depend exponentially on the age (note figure 1 is a semilog-plot). this finding is 9 . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.29.20222513 doi: medrxiv preprint in agreement with the fact that severe cases are more likely among the elderly patients. 16 the estimate for β is roughly 10x higher than the estimate for α. from the maximum likelihood estimate of the lower bound on the severe case rate β and from the number of severe cases t sev we obtain the maximum likelihood estimator for the upper bound of the total number of infectionst β (equation 5). we found an overall upper bound of 523 796 on covid-19 infections in poland (corresponding to 1.38% of the polish population), using a 10 days threshold for the severe cases (table 3) . this upper bound is around 14 times larger than the cumulative recorded number of cases. the 99 percentile of this upper bound estimator is 660 156 (corresponding to 1.74% of the population). using the 14 days threshold the upper bound is 585 147 (1.55%) with a 99 percentile of 779 118 (2.06%). the lower bound estimate for the total number of covid-19 cases in poland (t α ) is 64 595 using a 14 days threshold for the severe cases. this lower bound is 1.6 times larger than the recorded number of cases. the 1 percentile of the lower bound was equal to 50 901 and the 99-percentile was equal to 85 301. . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. figure 1 : partial dependence profiles for lower (beta) and upper (alpha) bound for the severity rate and death rate estimated with a linear tail-restricted cubic spline function. filled regions show 98% bootstrap intervals. the subscript stands for: 10 -severity calculated for 10 days, 14 -severity calculated for 14 days, d -death rate. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.29.20222513 doi: medrxiv preprint for this analysis, we split the before considered age group 0 − 39 into 0 − 19 and 20 − 39 and show that attack probability in the 0 − 19 age group was 0.057, around half as high as in the oldest two age groups (0.1273 for the 80+ group). the attack probability estimate for all secondary cases (age-independent) was λ * = 0.082. how significantly the data supports the hypothesis that the attack probability is age dependent is discussed in som e. the maximum likelihood estimate for the lower bound on the household attack ratio for the entire set of secondary cases was 11%, while the age dependent estimates ranged from 8.2% for the youngest to 16.9% for the oldest age group, respectively ( table 4 : age-dependent attack probability and